WO2021263194A1 - Inhibition of n-glycosylated grp94 - Google Patents

Inhibition of n-glycosylated grp94 Download PDF

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WO2021263194A1
WO2021263194A1 PCT/US2021/039230 US2021039230W WO2021263194A1 WO 2021263194 A1 WO2021263194 A1 WO 2021263194A1 US 2021039230 W US2021039230 W US 2021039230W WO 2021263194 A1 WO2021263194 A1 WO 2021263194A1
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grp94
cancer
substituted
halogen
aliphatic
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French (fr)
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Gabriela Chiosis
Pengrong YAN
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Memorial Sloan Kettering Cancer Center
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/40—Heterocyclic compounds containing purine ring systems with halogen atoms or perhalogeno-alkyl radicals directly attached in position 2 or 6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/26—Heterocyclic compounds containing purine ring systems with an oxygen, sulphur, or nitrogen atom directly attached in position 2 or 6, but not in both
    • C07D473/32—Nitrogen atom
    • C07D473/34—Nitrogen atom attached in position 6, e.g. adenine

Definitions

  • GRP94 Glucose regulated protein 94
  • GRP94 is one of four HSP90 paralogs and shares 50% amino acid homology with the cytosolic chaperone HSP90 (Marzec et al., 2012; McCaffrey and Braakman, 2016; Zhu and Lee, 2015).
  • GRP94 functions in ER quality control, buffers Ca 2 + levels, and is a key chaperone in the folding of “client” proteins.
  • TGF ⁇ associated protein GARP insulin like growth factors
  • TLRs Toll-like receptors
  • integrins integrins
  • GRP94 While primarily localized to the ER, GRP94 is also found in the cytosol, at the cell surface, and extracellularly (Ansa-Addo et al., 2016; Lee, 2014; Wiersma et al., 2015).
  • GRP94 maintains the stability of HER2 and its enhanced downstream signaling (Patel et al., 2013).
  • GRP94 hyperglycosylated GRP94 was also reported, but it is a non- functional form targeted for degradation in an OS-9–mediated, ERAD-independent, lysosomal- like mechanism (Cherepanova et al., 2019; Dersh et al., 2014).
  • stress is a common hallmark of disease, it is mostly studied as a damager of proteins and of their function (Solimini et al., 2007).
  • chaperones such as GRP94 are important in stress regulation as they may correct and influence such damage through folding or dis- aggregation and degradation (Brehme and Voisine, 2016).
  • chaperones Accordingly, changes in chaperone expression, have been extensively studied in disease, and GRP94 overexpression has been implicated in cancer (Buc Calderon et al., 2018; Lee, 2014). Stress however also alters how proteins interact (Harper and Bennett, 2016), a feature also influenceable by chaperones (Ellis, 2013). Accordingly, structurally modified chaperone pools, termed epichaperomes, may form under stress and act as scaffolds to pathologically remodel cellular processes by mediating aberrant protein-protein interactions, and in turn, creating a state of proteome-wide connectivity dysfunction (Dart, 2016; Joshi et al., 2018).
  • the present disclosure unveils a specific N- glycosylation pattern used by a chaperone, GRP94, to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane.
  • This ‘protein assembly mutation’ remodels protein networks and properties of the cell.
  • the present disclosure shows in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant.
  • the present disclosure provides biochemical evidence for stressor induced chaperone- mediated protein mis-assemblies and demonstrates how these alterations are actionable in disease.
  • the present disclosure provides a method of treating cancer, inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis, or diabetes, comprising the step of administering to a subject suffering therefrom an effective amount of a compound of Formula (I): or a pharmaceutically acceptable salt thereof, wherein X 1 , X 2 , X 3 , X 5 , Z 2 , and R 1 are as defined herein.
  • a compound of Formula (I): or a pharmaceutically acceptable salt thereof wherein X 1 , X 2 , X 3 , X 5 , Z 2 , and R 1 are as defined herein.
  • (A) Viability of cancer cell lines (n 64) treated for 72 hr with PU-WS13 (10 ⁇ M) or PU-29F (20 ⁇ M). Mean values of triplicate experiments are graphed. Negative values depict killing of the initial cell population.
  • (B) Correlative analysis between RTK (i.e. HER2 and EGFR) levels and cell viability for BC cells in (A). Pearson’s r, two-tailed, n 12.
  • a and B Native-PAGE (top) and SDS-PAGE (bottom) separation followed by immunoblot with the 9G10 anti-GRP94 antibody in un-treated cell lines (A) or in those treated for 4 hr with PU- WS13 (10 ⁇ M) (B).
  • Each data point is an individual cell line; 1-11: BT474, MDA-MB-468, SKBr3, AU565, MDA-MB-361, HCC1806, MCF7, MDA-MB-231, T47D, BT20, HMEC.
  • Graph mean. Error bar, SEM, unpaired t-test, **p ⁇ 0.01, ***p ⁇ 0.001.
  • FIG. 3A-3C describe a specific GRP94 conformation enables the formation of stable HMW GRP94 and HER2 pools.
  • A Anti-GRP94 antibody captured cargo from SKBr3 cell extracts and the remaining supernatant separated under native and denaturing conditions, and immunoblotted with the indicated antibodies.+, 1x, ++, 2x antibody amount. IgG, control; black arrow, unspecific signal.
  • FIG. 4A-4F describe that the N-glycan content of GRP94 regulates the stability and function of the HMW GRP94-HER2 PM-associated pool.
  • A Schematic of the experimental design to investigate the role of N-glycans on the conformation, stability and function of the HMW GRP94 pools.
  • WS13-B biotinylated PU-WS13.
  • PM pool of GRP94 and HER2 in SKBr3 extracts treated under native conditions as in (A) with the indicated enzymes.
  • FIG. 5A-5G describe that glycosylation at N62 of GRP94 is a key regulator in the formation of the HMW GRP94 variant and is important for its oncogenic activity.
  • A Schematic for clone generation and validation. MDA-MB-468 cells (WT) and clones containing the indicated GRP94 mutants were then used for the analyses below.
  • B The effect of GRP94 N- glycan mutagenesis or KO evaluated on glycosylation load.
  • (E) Baseline activity of EGFR-downstream signaling measured by western blot analysis of p- ERK and ERK. Graph, mean (n 3).
  • FIG. 6A-6E describe the accumulation of hgGRP94 at the PM is sufficient to augment HMW GRP94 levels, stabilize receptors at the PM and rewire protein networks in the cytosol.
  • FIG. 6A Biochemical profile of GRP94 in the indicated cell fractions obtained from cells containing the WT (1) or the TM96 (2) GRP94 construct. LRP6, control for PM proteins, p-p65, p- ERK control for signaling activity and HSP90-incorporating epichaperomes for oncogenic activation of cytosolic chaperomes.
  • Negative y-axis values depict killing of the initial cell population.
  • E Schematic of the findings.
  • Figures 7A-7L describe that the HMW GRP94 tumor variant is an actionable target.
  • a and B PD analyses at 24 hr after one dose of PU-WS13 (75 mg/kg, i.p.) administered to mice bearing AU565 (A) or MDA-MB-468 (B) orthotopic tumors.
  • mice 75MWF, 75 mg/kg given Mon-Wed-Fri; qod, every-other-day; qd, every day, Mon through Fri.
  • Graph mean ⁇ SEM, two-tailed Mann Whitney test, *p ⁇ 0.05.
  • Figures 8A-8E describe the sensitivity of cancer cells to PU-WS13 inhibition, Related to Figures 1A-1E.
  • A Viability (by Annexin staining) of breast cancer cell lines treated for 48 hr with PU-WS13 (WS, 10 ⁇ M) or Vehicle (V, DMSO).
  • D SDS-PAGE (top) and native PAGE (bottom) analysis of GRP94 knock-down in MDA- MB-468 cells treated for 72 hr with scramble or a GRP94-specific siRNA construct at the indicated concentrations.
  • cPARP cleaved PARP.
  • E Western blot analysis of GRP94 in cells differentiated by their RTK levels. GAPDH, loading control. Graph, mean ⁇ SEM, unpaired t-test, ns, p > 0.05. S, WS13-sensitive, R, WS13-resistant cell lines.
  • Figures 9A-9C describe the protein solubility tests and cell fractionation techniques used for the enrichment of the plasma membrane and ER-Golgi proteins, Related to Figures 2A- 2G.
  • A Ponceau stained and GRP94 (9G10) or concanavalin A (ConA) blotted membranes of experiments from Figure 2C. SDS and native PAGE separation for SKBr3 and MCF7 whole cell extracts are show; first column, vehicle treated and second column, PUWS13 treated (10 ⁇ M for 4 hr).
  • B Native PAGE analysis of protein unfolding induced by chemical denaturants such as urea.
  • FIG. 10A-10D describe that a biotinylated PU-WS13 has preferential capture for the plasma membrane resident GRP94 and isolates GRP94 in complex with the RTK in RTK- overexpressing cancer cells, Related to Figures 2A-2G.
  • A Western blot analysis of cell surface and intracellular GRP94. Cell fractions were obtained as indicated in the schematic. Black arrow indicates the GRP94 of MW higher than 100 kDa.
  • (B) Western blot of protein cargo isolated by PU-WS13-biotin from SKBr3 extracts prepared as indicated in the schematic. Graph, mean ⁇ SEM, n 3, unpaired t-test, **p ⁇ 0.01.
  • FIG. 11A-11C describe that GRP94 present at the plasma membrane has a higher N-glycan content than the ER resident GRP94, Related to Figures 4A-4F.
  • WCL whole cell lysate
  • F1 enriched in ER-Golgi and cytosol fractions
  • F2 enriched in plasma membrane.
  • B The effect of N-glycan removal on the interaction between GRP94 and RTKs as evidenced by the analysis of the protein cargo isolated by PU-WS13-biotin (WS13-B).
  • C Plasma membrane pool of GRP94 treated under native conditions as indicated in schematic with the indicated enzymes or for 4 hr with PU-WS13 (10 ⁇ M) (right), and western blot analysis of cells treated for 4 hr with PU-WS13 (10 ⁇ M) or vehicle (-) prior to Nglycan removal and capture with the WS13-B reagent (right). Gels are representative of three individual experiments.
  • Figures 12A-12C describe the effect of GRP94 N-glycan mutagenesis or KO on the HMW GRP94 species, Related to Figures 5A-5G.
  • A The effect of protein loading on the biochemical signature of GRP94 and its sensitivity to PU-WS13 (WS13), or the lack of, in the indicated clones treated with either vehicle or PU-WS13 (5 ⁇ M, 4 hr).
  • B Western blot analysis of whole cell extracts of indicated GRP94 mutant clones treated for 24 hr with PU-WS13 (0, 0.1, 0.25, 0.5 ⁇ M).
  • C Cellular localization of EGFR and GRP94.1, WCL; 2, F1; 3, F2 fractions.
  • FIGS. 13A-13B describe that TM96 GRP94 is N-glycosylated, participates in the formation of HMW GRP94 pools and is sensitive to PUWS13, Related to Figures 6A-6E.
  • A Western blot analysis of GRP94 in plasma membrane extracts of MethA WT and TM96 cells in which N-glycan removal was performed with the indicated enzymes under native conditions.
  • B Plasma membrane pool of HMW and total GRP94 in MethA TM96 extracts obtained from cells treated for 24 hr with PUWS13 (0, 5, 10 ⁇ M) prior to fractionation. Gels are representative of three independent experiments.
  • Figures 14A-14C describe that the HMW GRP94 tumor variant is an actionable target, Related to Figures 7A-7L.
  • A Schematic of the testing paradigm to evaluate the safety and efficacy of PU-WS13 in EGFR+ and HER2+ tumor bearing mice.
  • B and C Representative western blot analyses for the pharmacodynamic (PD) analyses of HMW GRP94 tumor markers following one dose of PU-WS13 (75 mg/kg) administered intraperitoneally (i.p.) to mice bearing AU565 (B) or MDA-MB-468 (C) orthotopic tumors. Each column, individual mouse. See Figure 7A, 7B for graphed data on all tumors.
  • Figure 16 depicts Native PAGE assays of PU-WS12, SO-IV-33A, and HJP-VI-110.
  • Figure 17 depicts Native PAGE assays of PU-WS13, SO-IV-33A, SO-IV-26A, and SO-III-116A.
  • FIG. 18 depicts Immunoprecipitation (IP) of PU-WS13, HJP-V-149, WS-12, Bnlm, and HJP-V-92 with the indicated GRP94 antibodies.
  • IP Immunoprecipitation
  • the present disclosure describes, among other things, the role of N-glycosylated forms of Grp94 in certain cancers.
  • the present disclosure also provides inhibitors of such N- glycosylated forms of Grp94.
  • Inhibitors of Grp94 are known, including those described in WO 2015/023976, which describes compounds that are particularly suited for inhibiting Grp94 selectively over Hsp90.
  • the present disclosure provides surprising evidence that only a subset of such compounds known to inhibit Grp94 are also capable of inhibiting N- glycosylated forms of Grp94 described herein.
  • compounds described herein as inhibitors of N-glycosylated Grp94 are unexpectedly less potent against Grp94 in biochemical assays and/or less selective for Grp94 over Hsp90 compared to other compounds known in the art.
  • the present disclosure encompasses the recognition that disease associated stresses may greatly modify the proteome creating intracellular pools of structurally and functionally heterogeneous proteins and protein assemblies.
  • the present disclosure describes that these protein pools are chaperone mediated assemblies which portend disease-associated activity by remodeling proteome-wide connectivity, and thus function.
  • the present disclosure combines chemical biology tools and complementary biochemical and functional approaches, with specific interest on functions and modifications induced by proteome stress associated with malignant transformation and mediated by GRP94 modifications.
  • the present disclosure uses established cancer cell lines, fresh patient biospecimens and cell- and patient-derived xenografts in mice and ex vivo as disease models.
  • the present disclosure identifies a biochemical mechanism whereby aberrant N- glycosylation of a fraction of the cellular pool of the chaperone GRP94 remodels its location and conformation, and in turn, its interaction strength and interaction partners (i.e. connectivity). The outcomes are aberrantly remodeled protein pathways and in turn, a pathologic cellular phenotype.
  • the present disclosure therefore provides a missing link in chaperone-mediated protein connectivity dysfunction by demonstrating how stress hijacks the customary role of a protein, turning it from a folder into a remodeler of protein connectivity.
  • GRP94 inhibition is lethal in a subset of tumor cells [0030]
  • PU-WS13 is a small molecule whose selectivity arises from its ability to bind to an allosteric pocket of GRP94 that only partly overlaps with the ATP-binding pocket and is not accessible in the closely related paralog, HSP90 (Gewirth, 2016; Patel et al., 2015; Patel et al., 2013; Shrestha et al., 2016; Stothert et al., 2017). It shows >100-fold selectivity over HSP90 and no interaction with kinases when tested at 10 ⁇ M in a 98-kinase panel (Patel et al., 2015).
  • the present disclosure used PU-29F, a selective inhibitor of cytosolic HSP90 (Patel et al., 2013).
  • the present disclosure provides that only a subset of these cell lines were vulnerable to PU-WS13 as measured by ATP levels and Annexin V staining ( Figures 1A and S1A).
  • RTK receptor tyrosine kinase
  • GRP94 is heterogeneous in cancer
  • Total GRP94 levels were comparable between the different cancer cell lines assessed for sensitivity to GRP94 inhibition ( Figure 8E), suggesting that chaperone concentration alone was not responsible for the different responses to inhibition.
  • the present disclosure analyzed the GRP94 isolated from sensitive and resistant cell lines for residence in stable protein complexes, cellular localization, conformation, and post-translational modification.
  • Cell homogenates from both inhibitor-sensitive and resistant cancer cells were run on native gels in buffers near the physiological pH (Figure 2A).
  • HMW high molecular weight
  • the present disclosure therefore proceeded to investigate the contribution of each factor to the observed HMW species: complexation, conformation and PTM.
  • GRP94 complexation contributes to its heterogeneity
  • HER2 an abundant RTK in SKBr3 cells, co-localizes with GRP94 at the PM in these cells ( Figure 2D and (Li et al., 2015; Patel et al., 2013)
  • the present disclosure probed the cellular fractions of this cell line for HER2.
  • HER2 detected by Western blotting on native gels was observed as a HMW species in the whole cell lysate (WCL), total membrane (TM), and PM fractions (Figure 2E).
  • HMW HER2 species absent in MCF7 cells, correspond to the same fractions that contained the HMW GRP94 species.
  • Short-term treatment of SKBr3 cells with PU- WS13 reduced the amount of HMW HER2 in the F2 pool (Figure 2D, immunofluorescence and Figure 2E, native PAGE) without majorly changing the overall levels of HER2 ( Figure 2E, WCL by SDS-PAGE), paralleling the observations seen with GRP94.
  • the present disclosure introduced a biotinylated PU-WS13 reagent, PU- WS13-B, which was found to preferentially isolate the GRP94 found in the F2, and not in the ER, Golgi, or cytosolic fractions (F1) (Figure 10B) and to enrich for the ⁇ 100 kDa GRP94 species ( Figure 2G, black arrow).
  • This probe pulled down HER2 along GRP94 ( Figure 2G).
  • Similar results were seen in EGFR+ BC cells, where EGFR, another RTK, was GRP94-bound ( Figure 10C).
  • the present disclosure found PU-WS13-B preferentially isolated GRP94 bound to HER2 over free GRP94 (Figure 10D).
  • the G4420 antibody which recognizes amino acids 733-750 in the C-terminal domain of GRP94, was able to capture both the HER2-bound and the free GRP94 ( Figure 10D). Conformation is key in HMW GRP94 formation [0041]
  • the present disclosure next determined if GRP94’s conformational state contributes to formation of the HMW protein pool.
  • the present disclosure probed the HMW pool with two anti-GRP94 antibodies, the conformation-specific 9G10 antibody, and the G4420 antibody, which is not known to discriminate between different conformational states of the chaperone.
  • a dose-dependent immunocapture of GRP94 and HER2 by the G4420 antibody was associated with a dose-dependent decrease in the HMW GRP94 and HER2 pools noted on Native PAGE and a decrease in both GRP94 and HER2 levels in the supernatant noted on Western blot ( Figure 3A).
  • the antibody 9G10 captured GRP94 but not HER2; nonetheless, both the GRP94 and HER2 HMW pools were diminished on native PAGE but only GRP94 was reduced in the supernatant following 9G10 immunocapture, consistent with a conformational change induced by this antibody which is associated with release of the bound cargo.
  • PU-WS13-B preferentially captured the PM-localized GRP94 which was bound to HER2. To see if this corresponds to a specific conformation of the chaperone, the present disclosure treated cells with PU-WS13 prior to immunocapture with the two GRP94 antibodies ( Figure 3B). Increasing amounts of PU-WS13 or increased duration of PU-WS13 exposure dramatically reduced the amount of GRP94 captured by the 9G10 antibody, indicating that the inhibitor changes GRP94 to a conformation that is no longer recognized by the antibody.
  • GRP94 is post-translationally modified by phosphorylation and glycosylation (Cala, 2000), and the present disclosure investigates whether these PTMs contribute to the formation or stability of the HMW GRP94 complexes.
  • the mobility shift due to glycosylation differed between cellular fractions.
  • the F2 fraction exhibited a greater mobility shift compared to the ER F1 fraction.
  • the present disclosure probed for GRP94 and HER2 HMW complexes on native gels, or captured the GRP94 complexes with immobilized PU-WS13-B, or with the two GRP94 antibodies G4420 or 9G10.
  • Glycan removal significantly reduced the amounts of HMW GRP94 and HER2 species seen on native gels, indicating that glycosylation is important for the stability of these complexes ( Figure 4B).
  • the amount of the GRP94 and HER2 (or EGFR) cargo captured by PU-WS13-B and G4420 pulldown was also decreased by deglycosylation treatment (Figure 4C and 11B).
  • GRP94 contains six potential N-glycan acceptor sites and under normal conditions the protein is predominantly monoglycosylated at N217 (Cloutier and Coulombe, 2013; Schwarz and Aebi, 2011).
  • the present disclosure describes glycosylation site mapping by mass spectrometry and identified N62, N217 and N502 as putative glycosylated Asn sites on the hgGRP94 variant.
  • N62 was a key residue needed for the formation of the HMW GRP94 pool, as evidenced by native PAGE (Figure 5C and 12A), insensitivity to PU-WS13 (see GRP94 pools on native PAGE, Figure 5C and p-ERK and EGFR on SDS-PAGE, Figure 5D and 12B), a decrease in RTK signaling activity (see ERK downstream signaling, Figure 5E and 12B), diminished interaction with the G4220 antibody ( Figure 5F) and a significant decrease in GRP94 and RTK localized at the PM (Figure 5G and S5C) in the N62Q-containing clones when compared to WT.
  • GRP94 KO mimicked the effects observed with the N62Q-containing mutants (i.e.
  • GRP94 ability to form long-lived, stable complexes with RTKs at the PM (as opposed to the dynamic interactions needed for RTK folding by GRP94 in the ER) (Eletto et al., 2010) is dependent on a specific hyperglycosylation pattern, with N62 being a key residue for the observed switch of GRP94 from a folding, ER chaperone, to an oncogenic protein that stabilizes and activates RTKs at the PM.
  • HMW GRP94 an oncogenic gain-of-function
  • the present disclosure investigates if the accumulation of GRP94 at the PM was sufficient to initiate such oncogenic signaling.
  • the present disclosure utilized a construct that directs myc tagged GRP94 to the PM by deletion of the KDEL sequence and incorporation of a transmembrane domain from platelet-derived growth factor receptor (Zheng et al., 2001).
  • the present disclosure used a Meth A fibrosarcoma cell line that was stably transfected with this construct, TM96, and compared the properties of this cell line to those of WT Meth A cells.
  • TM96 GRP94 construct was found only in the F2 fraction, but not the ER (F1) or cytoplasmic (C) fractions ( Figure 6A).
  • TM96 expressed GRP94 participated in the formation of stable HMW GRP94 complexes as evidenced by the characteristic electrophoretic migration pattern on native gels (Figure 6A), its glycosylation status suggestive of hyperglycosylation ( Figure 13A) and its sensitivity to PU-WS13 ( Figure 13B).
  • TM96 expressed GRP94 also augmented the formation of intracellular stable HMW complexes incorporating HSP90 (Figure 6A, HSP90 native PAGE), also referred to as HSP90-incorporating epichaperomes, which act as molecular scaffolding platforms that augment the activity of cytosolic protein pathways, including signaling pathways (Joshi et al., 2018; Kourtis et al., 2018; Rodina et al., 2016).
  • PU-WS13 treatment was sufficient to reverse these effects, as evidenced by inhibition of the activated but not baseline signaling (Figure 6B) and the loss of the HMW GRP94 pool located at the PM ( Figure 12B).
  • HMW GRP94 is an actionable target in cancer [0052] Because PU-WS13 exhibits preference for the GRP94 pool incorporated into stable HMW complexes located at the PM of cancer cells, it can be used to address the targetability and safety of inhibiting this unusual GRP94 variant in cancer. Treatment is a balance between target engagement and therapeutic index, and the present disclosure evaluated whether target suppression can be safely achieved by PU-WS13 in vivo. To understand target engagement during the study, the present disclosure measured tumor and tissue pharmacokinetics (PK) and pharmacodynamics (PD) after either a single dose of PU-WS13 administered intraperitoneally (i.p.) or at the end of a long- term treatment (see Figure 14A for study design).
  • PK tumor and tissue pharmacokinetics
  • PD pharmacodynamics
  • the present disclosure therefore investigated the efficacy of 75 mg/kg and 125 mg/kg PU-WS13 given three times weekly (M-W-F), every other day (qod) or daily (qd) with weekends off (Figure 7D).
  • the present disclosure provides significant, and dose- and schedule-dependent effects of PU-WS13 (Figures 7E-7G), with complete tumor growth suppression observed under the daily treatment paradigm. Similar results were also noted when tumors were established subcutaneously ( Figures 7H, 7I).
  • PU-WS13 was well tolerated. Even for the long treatment regimens that delivered 37 to 62 doses of PU-WS13 to mice over 87 days, no treatment-related toxicities were observed: mice retained a normal weight throughout treatment (Figure 7J).
  • the present disclosure conducted complete necropsies and analyzed hematology and serum chemistry panels on vehicle-treated mice and on mice receiving the 125mg/kg dose five times per week for 87 days (Figure 7K and Figure 15). All hematological and clinical chemistry findings were within normal parameters, and histopathology conducted on major organs showed no toxic changes induced by PU-WS13. [0055] The present disclosure also evaluated GI tract LRP6 levels after PU-WS13 administration ( Figure 7L).
  • Housekeeping GRP94 is essential for folding and regulating physiologic functions of the Wnt receptor LRP6 (Rachidi et al., 2015), and it is expected that compounds such as PU-WS13 selectively targeting the tumor-specific HMW GRP94 variant will act on tumor functions while leaving housekeeping GRP94 functions unaltered at similar or higher concentrations as those seen in the tumor. Because most small molecules, including PU-WS13, are largely cleared via the GI tract, it is a body site most exposed to such agents over the time they spend in the body.
  • the present disclosure identifies a GRP94 variant in cancer, whereby by altering N- glycosylation, a new protein conformationally, dynamically and functionally distinct from the GRP94 of normal cells is created.
  • a specific increase in N-glycosylation promotes a conformational state that allows for stable interactions with oncoproteins at the PM.
  • hyperglycosylation is a modality used by GRP94 to alter its conformational fitness and stabilize a state most permissive for stable interactions. Through this stabilization, these proteins’ functions are enhanced, and cellular protein pathways are aberrantly remodeled - N-glycosylation thus transforms a chaperone, GRP94, from a folding to a scaffolding protein that remodels protein connectivity, with an end result of proteome-wide dysfunction. Therefore, the N-glycosylation pattern of GRP94 the present disclosure identifies is a specific modification exploited by cancer cells to alter the customary role of a chaperone.
  • the aberrantly N-glycosylated GRP94 variant is present only in some tumors, is independent of total GRP94 levels, and is absent or scarce in non-transformed cells.
  • the present disclosure found that the functions of one class of oncoproteins, RTKs, are modified by this GRP94 variant, and only in cancer cells driven by RTK overexpression.
  • RTK overexpression is a form of proteome stress, and under these conditions GRP94 N-glycosylation at specific sites is key both to enhance the presence of these proteins at the plasma membrane by forming stable complexes with the RTKs, as well as maintain RTKs in a state that enables aberrant downstream signaling and a rewiring of cytosolic protein pathways.
  • N-linked glycosylation is among the most ubiquitous protein modifications in eukaryotes. It is implicated in a myriad of housekeeping functions, including modification of a protein’s folding capacity, stability, and oligomerization and aggregation status, ER quality control and protein trafficking, host cell-surface interactions, and modulation of enzyme activity (Lee et al., 2015). Changes in glycosylation are observed in cancer where they affect the interaction and subsequently activation capacity of RTKs (Mereiter et al., 2019). Conversely, there is no report of N-glycosylation increasing the oncogenic properties of a protein, indirectly, by modulating its complexation.
  • N- glycosylation does not induce significant changes in a protein’s structure, but decreases protein conformational dynamics, likely leading to an increase in protein stability (Lee et al., 2015).
  • N-glycans act like molecular glues, holding together residues around the glycosylation sites through favorable interactions made with nearby protein residues, thus resulting in the stabilization of a specific protein conformation or disfavoring others (Sola and Griebenow, 2006).
  • HSP90 the cytosolic paralog of GRP94
  • an oncogenic stress such as MYC hyperactivation or a neuronal damaging stress such as tau overexpression redistributes the cytosolic pool of molecular chaperones and helpers into complexes of enhanced stability.
  • These stable assemblies termed epichaperomes, function as multi-component scaffolds to provide a framework on which the cell’s complement of proteins can work more efficiently or differently than they would without chaperome participation.
  • a conformational switch changes the direct interaction of GRP94 with HER2 or whether it mediates the creation of a stable multimeric platform with co-chaperones and other factors that mediate HER2 stabilization, similarly to that seen for HSP90, remains to be seen.
  • the present disclosure shows that inhibition of the hgGRP94 variant with compounds such as PU-WS13 is feasible in cancer cells, human primary tumor specimens, and xenografted tumors in mice.
  • GRP94 is abundant in most cells of the mammalian body, it is clear that the housekeeping variant and the N-glycosylated variant targeted in cancers are different, rendering compounds such as PU- WS13 selective for the cancer form.
  • the present disclosure also demonstrates that the N-glycosylated GRP94 variant, and the specific aberrant proteins and cellular processes enabled by this variant, are targetable in disease.
  • inhibitors of the N- glycosylated GRP94 variant are an example of a ‘targeted protein degradation-based therapeutic’ that act specifically on dysfunctions, and protein networks, enabled by this variant, thus sparing the normal folding functions of GRP94.
  • the present disclosure specifically exemplifies the expression and significance of the hgGRP94 variant in BC, several lines of evidence suggest that it is implicated in other cancers as well.
  • RTK overexpression ex.
  • EGFR, HER2, MET and others is observed in a variety of cancer cells and in cells of a tumor supportive microenvironment (Butti et al., 2018; Contessa et al., 2008; Contessa et al., 2010; Siddals et al., 2011; Tan et al., 2018; Turrini et al., 2017).
  • RTK amplifications also allow tumor cells to escape therapeutic treatment (MET and HER2 amplification can be detected in EGFR-mutant lung cancers that become resistant to EGFR TKI therapy (Yu et al., 2013)). Because EGFR overexpression is often a side effect of radiation therapy (Cuneo et al., 2015), targeting the GRP94 variant with PU-WS13 may also radiosensitize tumors. [0063] It is noteworthy that inhibition of the GRP94 variant is more toxic to EGFR+ tumors than the direct inhibition of EGFR by kinase inhibitors or anti-EGFR antibodies. Approximately half of all triple- negative BCs (TNBCs) and inflammatory BCs overexpress EGFR.
  • TNBCs triple- negative BCs
  • inflammatory BCs overexpress EGFR.
  • the present disclosure reports that increasing the interaction strength between GRP94 and RTKs and other receptors at the plasma membrane, which the present disclosure found to be regulated by a specific N-glycosylation pattern, is a mechanism used by a chaperone to enhance the stabilization and interaction of certain proteins. Without wishing to be bound by any particular theory, it is believed that these findings identify a biochemical mechanism whereby stress remodels a chaperone from a folding to a scaffolding protein creating a state of chaperone- mediated protein connectivity dysfunction.
  • GRP94 Aberrant N-glycosylation of a fraction of the cellular pool of the chaperone GRP94 remodels GRP94 location and conformation, and in turn, its interaction strength and interaction partners, with the outcome being aberrantly remodeled protein pathways and a pathologic cellular phenotype.
  • the present disclosure describes that the HMW form of GRP94 is an example of a ‘protein assembly mutation’ (Nussinov et al., 2019), a proteome malfunction defined by defective protein-protein interaction that portends pathologic activity. This variant is a target for cancers and other diseases.
  • structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure.
  • the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomic, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure.
  • some structures depicted here show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and/or as a mixture.
  • the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value (except where such number would be less than 0% or exceed 100% of a possible value).
  • Aliphatic refers to a straight-chain (i.e., unbranched) or branched, optionally substituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation but which is not aromatic (also referred to herein as “carbocyclic” or “cycloaliphatic”), that has a single point of attachment to the rest of the molecule.
  • aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C 1-6 ).
  • aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C 1-3 ), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C 1-2 ). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof.
  • aliphatic refers to a straight- chain (i.e., unbranched) or branched, optionally substituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation that has a single point of attachment to the rest of the molecule.
  • Alkyl The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12, C1-10, C1-8, C1-6, C1-4, C1-3, or C 1-2 ).
  • Carbocyclyl The terms “carbocyclyl,” “carbocycle,” and “carbocyclic ring” as used herein, refer to saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein.
  • Carbocyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl.
  • “carbocyclyl” refers to an optionally substituted monocyclic C3-C8 hydrocarbon, or an optionally substituted C7-C10 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
  • the term “cycloalkyl” refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, cycloalkyl groups have 3–6 carbons.
  • Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • cycloalkenyl refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms.
  • Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.
  • Alkenyl refers to an optionally substituted straight or branched hydrocarbon chain having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C 2-12 , C 2-10 , C 2-8 , C 2-6 , C 2-4 , or C 2-3 ).
  • alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.
  • Alkynyl refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3).
  • exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.
  • Aryl refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C 6-14 ), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members.
  • the term “aryl” may be used interchangeably with the term “aryl ring”.
  • “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons.
  • Heteroaryl refers to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10- membered bicyclic heteroaryl); having 6, 10, or 14 ⁇ electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
  • heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2- a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl.
  • heteroaryl and “heteroar—”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms).
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H–quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3–b]–1,4– oxazin–3(4H)–one, and benzoisoxazolyl.
  • heteroaryl may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted.
  • Heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • Heterocycle As used herein, the terms “heterocycle”, “heterocyclyl”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • the nitrogen may be N (as in 3,4-dihydro- 2H-pyrrolyl), NH (as in pyrrolidinyl), or NR + (as in N-substituted pyrrolidinyl).
  • a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
  • saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl.
  • a heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic.
  • a bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings.
  • Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3- dihydrobenzofuranyl, and tetrahydroquinolinyl.
  • a bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)).
  • spirocyclic ring system e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)).
  • Partially Unsaturated when referring to a ring moiety, means a ring moiety that includes at least one double or triple bond between ring atoms.
  • Patient or subject refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient or subject is a human. In some embodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions.
  • a patient or subject displays one or more symptoms of a disorder or condition.
  • a patient or subject has been diagnosed with one or more disorders or conditions.
  • a patient or a subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition.
  • Substituted or optionally substituted As described herein, compounds of this disclosure may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • Substituted applies to one or more hydrogens that are either explicit or implicit from the structure (e.g., refers to at least ).
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein.
  • Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents.
  • Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.
  • treat refers to any administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
  • treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
  • such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
  • treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
  • the present disclosure provides compounds that are inhibitors of N-glycosylated Grp94.
  • Such compounds are represented schematically in Formula (I): or a pharmaceutically acceptable salt thereof, wherein: Z 2 is –N- or –CR 10 -, wherein R 10 is H or unsubstituted or substituted -(C1-C6)aliphatic; X 1 is –H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; X 2 is –H, halogen, or unsubstituted or substituted -(C 1 -C 6 )aliphatic; X 3 and X 5 are independently -halogen, unsubstituted or substituted -(C 1 -C 12 )aliphatic, unsubstituted or substitute
  • Z 2 is –N-. In some embodiments, Z 2 is –CR 10 -. In some embodiments, Z 2 is –CH-. [0084] In some embodiments, X 1 is –H. In some embodiments, X 1 is halogen. In some embodiments, X 1 is F. In some embodiments, X 1 is Cl. In some embodiments, X 1 is Br. In some embodiments, X 1 is I. In some embodiments, X 1 is F or Cl. [0085] In some embodiments, X 2 is -H. In some embodiments, X 2 is halogen.
  • X 2 is –H, halogen, or unsubstituted or substituted -(C1- C 6 )aliphatic; and X 3 and X 5 are independently -halogen, unsubstituted or substituted -(C 1 - C12)aliphatic, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 3- to 10- membered heterocyclic group having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or unsubstituted or substituted 3- to 10-membered cycloalkyl group.
  • both X 3 and X 5 are halogen.
  • the halogen is Cl.
  • the halogen is F.
  • the halogen is I.
  • the halogen is Br.
  • X 3 and X 5 are identical halogen.
  • neither X 3 nor X 5 are hydrogen.
  • one of X 3 and X 5 is halogen.
  • one of X 3 and X 5 is hydrogen.
  • X 3 and X 5 are both Cl.
  • X 3 and X 5 are Cl and Br.
  • X 3 and X 5 and Cl and I are halogen, and the other of X 3 and X 5 is unsubstituted or substituted -(C 1 -C 6 )aliphatic. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted -(C1-C6)aliphatic. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted -(C1-C6)aliphatic.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted -(C1-C6)alkyl.
  • the -(C1-C6)alkyl group is substituted with one or more halogen, -OH, -CN, -NH2, or unsubstituted C1-6 aliphatic.
  • the -(C 1 -C 6 )alkyl group is substituted with -OH.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted -(C2-C6)alkenyl.
  • the -(C2-C6)alkenyl group is substituted with halogen, -OH, -CN, -NH 2 , or unsubstituted C 1-6 aliphatic.
  • the -(C 2 -C 6 )alkenyl group is substituted with -OH.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted -(C2-C6)alkynyl.
  • the -(C2-C6)alkynyl group is substituted with halogen, -OH, -CN, -NH 2 , or unsubstituted C 1-6 aliphatic.
  • the -(C2-C6)alkynyl group is substituted with -OH.
  • X 3 and X 5 are independently selected from halogen or the following groups:
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted phenyl. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted phenyl. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted phenyl.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted phenyl, wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, -CF3, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO 2 , -CF 3 , or -NH 2 .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted phenyl, wherein the substituent is –NO 2 .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted phenyl, wherein the substituent is –OMe. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted phenyl, wherein the substituent is –CF 3 . [0095] In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted 5- to 6- membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C 1-6 aliphatic, or C 1-6 aliphatic substituted with halogen, -OH, -CN, -NO 2 , or -NH 2 .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C 1-6 aliphatic substituted with halogen, -OH, -CN, -NO 2 , or -NH 2 .
  • a substituent is –NO 2 .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is –-OMe. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is –CF 3 . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is –Me. [0097] In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein each substituent is independently halogen, -N(R) 2 , -OR, -CN, -NO 2 , unsubstituted C 1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is –NO2. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is -OMe. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is –CF 3 . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is –Me.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is –NO 2 . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is -OMe. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is –CF3. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted or , wherein the substituent is –Me.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted or , wherein the substituent is –NO 2 .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is -OMe.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the su 3 5 bstituent is –CF3.
  • one of X and X is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is –Me.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted or or . In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or or .
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted or or , wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted or , wherein the substituent is –NO2.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted , wherein the substituent is -OMe. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted wherein the substituent is –CF3. In some embodiments, one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted or or , wherein the substituent is –Me. [0101] In some embodiments, X 3 and X 5 are independently selected from halogen or the following groups:
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted or substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is unsubstituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
  • one of X 3 and X 5 is halogen, and the other of X 3 and X 5 is substituted 8- to 10- membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C 1-6 aliphatic, or C 1-6 aliphatic substituted with halogen, -OH, -CN, -NO 2 , or -NH 2 .
  • one of R 2 and R 3 is H. In some embodiments, both R 2 and R 3 are H.
  • neither of R 2 and R 3 is H.
  • one of R 2 and R 3 is H and the other is unsubstituted or substituted -(C 1 -C 8 )aliphatic. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted or substituted -(C1-C8)alkyl. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted -(C1-C8)alkyl. In some embodiments, one of R 2 and R 3 is H and the other is methyl. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted -(C 2 )alkyl.
  • one of R 2 and R 3 is H and the other is unsubstituted -(C 3 )alkyl. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted -(C4)alkyl. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted -(C 5 )alkyl. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted -(C 6 )alkyl. In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted -(C7)alkyl.
  • one of R 2 and R 3 is H and the other is unsubstituted -(C 8 )alkyl. In some embodiments, one of R 2 and R 3 is H and the other is substituted -(C 1 -C 8 )alkyl, wherein each substituent is independently halogen, -OH, -CN, -NH2, or unsubstituted C1-6 aliphatic. [0106] In some embodiments, one of R 2 and R 3 is H and the other is unsubstituted or substituted -(C 2 -C 8 )alkenyl.
  • R 2 and R 3 are H and the other is unsubstituted or substituted -(C2-C8)alkynyl.
  • R 2 and R 3 are independently selected from the following groups: [ , [0110]
  • compounds of Formula (I) have the structure of Formula (Ia): or a pharmaceutically acceptable salt thereof, wherein each of X 1 , X 2 , X 3 , X 5 , Z 2 , and R 3 is as defined and described herein, both singly and in combination.
  • compounds of Formula (I) have the structure of Formula (Ib): Ib or a pharmaceutically acceptable salt thereof, wherein each of X 1 , X 3 , X 5 , Z 2 , and R 1 is as defined and described herein, both singly and in combination.
  • compounds of Formula (I) have the structure of Formula (Ic- i) or (Ic-ii): Ic-ii or a pharmaceutically acceptable salt thereof, wherein each of X 1 , X 3 , Z 2 , and R 1 is as defined and described herein, both singly and in combination.
  • compounds of Formula (I) have the structure of Formula (Id): or a pharmaceutically acceptable salt thereof, wherein each of X 1 , X 3 , X 5 , Z 2 , and R 3 is as defined and described herein, both singly and in combination.
  • compounds of Formula (I) have the structure of Formula (Ie- i) or (Ie-ii): Ie-i
  • the present disclosure provides a method of treating cancer, inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis, or diabetes, comprising the step of administering to a subject suffering therefrom an effective amount of a compound having the structure of Formula I, Ia, Ib, Ic-i, Ic-ii, Id, Ie-i, or Ie-ii, or a pharmaceutically acceptable salt thereof.
  • compositions [0117] The present disclosure also provides compositions comprising a compound provided herein with one or more other components.
  • provided compositions comprise and/or deliver a compound described herein (e.g., compounds of Formulae I, Ia, Ib, Ic-i, Ic-ii, Id, Ie-i, and Ie-ii ).
  • a provided composition is a pharmaceutical composition that comprises and/or delivers a compound provided herein (e.g., compounds of Formulae I, Ia, Ib, Ic-i, Ic-ii, Id, Ie-i, and Ie-ii ) and further comprises a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions typically contain an active agent (e.g., a compound described herein) in an amount effective to achieve a desired therapeutic effect while avoiding or minimizing adverse side effects.
  • provided pharmaceutical compositions comprise a compound described herein and one or more fillers, disintegrants, lubricants, glidants, anti-adherents, and/or anti-statics, etc.
  • Provided pharmaceutical compositions can be in a variety of forms including oral dosage forms, topical creams, topical patches, iontophoresis forms, suppository, nasal spray and/or inhaler, eye drops, intraocular injection forms, depot forms, as well as injectable and infusible solutions. Methods of preparing pharmaceutical compositions are well known in the art. [0119] In some embodiments, provided compounds are formulated in a unit dosage form for ease of administration and uniformity of dosage.
  • unit dosage form refers to a physically discrete unit of an active agent (e.g., a compound described herein) for administration to a subject.
  • each such unit contains a predetermined quantity of active agent.
  • a unit dosage form contains an entire single dose of the agent.
  • more than one unit dosage form is administered to achieve a total single dose.
  • administration of multiple unit dosage forms is required, or expected to be required, in order to achieve an intended effect.
  • a unit dosage form may be, for example, a liquid pharmaceutical composition containing a predetermined quantity of one or more active agents, a solid pharmaceutical composition (e.g., a tablet, a capsule, or the like) containing a predetermined amount of one or more active agents, a sustained release formulation containing a predetermined quantity of one or more active agents, or a drug delivery device containing a predetermined amount of one or more active agents, etc.
  • a liquid pharmaceutical composition containing a predetermined quantity of one or more active agents
  • a solid pharmaceutical composition e.g., a tablet, a capsule, or the like
  • a sustained release formulation containing a predetermined quantity of one or more active agents
  • a drug delivery device containing a predetermined amount of one or more active agents
  • provided compounds and compositions are useful in research as, for example, analytical tools and/or control compounds in biological assays.
  • the present disclosure provides methods of administering provided compounds or compositions to a subject in need thereof.
  • the present disclosure provides methods of administering provided compounds or compositions to a subject suffering from or susceptible to a disease, disorder, or condition associated with N- glycosylated Grp94.
  • provided compounds are useful as N-glycosylated Grp94 inhibitors.
  • the present disclosure provides methods of inhibiting N- glycosylated Grp94 in a subject comprising administering a provided compound or composition.
  • the present disclosure provides methods of inhibiting N-glycosylated Grp94 in a biological sample comprising contacting the sample with a provided compound or composition. [0124] In some embodiments, the present disclosure provides methods of treating cancer, comprising administering a provided compound or composition to a subject in need thereof. In some embodiments, the present disclosure provides methods of treating proliferative diseases, comprising administering a provided compound or composition to a subject in need thereof.
  • the cancer is colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, leukemias, myelomas, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, or endometrial cancer.
  • the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is non-small-cell lung cancer. [0125] In some embodiments, the present disclosure provides methods of treating a hematological malignancy, comprising administering a provided compound or composition to a subject in need thereof.
  • a hematological malignancy is leukemia (e.g., chronic lymphocytic leukemia, acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, or acute monocytic leukemia).
  • a hematological malignancy is lymphoma (e.g., Burkitt’s lymphoma, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma).
  • a hematological malignancy is myeloma (e.g., multiple myeloma).
  • a hematological malignancy is myeloproliferative neoplasm (e.g., polycythemia vera, essential thrombocytopenia, or myelofibrosis). In some embodiments, a hematological malignancy is myelodysplastic syndrome. [0126] In some embodiments, the present disclosure provides methods of treating an inflammatory disease, disorder, or condition (e.g., acute respiratory syndrome, hyperinflammation, and/or cytokine storm syndrome (including those associated with COVID-19) or atopic dermatitis), neurodegenerative diseases, rheumatoid arthritis, or diabetes comprising administering a provided compound or composition to a subject in need thereof.
  • an inflammatory disease, disorder, or condition e.g., acute respiratory syndrome, hyperinflammation, and/or cytokine storm syndrome (including those associated with COVID-19) or atopic dermatitis
  • neurodegenerative diseases rheumatoid arthritis, or diabetes
  • a provided compound or composition is administered as part of a combination therapy.
  • combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic or prophylactic regimens (e.g., two or more therapeutic or prophylactic agents).
  • the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
  • “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
  • combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.
  • a provided compound or composition is administered to a subject who is receiving or has received one or more additional therapies (e.g., an anti-cancer therapy and/or therapy to address one or more side effects of such anti-cancer therapy, or otherwise to provide palliative care).
  • Exemplary additional therapies include ERBB2 inhibitors, EGFR inhibitors, CDK4 inhibitors, CRAF inhibitors, BRAF inhibitors, AKT inhibitors, MET inhibitors, BCR-ABL inhibitors, JAK inhibitors, HIF-1 ⁇ inhibitors, and p53 inhibitors.
  • the present disclosure contemplates, among other things, the following numbered embodiments: 1. A method of treating cancer comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N-glycosylated Grp94. 2. A method of treating cancer characterized by the presence of N-glycosylated Grp94 comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N-glycosylated Grp94. 3.
  • a method of treating a disease associated with N-glycosylated Grp94 comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N- glycosylated Grp94.
  • the compound has the structure of Formula (I): or a pharmaceutically acceptable salt thereof, wherein: (b) each of Z 1 and Z 3 is independently -CH- or -N-; (c) Z 2 is -N- or –CR 10 -, wherein R 10 is H or unsubstituted or substituted -(C1-C6)aliphatic; (d) each of Z 4 , Z 5 , Z 6 , Z 7 and Z 8 is independently -C- or -N-, with the provisos that at least one of Z 4 , Z 6 and Z 7 is -C- and no three consecutive Z 4 through Z 8 are N; (e) X 1 is -H, -halogen, -N(R) 2 , -
  • each of Z 1 and Z 3 is independently -CH- or -N-;
  • Z 2 is -CH-, -N-, or –CR 10 -, wherein R 10 is -(C1-C6)alkyl;
  • each of Z 4 , Z 5 , Z 6 , Z 7 and Z 8 is independently -C- or -N-, with the provisos that at least one of Z 4 , Z 6 and Z 7 is -C- and no three consecutive Z 4 through Z 8 are N;
  • X 1 is -H, -halogen, -NH2, -CN, -(C1-C6)alkyl, -O(C1-C6)alkyl, -CH2OH, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -OC(halogen)3, -OCH(halogen)2, or -OCH2(halogen);
  • each of Z 1 and Z 3 is independently -CH- or -N-
  • each of Z 1 and Z 3 is independently -CH- or -N-;
  • Z 2 is -N- or –CR 10 -, wherein R 10 is H or unsubstituted or substituted -(C 1 -C 6 )aliphatic;
  • X 1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic;
  • each of X 3 and X 5 is independently -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, - CN, -C(O)R, -C(O) 2 R, -S(O)R, -S(O) 2 R, -C(O)N(R) 2 , -SO 2 N(R) 2 , -OC(O)R, -
  • each of Z 1 and Z 3 is independently -CH- or -N-;
  • Z 2 is -CH-, -N-, or –CR 10 -, wherein R 10 is -(C 1 -C 6 )alkyl;
  • X 1 is -H, -halogen, -NH2, -CN, -(C1-C6)alkyl, -O(C1-C6)alkyl, -CH2OH, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -OC(halogen)3, -OCH(halogen)2, or -OCH2(halogen);
  • each of X 3 and X 5 is independently -H, -halogen, -NH 2 , -CN, -(C 1 -C 6 )alkyl, -O(C 1 - C6)alkyl, -CH2OH, -C(halogen);
  • each of Z 1 and Z 3 is independently -CH- or -N-;
  • Z 2 is -N- or –CR 10 -, wherein R 10 is H or unsubstituted or substituted -(C 1 -C 6 )aliphatic;
  • each of Z 6 , Z 7 and Z 8 is independently -C- or -N-, with the proviso that at least one of Z 6 - Z 8 is -C-;
  • X 1 is -H, -halogen, -N(R) 2 , -OR, -CN, or unsubstituted or substituted -(C 1 -C 6 )aliphatic;
  • each of X 4 , X 5 , and X 6 is independently -H, -halogen, -SR, -N(R) 2 , -
  • each of Z 1 and Z 3 is independently -CH- or -N-;
  • Z 2 is -N- or –CR 10 -, wherein R 10 is H or unsubstituted or substituted -(C1-C6)aliphatic;
  • each of Z 4 , Z 5 , Z 6 , Z 7 and Z 8 is independently -C- or -N-, with the proviso that no three consecutive Z 4 through Z 8 are N;
  • X 1 is -H, -halogen, -N(R) 2 , -OR, -CN, or unsubstituted or substituted -(C 1 -C 6 )aliphatic;
  • each of X 4 , X 5 , and X 6 is independently -H, -halogen, -SR, -N(R)2, -
  • the compound is part of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient.
  • the N-glycosylated Grp94 comprises two or more N-glycan modified residues.
  • the N-glycosylated Grp94 comprises two or more N-glycan modified Asn residues.
  • the N-glycosylated Grp94 comprises glycosylated Asn at the N62 residue. 46.
  • the N-glycosylated Grp94 alters the function of one or more aberrant oncogenic proteins.
  • RTK receptor tyrosine kinase
  • the aberrant oncogenic protein is a receptor tyrosine kinase (RTK). 50.
  • the cancer is colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, leukemias, myelomas, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, or endometrial cancer.
  • the cancer is colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal
  • Reagents and conditions (a) CS 2 , NaHCO 3 , H 2 O, EtOH, reflux, 4 days; (b) neocuproine hydrate, CuI, NaOtBu, 3,5-dichloroiodobenzene, DMF, 115°C, 24 h; (c) 1,3- dibromopropane, Cs 2 CO 3 , DMF, rt, 2 h; (d) amines, DMF, rt, 18-48 h.
  • Athymic nude mice (Hsd:Athymic Nude-Foxn1 nu , female, 20-25 g, 6 weeks old; RRID:MGI:5652489) were obtained from Envigo and NSG mice (NOD.Cg-Prkdc scid female, 20-25 g, 8 weeks old, IMSR Cat# JAX:005557, RRID:IMSR_JAX:005557) were obtained from the Jackson Laboratory.
  • Human Cell Lines [0161] Cell lines were obtained from laboratories at MSKCC, or purchased from the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Cells were cultured as per the providers’ recommended culture conditions.
  • the breast cancer cell lines (MDA-MB-468 (HTB-132), HCC1806 (CRL- 2335), MDA-MB-231 (HTB-26), MDA-MB-415 (HTB-128), MCF-7 (HTB-22), BT474 (HTB- 20), BT20 (HTB-19), MDA- MB-361 (HTB-27), SKBr3 (HTB-30), MDA-MB-453 (HTB-131), T47D (HTB-133), AU565 (CRL-2351) and the non-transformed cell line HMEC (human primary mammary epithelial cells, PCS- 600-010) were obtained from the American Type Culture Collection (ATCC).
  • ATCC American Type Culture Collection
  • the pancreatic cancer cell lines include: MiaPaCa2 (CRL-1420), Panc-1 (CRL-1469), BxPc-3 (CRL-1687), Capan-1 (HTB-79), SU.86.86 (CRL-1837), HPAF2 (CRL- 1997), ASPC-1 (CRL-1682), PL45 (CRL-2558), CFPAC (CRL-1918), Capan-2 (HTB-80) were purchased from ATCC; 931102 and 931019 are patient derived cell lines provided by Dr. Y. Janjigian, MSKCC. The lung cancer cell lines NCI-H3122, NCI-H299 were kindly provided by Dr. M.
  • Neuroblastoma cells SY5Y (CRL-2266) was purchased from ATCC; LAN5 and SMS-KCNR were obtained from the Children’s Oncology Group (COG). Ewing’s sarcoma cells TC71 and A673 were kindly provided by Dr. S. Ambati, MSKCC. Lymphoma cell lines include: SU-DHL-6 (CRL- 2959), Toledo (CRL-2631), Farage (CRL-2630) and BC3 (CRL-2277) were purchased from ATCC; HBL-1, MD901 and U2932 were kindly provided by J.
  • Murine Cell Lines Wild type and TM96 expressing MethA fibrosarcoma cells were kindly provided by Dr. Z. Li, OSU. The cells were established as previously reported (Zheng et al., 2001) and cultured in RPMI medium with 10% heat-inactivated FBS (VWR) and 1% Penicillin/Streptomycin. Reagents [0163] PU-WS13, PU-WS13-biotin, inactive-WS13-biotin, PU29F, PU-H71, HJP-149 and SO-33 were synthesized using a previously reported protocol (Patel et al., 2015; Patel et al., 2013; Rodina et al., 2016).
  • PU-WS13 was synthesized via CuI-catalyzed coupling of 8- mercaptoadenine with 3,5-dichloroiodobenzene at 110 °C resulting in 8-(3,5-dichloro- phenylsulfanyl)adenine in 72% yield, which was heated with 3-(tertbutoxycarbonyl-isopropyl- amino)-propyl tosylate in DMF at 80 °C under nitrogen protection for 30 min.
  • PU-WS13-biotin was synthesized through alkylation of 8-(3,5-dichloro-phenyl sulfanyl)adenine at position N9 with N-(8- bromooctyl)phthalimide in the presence of Cs 2 CO 3 in DMF at room temperature to obtain 2-(8-(6- amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9- yl)octyl)isoindoline-1,3-dione in 21% yield.
  • the synthetic route to inactive-WS13- biotin comprises S-alkylation of 8-mercaptoadenine with 1-iodo-2-methoxyethane in aqueous KOH solution providing 87% yield of 8-((2- methoxyethyl)thio)-9H-purin-6-amine. Further reaction with N-(8-bromooctyl)phthalimide, followed by phathalimide-deprotection and coupling with NHS-active ester of biotin in DMF gave crude inactive-WS13-biotin. This resulting residue was purified by preparatory TLC (CH 2 Cl 2 :MeOH-NH 3 (7N), 10:1) to give in 72% yield the inactive- WS13-biotin.
  • Synthesis of PU29F commenced with the coupling of 2,4,5,6-tetraaminopyrimidine with the acid fluoride of the 3,4,5- trimethoxyphenylacetic acid resulting in N-(2,4,6-triaminopyrimidin-5-yl)-2-(3,4,5- trimethoxyphenyl)acetamide.
  • the acid fluoride was generated by treating phenylacetic acid derivative with cyanuric fluoride and pyridine in CH 2 Cl 2 .
  • the acetamide derivative was cyclized to 8-(3,4,5-trimethoxybenzyl)-9H-purine-2,6-diamine by heating it in alcoholic NaOMe.
  • Taxol (S1150), Erlotinib (S1023) and Lapatinib (S2111) were purchased from Selleckchem. Cetuximab was received as leftover from the MSKCC Clinical Pharmacy. Lambda protein Phosphatase (Lambda PPase, P0753S), Endo H (P0703S) and PNGase F (P0709S) were purchased from NEB Inc. High capacity Streptavidin Agarose (20361) was purchased from ThermoFisher Scientific.
  • Cell Fractionation and Immunoblotting [0164] Cells were either treated with DMSO (vehicle) or indicated compounds and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 0.5% NP40) supplemented with cocktail protease inhibitors (Roche) to produce whole-cell lysates. Lysates for cytosol and total membrane fractions were harvested and processed using ProteoExtract Subcellular Proteome Extraction Kit (Millipore Sigma) following the manufacturer's instructions. Plasma membrane proteins were prepared using the Minute Kit (Invent Biotechnologies Inc.) according to manufacturer’s instructions. Protein concentrations were determined using the BCA kit (Pierce).
  • HSP90 SMC-107 from Stressmarq
  • HER2 28-0004
  • myc R950-25
  • Calnexin 610523
  • Calnexin 610523
  • Calnexin 610523
  • HSP70 SPA-810
  • GRP94 SPA-850
  • GAPDH ab8245
  • GRP78 ab21685
  • HSP90 ⁇ ab2928
  • cleaved PARP G7341) from Promega
  • EGFR 4267
  • LRP6 2560
  • p-AKT S473
  • AKT 4691
  • Caspase 7 9494
  • p-ERK1/2 T202/Y204
  • ERK1/2 4695
  • p-STAT3 9145
  • STAT3 (12640
  • p-p65 S536) (3033
  • p65 8242
  • Flotillin-1 3253
  • Protein extracts were prepared in the indicated buffers and diluted in Felts buffer. Samples were incubated with D-biotin (Control), Inactive-WS13-biotin (Control), PU-WS13-biotin (GRP94 bait) or GRP94 antibodies for 3 hr at 4°C, followed by incubation with High Capacity Streptavidin agarose beads (ThermoFisher Scientific) or Protein A/G agarose beads (Roche) for another 2 hr at 4°C.
  • PU-WS13-biotin beads were prepared by incubating 20 ⁇ M PU-WS13-biotin (chemical bait) with High Capacity Streptavidin agarose beads (ThermoFisher Scientific) for 3 hr at 4 °C followed by washing with Felts buffer for three times.
  • Antibody beads were prepared by incubating 9G10 or G4420 anti-GRP94 antibodies with protein A/G agarose beads (Roche) for 2 hr at 4 °C followed by washing with Felts buffer for three times.
  • the pre-formed chemical bait or antibody bait was then added into the cell lysate and the mixture was incubated on a rotator for 3 hr at 4 °C. After separating the beads by centrifugation, the supernatant was collected and incubated with new pre-formed chemical bait or antibody bait.
  • the sequential capture experiment was carried out by repeating the chemical precipitation (CP)/immunoprecipitation (IP) three times before the final IP with the indicated antibody bait. Captured cargos at each step were washed with Felts buffer three times before loading onto SDS-PAGE and subjecting to immunoblotting.
  • siRNA knock-down of GRP94 Transient transfections were carried out using Lipofectamine RNAiMax reagent (ThermoFisher) according to the manufacturer's instructions.
  • siGRP94 Gene HSP90B1
  • scramble siRNA were purchased from Qiagen. Cells were transfected with 5 nM or 20 nM siRNA. The knockdown efficiency and other cellular markers were evaluated at 72 hr post transfection by immunoblotting.
  • Immunofluorescence [0170] Cells were seeded and grown onto Lab-Tek II chamber slides for 24 hr before the experiment.
  • HER2 Zymed; 28004; 1:50
  • HER2-FITC BD; 340553; 1:200
  • GRP94 Sigma; G4420; 1:100
  • EEA1 Abcam; ab70521; 1:100
  • Calnexin Abcam; 22595; 1:100
  • Enzymatic Deglycosylation and Dephosphorylation [0171] Cell lysates were treated with Endo H or PNGase F according to the manufacturer’s instructions. After reacted at 37°C for 1h, the samples were mixed with protein loading buffer and subjected to immunoblotting.
  • the lysates were firstly diluted in Felts buffer and then incubated with the enzymes without protein denaturing on ice overnight.
  • the deglycosylated samples were further used for detatured or native gel electrophoresis, chemical or immuno precipitation experiments.
  • 1 ⁇ enzyme reaction buffer (1 ⁇ Protein MetalloPhosphatase (PMP) buffer for lambda PPase, 1 ⁇ glyco buffer 3 for Endo H or 1 ⁇ glyco buffer 2 for PNGase F.
  • PMP Protein MetalloPhosphatase
  • Enzymes were added into the reaction tubes and incubated for 1hr at 30 o C for PPase, at 37 o C for Endo H and PNGase F. Immediately after the reaction was finished, the samples were mixed with the protein loading buffer, heated at 95 o C for 5 min, stored on ice before loading into the gels.
  • Cell surface protein isolation kit (Pierce) was used to purify the cell surface proteins according to the manufacturer’s instructions. Briefly, cell surface proteins were biotinylated by incubating the live cells with Sulfo-NHS-SS-biotin for 30 min at 4 °C. The reaction was quenched and cells were lysed.
  • CRISPR/Cas9 mediated Knock-Out and targeted Mutagenesis of endogenous GRP94 were designed using the online tools CRISPOR (http://crispor.tefor.net/) and CHOPCHOP (https://chopchop.cbu.uib.no/).
  • sgRNAs against hGRP94 used in this study are: sg1948, GAAGAAGCTATTCAGTTGGA; sg3859, CAACGATACCCAGCACATCT.
  • the single- stranded oligos were synthesized by Intergrated DNA Technologies, cloned into PX458 (pSpCas9(BB)-2A- GFP, Addgene plasmid #48138, (Ran et al., 2013)) via BsaI, and the positive clones were validated by plasmid sequencing (Genewiz).
  • T7 endonuclease I assay Genomic regions flanking the CRISPR sgRNA target sites were PCR amplified with Fusion Flash High-Fidelity PCR Master Mix (F548S, ThermoFIsher Scientifics) using gene-specific primers. PCR products were purified with MinElute PCR Purification Kit (Qiagen) and hybridized in PCR buffer (95°C, 5 min; 95-85°C at ⁇ 2°C/s; 85- 25°C at ⁇ 0.1°C/s; hold at 4°C).
  • the sequencing primers used in this study are: N62-F, ccattttaacccccaagaca; N62-R, atcaggccgtgaacctattt; N217-F, cactttcagaaaaggccataaaa; N217-R, caggaaaattaaggcccaga.
  • Whole cell lysates were also validated by immunoblotting with GRP94 antibodies.
  • Soft Agar Colony Formation Assay [0178] 6-well plates were coated with a bottom layer of 2 mL 1% low-melting-point agarose (Invitrogen) dissolved in the complete culture medium. Cell suspension in culture medium containing 0.4% low-melting-point agarose was then added on the top of the layer.
  • Example 1 Ex vivo Studies [0181] The fresh tissue slicing method maintains tissue integrity and architecture within an intact tumor- microenvironment-macroenvironment context throughout treatment, providing a more clinically- relevant means to assess the inhibitors’ effects. This is important because interactions among tumor and stromal cells are known to play a major role in cancer growth and progression and in the anti-tumor efficacy of agents.
  • De-identified pathology discarded specimens were obtained in accordance with the guidelines and approval of the Institutional Review Board# 09-121 (PI: Dr. Modi).
  • the primary breast cancer specimens or fresh esophagogastric PDX samples were processed as reported before (Corben et al., 2014). Briefly, the sample was delivered in a fresh state, harvested in a sterile environment under 30 minutes from the surgical procedure. Tumor tissue was chosen from the periphery of the index lesion to avoid potential frank central necrosis (cell death). The necrotic tissue may be grossly recognizable by any of the following criteria: loss of color or paleness of the tissue; loss of strength in which necrotic tissue is soft and friable; a distinct demarcation between the necrotic and viable tissue.
  • the sample was placed in wet ice and transported to the laboratory for ex vivo fresh tissue sectioning.
  • Samples were then embedded into 5% Agarose gel and cut into 200 ⁇ m thick sections on a Leica VT 1000S vibratome.
  • the live sections were transferred into 24-well tissue culture plates and treated for 24 hr or 48 hr with the indicated concentration of PU-WS13.
  • Sections were then fixed in 4% formalin for 1hr at room temperature, and transferred into 70% ethanol. Following paraffin embedding, sectioning and mounting, the sections were stained with hematoxylin and eosin (H&E) and evaluated by the pathologists.
  • H&E hematoxylin and eosin
  • Example 2 In vivo Studies in Mice [0182] For the breast cancer model: Athymic nude mice (Hsd:Athymic Nude-Foxn1 nu , female, 20-25 g, 6 weeks old; RRID:MGI:5652489) were obtained from Envigo and were allowed to acclimatize at the MSKCC vivarium for 1 week prior to implanting tumors.
  • Tumor xenografts were established subcutaneously into the dorsal flank or orthotopically into the 4 th mammary fat pad. Tumors were initiated by subcutaneous injection of 5 ⁇ 10 6 cells for MDA-MB-468 or orthotopic injection of 5 ⁇ 10 6 cells for AU565 in a 200 ⁇ L cell suspension of a 1:1 v/v mixture of PBS with reconstituted basement membrane (BD matrigel, Collaborative Biomedical Products Inc.).
  • Tumor Volume was determined by measurement with Vernier calipers, calculated using the formula - length ⁇ width 2 ⁇ 0.5 and analyzed on indicated days as the median tumor volume ⁇ SD Mice were randomized prior to treatments, and euthanized after similar PU-WS13 treatment periods and at a time before tumor reached a size that resulted in discomfort or difficulty in physiological functions in the individual treatment group, in accordance with the IUCAC protocol.
  • Esophagogastric PDX model was generated as previously described (Mattar et al., 2018). Briefly, patient specimens ( ⁇ 0.5 g) collected under the approved IRB protocol (10-018, PI: Dr.
  • PU-WS13 was extracted in methylene chloride, and the organic layer was separated and dried under vacuum. Samples were reconstituted in mobile phase. Concentrations of PU-WS13 in tissue or plasma were determined by high-performance LC-MS/MS. PU-H71 was added as the internal standard. Compound analysis was performed on the 6410 LC-MS/MS system (Agilent Technologies) in multiple reaction monitoring mode using positive-ion electrospray ionization.
  • a Zorbax Eclipse XDB-C18 column (2.1 ⁇ 50 mm, 3.5 ⁇ m) was used for the LC separation, and the analyte was eluted under an isocratic condition (80% H 2 O + 0.1% HCOOH: 20% CH 3 CN) for 3 min at a flow rate of 0.4 mL/min.
  • a Zorbax Eclipse XDB-C18 column (4.6 x 50 mm, 5 ⁇ m) was used for the LC separation, and the analyte was eluted under a gradient condition (H 2 O+0.1% HCOOH:CH 3 CN, 95:5 to 70:30) at a flow rate of 0.35 mL/min.
  • PD study Tumors or tissues were homogenized in tissue lysis buffer (50 mM Tris- HCl pH 7.5, 50 mM KCl, 150 mM NaCl, 2 mM EDTA, 0.1% Sodium deoxycholate, 0.5% NP40, 0.5% Triton X-100, 0.5% SDS) using Bullet Blender Tissue Homogenizer (Next Advance Inc.). Protein concentrations were determined using the BCA kit (Pierce) according to the manufacturer’s instructions. Protein lysates (20-100 ⁇ g) were electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated antibodies.
  • tissue lysis buffer 50 mM Tris- HCl pH 7.5, 50 mM KCl, 150 mM NaCl, 2 mM EDTA, 0.1% Sodium deoxycholate, 0.5% NP40, 0.5% Triton X-100, 0.5% SDS
  • Example 4 Efficacy Studies
  • Mice bearing MDA-MB-468 or AU565 xenograft tumors reaching a volume of 100–150 mm 3 were treated i.p. with PU-WS13 (75mg/kg or 125mg/kg, dissolved in 60mM citrate buffer (pH 4.0) with 30% Captisol) or vehicle, on a 3-times or 5 times per week schedule, as indicated.
  • Tumor volume (in mm 3 ) was determined by measurement with Vernier calipers, and was calculated as the product of its length ⁇ width 2 ⁇ 0.5.
  • mice were sacrificed after similar PU-WS13 treatment periods, and at a time before tumors reached a size that resulted in a discomfort or difficulty in physiological functions of mice in the individual treatment group, in accordance with our IUCAC protocol. All animals were observed daily for mortality from the time of animal receipt through the end of the study. Body weights for all animals were recorded no more than three times, but no fewer than once per week during the administration of the test article. All mice were observed for clinical symptoms at the time the animals were received and on all days in which the test article was administered.
  • Example 5 Toxicology Studies [0186] Toxicology studies: The study assessed the safety and relevant toxicities of PU- WS13 administered by i.p.
  • mice were anesthetized with isoflurane and approximately 100 ⁇ L of whole blood was collected from the orbital plexus of each mouse into a labeled tube containing EDTA anticoagulant.
  • EDTA anticoagulant EDTA anticoagulant
  • complete necropsies hematology and clinical chemistry were analyzed.
  • a necropsy was performed on each animal.
  • Gross examinations of each animal including internal organs were performed by a pathologist and any macroscopic lesions or other abnormal findings were recorded using standard terminology. For histopathology, tissues were collected and preserved in formalin.
  • Example 6 Identification of N-Glycosylation sites using nano LC-Mass Spectrometry (LC- MS/MS) [0187] Samples were treated with EndoH as described. Initially, non-treated samples, as well as samples treated with EndoH were utilized to develop the mass spectrometric workflow used.
  • Protein samples were separated by SDS-PAGE and processed using standard published protocols (Rodina et al., 2016) with the following modifications: Gel regions containing endoplasmin were generously excised and subjected to in-gel tryptic digestion with 200-300ng Trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) overnight, and after acidification with 10% formic acid (final concentration of 0.5-1% formic acid) resulting peptides were desalted using hand packed reversed phase Empore C18 Extraction Disks (3M, St. Paul, MN, USA) using a method described before (Rappsilber et al., 2007).
  • Desalted peptides were concentrated to a very small droplet by vacuum centrifugation and reconstituted in 10 ⁇ L 0.1% formic acid in water. Approximately 90% of the peptide material was used for liquid chromatography followed by tandem mass spectrometry (LC-MS/MS).
  • a Q Exactive HF mass spectrometer was coupled directly to an EASY-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a self-packed 75 ⁇ m x 20-cm reverse phase column (360 mm OD, 75 mm ID, 10 mm ID tip Picotip emitter, New Objective, Woburn MA column packed with ReproSil-Pur C18, 3 ⁇ M beads, Dr. Maisch GmbH, Germany) for peptide separation.
  • Peptides were eluted with a 3-40% acetonitrile gradient over 110 min at a flow rate of 250 nL/min.
  • the mass spectrometer was operated in DDA mode with survey scans acquired at a resolution of 120,000 (at m/z 200) over a scan range of 300- 1750 m/z. Up to 15 most abundant precursors from the survey scan were selected with an isolation window of 1.6 Th for fragmentation by higher-energy collisional dissociation with normalized collision energy (NCE) of 27.
  • NCE normalized collision energy
  • the maximum injection time for the survey and MS/MS scans was 60 ms and the ion target value (AGC) for both scan modes was set to 3e6.
  • Mass Spectrometry Data Processing All mass spectra were first converted to mgf peak list format using Proteome Discoverer 1.4 (Thermo Fisher Scientific, Waltham, MA, USA) and the resulting mgf files searched against a human UniProt protein database using Mascot (Matrix Science, London,UK; version 2.5.0; www.matrixscience.com). Decoy protein sequences with reversed sequence were added to the database to allow for the calculation of false discovery rates (FDR).
  • FDR false discovery rates
  • a stock of 10 ⁇ M cy3B-GM and PU-FITC3 was prepared in DMSO and diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl 2 , 20 mM Na 2 MoO 4 and 0.01% NP40 with 0.1 mg/mL BGG). To each well was added the fluorescent dye–labeled FP ligand, protein and tested inhibitor (initial stock in DMSO) in a final volume of 100 ⁇ L Felts buffer. Compounds were added in duplicate or triplicate wells.
  • FP refers to the fluorescent polarization binding assay with purified Grp94 protein – equilibrium binding.
  • cyt refers to alamar blue cytotoxicity assay in MDA-MB-468 breast cancer cells – N-glyc GRP94 dependent cancer cell. See Yan et al., Cell Reports 2020, 31, 107840, June 30, 2020, incorporated herein by reference. [0193] Results of FP and cyt Assays:
  • Example 9 Compound Profiles in Native Page and co-IP Studies
  • DMSO vehicle
  • RIPA buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 0.5% NP40
  • protease inhibitors Roche
  • Protein concentrations were determined using the BCA kit (Pierce).
  • the protein lysates (10–50 ⁇ g) were electrophoretically resolved by SDS-PAGE, transferred onto nitrocellulose membranes and probed with the indicated antibodies.
  • EGFR 4267) from Cell Signaling Technology; cleaved PARP (G7341) from Promega; HSP70 (SPA-810), GRP94 (SPA-850) from Enzo; GAPDH (ab8245) from Abcam; HER2 (28-0004) from Invitrogen.
  • the membranes were washed with TBS/0.1% Tween-20 and incubated with appropriate HRP-conjugated secondary antibodies.
  • Chemiluminescent signal was detected with Enhanced Chemiluminescence Detection System (GE Healthcare) according to the manufacturer’s instructions.
  • Native Gel Electrophoresis Native Gel Electrophoresis (Native PAGE) and Coomassie stain. Cells were lysed in the RIPA buffers.
  • Cells were either treated with DMSO (vehicle) or compounds for indicated time and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 0.5% NP40) supplemented with cocktail protease inhibitors (Roche) to produce cell lysates.
  • the lysates were diluted in Felts buffer (20 mM Hepes pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4 and 0.01 % NP40 with 0.1 mg/mL BGG).
  • p185erbB2 binds to GRP94 in vivo. Dissociation of the p185erbB2/GRP94 heterocomplex by benzoquinone ansamycins precedes depletion of p185erbB2.
  • the epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.
  • Nature Communications s41467-019-14082-5. Joshi, S., Wang, T., Araujo, T.L.S., Sharma, S., Brodsky, J.L., and Chiosis, G. (2018). Adapting to stress - chaperome networks in cancer. Nat Rev Cancer 18, 562-575. Kim, J.Y., Jung, H.H., Do, I.G., Bae, S., Lee, S.K., Kim, S.W., Lee, J.E., Nam, S.J., Ahn, J.S., Park, Y.H., et al. (2016).
  • Fibroblast-secreted hepatocyte growth factor mediates epidermal growth factor receptor tyrosine kinase inhibitor resistance in triple-negative breast cancers through paracrine activation of Met.
  • Neuropathology official journal of the Japanese Society of Neuropathology 31, 583-588. Nussinov, R., Tsai, C.J., and Jang, H. (2019). Protein ensembles link genotype to phenotype.
  • Hsp90 inhibitors define tumor-specific regulation of HER2. Nat Chem Biol 9, 677-684. Pillarsetty, N., Jhaveri, K., Taldone, T., Caldas-Lopes, E., Punzalan, B., Joshi, S., Bolaender, A., Uddin, M.M., Rodina, A., Yan, P., et al. (2019).
  • Endoplasmic reticulum heat shock protein gp96 maintains liver homeostasis and promotes hepatocellular carcinogenesis.
  • a combination of Trastuzumab and 17- AAG induces enhanced ubiquitinylation and lysosomal pathway-dependent ErbB2 degradation and cytotoxicity in ErbB2-overexpressing breast cancer cells.
  • Selective compounds define Hsp90 as a major inhibitor of apoptosis in small-cell lung cancer. Nat Chem Biol 3, 498-507.
  • the epichaperome is an integrated chaperome network that facilitates tumour survival. Nature 538, 397-401. Schwarz, F., and Aebi, M. (2011). Mechanisms and principles of N-linked protein glycosylation. Curr Opin Struct Biol 21, 576-582. Shi, J., Yao, D., Liu, W., Wang, N., Lv, H., He, N., Shi, B., Hou, P., and Ji, M. (2012). Frequent gene amplification predicts poor prognosis in gastric cancer. International journal of molecular sciences 13, 4714-4726. Shrestha, L., Patel, H.J., and Chiosis, G. (2016).

Abstract

The present disclosure provides, among other things, methods of treating cancer. In some embodiments, the present disclosure provides methods of treating cancer comprising administering an inhibitor of N-glycosylated Grp94.

Description

INHIBITION OF N-GLYCOSYLATED GRP94 CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of and priority to U.S. Provisional Application No.63/045,021, filed June 26, 2020, the contents of which is hereby incorporated herein by reference in its entirety. BACKGROUND [0002] Changes in the fitness of the endoplasmic reticulum (ER) and the function of the proteins within it are associated with various diseases including cancer, autoimmune disorders, neurodegeneration and cardiac dysfunction (Giampietri et al., 2015; Luoma, 2013; Mekahli et al., 2011; Ozcan and Tabas, 2012; Yadav et al., 2014). Understanding the nature of such dysfunctions is central to how to treat these diseases. [0003] An important component and regulator of ER function is Glucose regulated protein 94 (GRP94) also called gp96 or endoplasmin. GRP94 is one of four HSP90 paralogs and shares 50% amino acid homology with the cytosolic chaperone HSP90 (Marzec et al., 2012; McCaffrey and Braakman, 2016; Zhu and Lee, 2015). In normal cells, GRP94 functions in ER quality control, buffers Ca 2 + levels, and is a key chaperone in the folding of “client” proteins. These clients include the TGFβ associated protein GARP, insulin like growth factors, Toll-like receptors (TLRs) and integrins (Ansa-Addo et al., 2016; Eletto et al., 2010). [0004] While primarily localized to the ER, GRP94 is also found in the cytosol, at the cell surface, and extracellularly (Ansa-Addo et al., 2016; Lee, 2014; Wiersma et al., 2015). This phenomenon is often associated with and enhanced under conditions of chronic stress, such as associated with disease (Altmeyer et al., 1996; Booth and Koch, 1989; Lee, 2014; Wiersma et al., 2015; Wu et al., 2016; Zheng et al., 2001). For example, in dendritic cells, forced overexpression of cell-surface GRP94 results in spontaneous autoimmune disease due to the GRP94-mediated enhancement of TLR function and its subsequent downstream signaling through MyD88 (Liu et al., 2003). Similarly, pathogens use surface GRP94 to infect host cells (Martins et al., 2012), and extracellular GRP94 complexed with IgGs plays a pathologic role in type I diabetes (Pagetta et al., 2014). Finally, in breast cancer (BC) cells, chronic proteome stress induced by overexpression of HER2 kinase leads to enhanced translocation of GRP94 to the plasma membrane (PM) (Chavany et al., 1996; Li et al., 2015; Patel et al., 2013). There, GRP94 maintains the stability of HER2 and its enhanced downstream signaling (Patel et al., 2013). While the function of these disease- associated GRP94 pools is becoming better understood, little is known on how stress structurally modifies the ER chaperone to alter its function. [0005] Stress also appears to modulate post-translational modifications (PTMs) on GRP94 (Cloutier and Coulombe, 2013). While GRP94 isolated from normal tissues is primarily monoglycosylated (Wearsch and Nicchitta, 1996), the heterologous overexpression of murine GRP94 in COS cells or of canine GRP94 in Sf21 insect cells results in an altered GRP94 glycosylation pattern, with several hyperglycosylated forms observed (Qu et al., 1994). Similar species were also noted in preparations of endogenous GRP94 isolated from mouse sarcomas (Feldweg and Srivastava, 1995). The functional significance of such modifications is however poorly understood. Earlier studies showed that both non-glycosylated and glycosylated GRP94 species associate with various client proteins, including Ig light chain, α1-antitrypsin and plasma IgGs (Marzec et al., 2012; Pagetta et al., 2014). A hyperglycosylated GRP94 was also reported, but it is a non- functional form targeted for degradation in an OS-9–mediated, ERAD-independent, lysosomal- like mechanism (Cherepanova et al., 2019; Dersh et al., 2014). [0006] While stress is a common hallmark of disease, it is mostly studied as a damager of proteins and of their function (Solimini et al., 2007). In this context, chaperones such as GRP94 are important in stress regulation as they may correct and influence such damage through folding or dis- aggregation and degradation (Brehme and Voisine, 2016). Accordingly, changes in chaperone expression, have been extensively studied in disease, and GRP94 overexpression has been implicated in cancer (Buc Calderon et al., 2018; Lee, 2014). Stress however also alters how proteins interact (Harper and Bennett, 2016), a feature also influenceable by chaperones (Ellis, 2013). Accordingly, structurally modified chaperone pools, termed epichaperomes, may form under stress and act as scaffolds to pathologically remodel cellular processes by mediating aberrant protein-protein interactions, and in turn, creating a state of proteome-wide connectivity dysfunction (Dart, 2016; Joshi et al., 2018). While first reported in cancer (Pillarsetty et al., 2019; Rodina et al., 2016), recent evidence in Parkinson’s and Alzheimer’s (Inda et al., 2020; Kishinevsky et al., 2018) proposes that chaperone-mediated protein mis-assembly, and in turn protein connectivity dysfunction, has broad disease relevance. Yet a biochemical mechanism for this phenomenon has been difficult to pinpoint, and evidence for how a chaperone may switch from a folding protein into a maladaptive scaffolder remains to be provided. SUMMARY [0007] Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. The present disclosure unveils a specific N- glycosylation pattern used by a chaperone, GRP94, to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This ‘protein assembly mutation’ remodels protein networks and properties of the cell. The present disclosure shows in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In sum, the present disclosure provides biochemical evidence for stressor induced chaperone- mediated protein mis-assemblies and demonstrates how these alterations are actionable in disease. [0008] In some embodiments, the present disclosure provides a method of treating cancer, inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis, or diabetes, comprising the step of administering to a subject suffering therefrom an effective amount of a compound of Formula (I):
Figure imgf000004_0001
or a pharmaceutically acceptable salt thereof, wherein X1, X2, X3, X5, Z2, and R1 are as defined herein. BRIEF DESCRIPTION OF THE DRAWINGS [0009] Figures 1A-1E describe the sensitivity of cancer cells and primary specimens to GRP94 inhibition by PU- WS13. (A) Viability of cancer cell lines (n = 64) treated for 72 hr with PU-WS13 (10 μM) or PU-29F (20 μM). Mean values of triplicate experiments are graphed. Negative values depict killing of the initial cell population. (B) Correlative analysis between RTK (i.e. HER2 and EGFR) levels and cell viability for BC cells in (A). Pearson’s r, two-tailed, n = 12. (C) Ex vivo sensitivity of primary BC explants treated for 48 hr with PU-WS13 (10 μM), PU-29F (20 μM) or taxol (1 μM) evaluated as indicated in the associated schematic. Mean values from 2 slices per condition are shown. (D) Western blot analysis of MDA-MB-468 cells treated for 24 hr with PU-WS13 (0, 0.1, 0.25, 0.5, 1, 2.5, 5, 10 μM). PU-WS13, WS13. Graph, mean of 3 independent experiments; error bars, SEM. (E) Same as in C for an EGFR+ BC specimen treated with PU-WS13 (20 μM) or Vehicle for 24 hr. Size bar, 200 μm. [0010] Figures 2A-2G describe that GRP94 is biochemically and functionally heterogeneous in cancer cells. (A and B) Native-PAGE (top) and SDS-PAGE (bottom) separation followed by immunoblot with the 9G10 anti-GRP94 antibody in un-treated cell lines (A) or in those treated for 4 hr with PU- WS13 (10 μM) (B). Each data point is an individual cell line; 1-11: BT474, MDA-MB-468, SKBr3, AU565, MDA-MB-361, HCC1806, MCF7, MDA-MB-231, T47D, BT20, HMEC. Graph, mean. Error bar, SEM, unpaired t-test, **p < 0.01, ***p < 0.001. (C) Biochemical signature of GRP94 in distinct cellular compartments of a PU-WS13-sensitive (SKBr3) and a resistant (MCF7) cell line. Cells were treated as in (B) prior to fractionation. WCL, whole cell lysate; C, cytosol; TM, total membrane; F1, cytosol and ER/Golgi; F2, plasma membrane. HSP70, cellular fractionation and loading control. LE and HE, low and high exposure, respectively. black arrow, lower motility GRP94 specific to F2; grey arrow, higher motility GRP94 specific to F1. Graph, mean, n = 4. Error bar, SEM, unpaired t-test, ***p < 0.001. (D) Representative immunofluorescence (IF) staining of SKBr3 cells (n = 50) treated as in (B). Calnexin, ER marker; DAPI, grey, nuclear stain; PU-WS13, WS13. Size bar, 10 μm. Graph, mean. Error bar, SEM, unpaired t-test, ****p < 0.0001. (E) Same as in (C) for HER2 normalized to vehicle treated cells. Graph, mean, n = 9 for native- PAGE and n = 5 for SDS-PAGE. Error bar, SEM, unpaired t-test, ***p < 0.001. (F) IF staining of SKBr3 cells (n = 20) treated for 4 hr with vehicle (DMSO), PU-WS13 (WS13, 10 μM) or PU-29F (29F, 20 μM). Mean Pearson’s correlation coefficient (PCC) R value is shown corresponding to the colocalization of HER2 and EEA1. Size bar, 10 μm. Graph, mean. Error bar, SEM, one-way ANOVA with Dunnett's post-hoc, ***p < 0.001, ****p < 0.0001. (G) Western blot analysis of the protein cargo isolated by streptavidin- immobilized WS13-B (a biotinylated version of PU-WS13) from SKBr3 total membrane extracts. [0011] Figures 3A-3C describe a specific GRP94 conformation enables the formation of stable HMW GRP94 and HER2 pools. (A) Anti-GRP94 antibody captured cargo from SKBr3 cell extracts and the remaining supernatant separated under native and denaturing conditions, and immunoblotted with the indicated antibodies.+, 1x, ++, 2x antibody amount. IgG, control; black arrow, unspecific signal. (B) Immuno-capture as in (A) of SKBr3 cells treated with PU-WS13 in a dose- and time- dependent manner. Graph, mean of 3 independent experiments; error bars, SEM. (C) Summary of the findings. [0012] Figures 4A-4F describe that the N-glycan content of GRP94 regulates the stability and function of the HMW GRP94-HER2 PM-associated pool. (A) Schematic of the experimental design to investigate the role of N-glycans on the conformation, stability and function of the HMW GRP94 pools. WS13-B, biotinylated PU-WS13. PM pool of GRP94 and HER2 in SKBr3 extracts treated under native conditions as in (A) with the indicated enzymes. Graph, mean; error bars, SEM, n = 5, unpaired t-test, ***p < 0.001, ****p < 0.0001. (B) Western blot analysis of GRP94 and HER2 isolated from extracts treated as in (A and B). Graph, mean; error bars, SEM, n = 3 to 5, unpaired t-test, **p < 0.01, *p < 0.05. (C) Western blot analysis of cells treated for 4 hr with PU- WS13 (10 μM) prior to N-glycan removal and immuno-capture with the G4420 anti-GRP94 antibody as in (A). Graph, mean; error bars, SEM, n = 3, unpaired t-test, **p < 0.01. (D) Biochemical signature of HER2 in extracts as in (C and D). (E) Summary of the findings. [0013] Figures 5A-5G describe that glycosylation at N62 of GRP94 is a key regulator in the formation of the HMW GRP94 variant and is important for its oncogenic activity. (A) Schematic for clone generation and validation. MDA-MB-468 cells (WT) and clones containing the indicated GRP94 mutants were then used for the analyses below. (B) The effect of GRP94 N- glycan mutagenesis or KO evaluated on glycosylation load. (C) GRP94 HMW formation (measured by blotting for GRP94 HMW species on native-PAGE and ERK activity on SDS- PAGE) and its sensitivity to PU-WS13, in cells treated for 24 hr with 0, 0.25, 1μM PU-WS13. (D) Steady-state levels of EGFR and activity of its downstream signaling (measured by p-ERK levels) in cells treated for 24 hr to PU-WS13. Graph, mean±SEM; quantification of 3 western blot analyses. (E) Baseline activity of EGFR-downstream signaling measured by western blot analysis of p- ERK and ERK. Graph, mean (n = 3). Error bar, SEM, one-way ANOVA with Dunnett's post- hoc, *p < 0.05, **p < 0.01. (F) GRP94 immunocapture in clones pre-treated for 4 hr with vehicle (-) or PU-WS13 (2.5 μM).4x times more lysate from the N62Q-containing clone was loaded to normalize for the GRP94 input. (G) Cellular localization of EGFR and GRP94.1, WCL; 2, F1; 3, F2 fractions. Tubulin, HSP70 and Flotillin-1 are controls for equal loading and for cellular fractionation purity; NA, not available. Graph, mean±SEM; one-way ANOVA with Dunnett's post- hoc, n = 6 individual experiments. ***p < 0.001, **p < 0.01. [0014] Figures 6A-6E describe the accumulation of hgGRP94 at the PM is sufficient to augment HMW GRP94 levels, stabilize receptors at the PM and rewire protein networks in the cytosol. (A) Biochemical profile of GRP94 in the indicated cell fractions obtained from cells containing the WT (1) or the TM96 (2) GRP94 construct. LRP6, control for PM proteins, p-p65, p- ERK control for signaling activity and HSP90-incorporating epichaperomes for oncogenic activation of cytosolic chaperomes. myc, control for the presence of TM96. Gels are representative of three individual experiments. (B) Western blot of indicated cells treated for 24 hr with PU- WS13 (10 μM). Graphs, mean±SEM, n = 3, one-way ANOVA with Dunnett's post-hoc, ***p < 0.001. (C) Anchorage-independent growth in soft agar examined for MethA WT and TM96 cells at day 21. Graph, mean±SEM, n = 3 experiments, unpaired t-test, **p < 0.01. (D) Viability of cells treated with PU-WS13 for 72 hr. Graph, mean±SEM, n = 4, two-way ANOVA, ****p < 0.0001. Negative y-axis values depict killing of the initial cell population. (E) Schematic of the findings. [0015] Figures 7A-7L describe that the HMW GRP94 tumor variant is an actionable target. (A and B) PD analyses at 24 hr after one dose of PU-WS13 (75 mg/kg, i.p.) administered to mice bearing AU565 (A) or MDA-MB-468 (B) orthotopic tumors. Graphs, mean ± SEM, n = 6, Vehicle (V), n = 4, PU-WS13 (WS13) for AU565 and n = 11, V, n = 9, WS13, for MDA-MB-468, unpaired two tailed t-test, ***p < 0.001, *p < 0.05. See Figures 13A-13B for representative western blot analyses. (C) Molar concentration of PU-WS13 in individual tumors and tissues for mice (n = 12) as in B. Graphs, mean ± SEM. T, tumor; H, heart; L, lung; 4, Stomach; LI, large intestine. (D) Dose and schedule paradigms used in testing the efficacy and safety of PU-WS13. 75MWF, 75 mg/kg given Mon-Wed-Fri; qod, every-other-day; qd, every day, Mon through Fri. (E) Tumor weight measurements of mice as in (A) after 64 days of treatment as indicated in D.1, n = 12; 2, n = 9; 3, n = 6; 4, n = 9; 5, n = 6; 6, n = 6, 7, n = 6, pooled data from two experiments. Graph, mean ± SEM, two-tailed Mann Whitney test, *p < 0.05. (F) Tumor weight measurements of mice as in (B) after 87 days of treatment as indicated in D.1, n = 6; 2, n = 8; 4, n = 10; 5, n = 6; 7, n = 8, pooled data from two experiments. Graph, mean ± SEM, one-way ANOVA, Dunnett’s multiple comparisons, *p < 0.05, ****p < 0.0001. (G) Concentration of PU-WS13 in individual tumors as in F. (H) Tumor volume monitoring during treatment as in D of mice bearing subcutaneous MDA-MB- 468 tumors. Graph, mean ± SEM, n = 7 per cohort, two-way ANOVA, ****p < 0.0001. (I) Tumor weight measurements of mice as in (H). Graph, mean ± SEM, one- way ANOVA, Dunnett’s multiple comparisons, *p < 0.05, **p < 0.01. Picture of individual tumors is also shown. No tumor, no macroscopic tumor evident at sacrifice. Size bar, 2.5 cm. (J) Body weight monitoring of mice as in (F). Graph, mean ± SD. (K) H&E stained tissues and organs from cohorts 1 and 7 as in (F). Size bar, 500 μm. (L) Western blot of indicated tissues from individual mice treated as in (B). LRP6, control for HMW GRP94 selectivity; HSP70, control for GRP94 over HSP90 selectivity. [0016] Figures 8A-8E describe the sensitivity of cancer cells to PU-WS13 inhibition, Related to Figures 1A-1E. (A) Viability (by Annexin staining) of breast cancer cell lines treated for 48 hr with PU-WS13 (WS, 10 μM) or Vehicle (V, DMSO). (B) Ex vivo sensitivity of primary esophagogastric cancer tumors treated for 24 hr with PU-WS13 (10 μM) or vehicle (V, DMSO) and evaluated as indicated in the associated schematics. Error bars, mean±SEM, unpaired t-test, n = 2 - 3 slices per condition; * p <0.05, ** p < 0.01. Size bar, 500 μm. (C) Western blot analysis of cell lines treated for 24 hr with the PU-WS13 or its analogues, HJP-149 and SO-33 (10 μM), or with the HER2 inhibitor lapatinib (1 μM), EGFR inhibitor erlotinib (1 μM) or the anti-EGFR antibody cetuximab (5 μg/mL). HMEC, human mammary epithelial cells. HSP70 serves as GRP94 over HSP90-inhibition specificity control; GAPDH and HSP90 loading controls. cPARP, cleaved PARP. (D) SDS-PAGE (top) and native PAGE (bottom) analysis of GRP94 knock-down in MDA- MB-468 cells treated for 72 hr with scramble or a GRP94-specific siRNA construct at the indicated concentrations. cPARP, cleaved PARP. (E) Western blot analysis of GRP94 in cells differentiated by their RTK levels. GAPDH, loading control. Graph, mean±SEM, unpaired t-test, ns, p > 0.05. S, WS13-sensitive, R, WS13-resistant cell lines. [0017] Figures 9A-9C describe the protein solubility tests and cell fractionation techniques used for the enrichment of the plasma membrane and ER-Golgi proteins, Related to Figures 2A- 2G. (A) Ponceau stained and GRP94 (9G10) or concanavalin A (ConA) blotted membranes of experiments from Figure 2C. SDS and native PAGE separation for SKBr3 and MCF7 whole cell extracts are show; first column, vehicle treated and second column, PUWS13 treated (10 μM for 4 hr). (B) Native PAGE analysis of protein unfolding induced by chemical denaturants such as urea. (C) Schematic of the fractionation methods and representative western blots are shown to indicate the distribution of cellular proteins in the respective cellular fractions. Controls for fraction purity and identity: Flotilin-1 for plasma membrane; HSP90 and tubulin for cytosol. [0018] Figures 10A-10D describe that a biotinylated PU-WS13 has preferential capture for the plasma membrane resident GRP94 and isolates GRP94 in complex with the RTK in RTK- overexpressing cancer cells, Related to Figures 2A-2G. (A) Western blot analysis of cell surface and intracellular GRP94. Cell fractions were obtained as indicated in the schematic. Black arrow indicates the GRP94 of MW higher than 100 kDa. (B) Western blot of protein cargo isolated by PU-WS13-biotin from SKBr3 extracts prepared as indicated in the schematic. Graph, mean±SEM, n = 3, unpaired t-test, **p < 0.01. (C) Western blot of protein cargo isolated by PU-WS13-biotin (PU-WS13-B) from MDA-MB-468 (EGFR-overexpressing breast cancer cell line) plasma membrane extracts. Biotin and an inactive PU-WS13 biotin derivative were used as specificity controls for the affinity purification. (D) Western blot of protein cargo from sequential capture steps performed with PU-WS13-B or the G4420 antibody as indicated. WS13-B beads, PU-WS13- biotin immobilized onto streptavidin beads; G4420 or 9G10 beads, protein G immobilized beads; black arrow, non-specific signal. [0019] Figures 11A-11C describe that GRP94 present at the plasma membrane has a higher N-glycan content than the ER resident GRP94, Related to Figures 4A-4F. (A) The effect of Endoglycosidase H (Endo H), Peptide:N-glycosidase F (PNGase F) and lambda-protein phosphatase (PPase) on GRP94 analyzed by western blot. ERK and HSP90 are used as controls. WCL, whole cell lysate; F1, enriched in ER-Golgi and cytosol fractions and F2, enriched in plasma membrane. (B) The effect of N-glycan removal on the interaction between GRP94 and RTKs as evidenced by the analysis of the protein cargo isolated by PU-WS13-biotin (WS13-B). (C) Plasma membrane pool of GRP94 treated under native conditions as indicated in schematic with the indicated enzymes or for 4 hr with PU-WS13 (10 μM) (right), and western blot analysis of cells treated for 4 hr with PU-WS13 (10 μM) or vehicle (-) prior to Nglycan removal and capture with the WS13-B reagent (right). Gels are representative of three individual experiments. [0020] Figures 12A-12C describe the effect of GRP94 N-glycan mutagenesis or KO on the HMW GRP94 species, Related to Figures 5A-5G. (A) The effect of protein loading on the biochemical signature of GRP94 and its sensitivity to PU-WS13 (WS13), or the lack of, in the indicated clones treated with either vehicle or PU-WS13 (5 μM, 4 hr). (B) Western blot analysis of whole cell extracts of indicated GRP94 mutant clones treated for 24 hr with PU-WS13 (0, 0.1, 0.25, 0.5 μM). (C) Cellular localization of EGFR and GRP94.1, WCL; 2, F1; 3, F2 fractions. Gels are representative of three independent experiments. [0021] Figures 13A-13B describe that TM96 GRP94 is N-glycosylated, participates in the formation of HMW GRP94 pools and is sensitive to PUWS13, Related to Figures 6A-6E. (A) Western blot analysis of GRP94 in plasma membrane extracts of MethA WT and TM96 cells in which N-glycan removal was performed with the indicated enzymes under native conditions. (B) Plasma membrane pool of HMW and total GRP94 in MethA TM96 extracts obtained from cells treated for 24 hr with PUWS13 (0, 5, 10 μM) prior to fractionation. Gels are representative of three independent experiments. [0022] Figures 14A-14C describe that the HMW GRP94 tumor variant is an actionable target, Related to Figures 7A-7L. (A) Schematic of the testing paradigm to evaluate the safety and efficacy of PU-WS13 in EGFR+ and HER2+ tumor bearing mice. PU-WS13, WS13. (B and C) Representative western blot analyses for the pharmacodynamic (PD) analyses of HMW GRP94 tumor markers following one dose of PU-WS13 (75 mg/kg) administered intraperitoneally (i.p.) to mice bearing AU565 (B) or MDA-MB-468 (C) orthotopic tumors. Each column, individual mouse. See Figure 7A, 7B for graphed data on all tumors. [0023] Figure 15 is related to Figures 7A-7L and describes hematology and clinical chemistry analyses of mice (n = 7, bearing subcutaneously established MDA-MB-468 tumors) treated with Vehicle or 125 mg/kg PU-WS13 daily for 87 days. [0024] Figure 16 depicts Native PAGE assays of PU-WS12, SO-IV-33A, and HJP-VI-110. [0025] Figure 17 depicts Native PAGE assays of PU-WS13, SO-IV-33A, SO-IV-26A, and SO-III-116A. [0026] Figure 18 depicts Immunoprecipitation (IP) of PU-WS13, HJP-V-149, WS-12, Bnlm, and HJP-V-92 with the indicated GRP94 antibodies. DETAILED DESCRIPTION [0027] The present disclosure describes, among other things, the role of N-glycosylated forms of Grp94 in certain cancers. The present disclosure also provides inhibitors of such N- glycosylated forms of Grp94. Inhibitors of Grp94 are known, including those described in WO 2015/023976, which describes compounds that are particularly suited for inhibiting Grp94 selectively over Hsp90. Against this backdrop, the present disclosure provides surprising evidence that only a subset of such compounds known to inhibit Grp94 are also capable of inhibiting N- glycosylated forms of Grp94 described herein. In particular, compounds described herein as inhibitors of N-glycosylated Grp94 are unexpectedly less potent against Grp94 in biochemical assays and/or less selective for Grp94 over Hsp90 compared to other compounds known in the art. [0028] The present disclosure encompasses the recognition that disease associated stresses may greatly modify the proteome creating intracellular pools of structurally and functionally heterogeneous proteins and protein assemblies. The present disclosure describes that these protein pools are chaperone mediated assemblies which portend disease-associated activity by remodeling proteome-wide connectivity, and thus function. The present disclosure combines chemical biology tools and complementary biochemical and functional approaches, with specific interest on functions and modifications induced by proteome stress associated with malignant transformation and mediated by GRP94 modifications. The present disclosure uses established cancer cell lines, fresh patient biospecimens and cell- and patient-derived xenografts in mice and ex vivo as disease models. [0029] The present disclosure identifies a biochemical mechanism whereby aberrant N- glycosylation of a fraction of the cellular pool of the chaperone GRP94 remodels its location and conformation, and in turn, its interaction strength and interaction partners (i.e. connectivity). The outcomes are aberrantly remodeled protein pathways and in turn, a pathologic cellular phenotype. The present disclosure therefore provides a missing link in chaperone-mediated protein connectivity dysfunction by demonstrating how stress hijacks the customary role of a protein, turning it from a folder into a remodeler of protein connectivity. GRP94 inhibition is lethal in a subset of tumor cells [0030] To better understand how oncogenic stress alters the proteome through chaperone- mediated modifications, the present disclosure investigated the vulnerability for apoptosis (i.e. sensitivity) of a panel of cancer cell lines (n = 64) encompassing eleven distinct tumor types (Figure 1A) to GRP94 inhibition by PU-WS13. PU-WS13 is a small molecule whose selectivity arises from its ability to bind to an allosteric pocket of GRP94 that only partly overlaps with the ATP-binding pocket and is not accessible in the closely related paralog, HSP90 (Gewirth, 2016; Patel et al., 2015; Patel et al., 2013; Shrestha et al., 2016; Stothert et al., 2017). It shows >100-fold selectivity over HSP90 and no interaction with kinases when tested at 10 μM in a 98-kinase panel (Patel et al., 2015). As a control, the present disclosure used PU-29F, a selective inhibitor of cytosolic HSP90 (Patel et al., 2013). [0031] The present disclosure provides that only a subset of these cell lines were vulnerable to PU-WS13 as measured by ATP levels and Annexin V staining (Figures 1A and S1A). To understand the basis of the differential response the present disclosure focused further on BC where vulnerability correlated with receptor tyrosine kinase (RTK) status (i.e. HER2 or EGFR), and most sensitive samples exhibited greater RTK levels than the insensitive ones (n = 12 cell lines, Figure 1B and S1E). Sensitivity to PU-WS13 was retained in RTK-overexpressing (RTK+) primary breast tumors (n=5 fresh BC tissue slices, Figure 1C) and esophagogastric tumors (n=2 patient- derived xenografted tumors, Figure 8B). These effects were GRP94 specific as there was no overlap with sensitivity to inhibition of cytosolic HSP90 or to taxol, a standard-of-care chemotherapy in BC (Figure 1A, 1C). PU-WS13 treatment of these cell lines and primary specimens was sufficient to reduce the steady-state level of the RTKs and impair downstream signaling through these receptors (Figures 1D, S1B and S1C, see p-ERK and p-STAT3). This is consistent with previous findings for GRP94 knock-down or inhibition in HER2+ BC cells (Li et al., 2015; Patel et al., 2013) and with GRP94 knock-down in EGFR+ BC cell lines (Figure 8D). [0032] The present disclosure found that suppressing GRP94 function by compounds described herein (e.g., PU-WS13, or analogues HJP-149 and SO-33), was more toxic to BC cell lines overexpressing HER2 and EGFR than was direct inhibition of the RTK by either a kinase inhibitor or an anti-RTK antibody as judged using PARP cleavage as a marker of cell death (Figure 8C). Conversely, non-transformed human mammary epithelial cells remained unaffected by treatment with PU-WS13. In the fresh breast cancer tissue explants (Corben et al., 2014; Rodina et al., 2016), normal cells adjacent to the cancer cells remained unaltered at concentrations of PU- WS13 that induced apoptosis in >70% of the tumor cells. This was seen in the benign lobules with associated acini and ducts (white arrows, Figure 1E) that remained unaltered in the same treated section where treated tumor cells showed pyknotic nuclei and apoptotic debris, nuclear morphological changes that are indicative of apoptosis (black arrows, Figure 1E). GRP94 is heterogeneous in cancer [0033] Total GRP94 levels were comparable between the different cancer cell lines assessed for sensitivity to GRP94 inhibition (Figure 8E), suggesting that chaperone concentration alone was not responsible for the different responses to inhibition. To understand the cause for heightened sensitivity to GRP94 inhibition in some cell lines, the present disclosure analyzed the GRP94 isolated from sensitive and resistant cell lines for residence in stable protein complexes, cellular localization, conformation, and post-translational modification. [0034] Cell homogenates from both inhibitor-sensitive and resistant cancer cells were run on native gels in buffers near the physiological pH (Figure 2A). In addition to the ~242 kDa dimer that is characteristic of non-transformed cells (Wearsch and Nicchitta, 1996), the present disclosure also observed a number of distinct and indistinct high molecular weight (HMW) GRP94 species above the main 242 kDa band when blotting with the 9G10 antibody which recognizes the charged linker region (residues 290-350) and is sensitive to the conformation of GRP94 (Edwards et al., 1984; Vogen et al., 2002). These species were significantly enriched in the PU-WS13-sensitive cell lines (Figure 2A) indicating an enrichment of the 9G10-recognized conformation in the inhibitor sensitive cell lines even though the total amount of GRP94 in all cell lines was comparable. This difference was not due to protein unfolding or degradation under the experimental conditions (Figures 9A, 9B). In native PAGE carried out near physiological pH (to avoid denaturation), the signal may reflect both binding of other proteins and the protein’s own conformation. Such complexity influences the subsequent immunoblotting step. The signal observed in native PAGE may therefore reflect both complexation and retained native conformation of the proteins, which is recognized by the antibody. When cells were treated with PU-WS13 prior to native PAGE analysis, a complete disappearance of the HMW GRP94 species was observed, but no change in total GRP94, as detected under denaturing conditions (Figure 2B). [0035] The present disclosure next profiled the subcellular localization of GRP94 (Figure 9C) in SKBr3 cells, a PU-WS13 sensitive cell line, and in MCF7 cells, an insensitive cell line. While the total (WCL) GRP94 levels in these two cell lines were comparable, as judged by Western blot of SDS PAGE separations, substantially more GRP94 was located in the PM fraction (F2) in SKBr3 cells compared to the MCF7 cells (Figure 2C, WCL vs F2/F1 where F1 is the GRP94 pool found in the ER, Golgi and cytosol). [0036] In SKBr3 cells, it was also observed that F2 was enriched in a ~100 kDa GRP94 species compared to the same fraction in MCF7 cells. This species ran slower than the normal 94 kDa species on SDS gels and may reflect additional PTMs (Figure 2C, black vs. grey arrows), as the present disclosure demonstrate further. The distinctive mobility of this species was also apparent in the F2 fraction of SKBr3 cells under native PAGE separation and in experiments where surface proteins were isolated by biotinylation (Figure 10A). Finally, PU-WS13 treatment resulted in a decrease in the HMW species noted in F2 but had no effect on the GRP94 pool in F1 (Figure 2C, Native separation). [0037] In sum, the viability data presented above indicate that GRP94 is essential in only some cancer cells. Essentiality correlates with RTK overexpression and potentially, with an increase in the translocation of GRP94 to the PM, the formation of GRP94-containing HMW electrophoretic complexes, a preference for particular GRP94 conformations, and increased GRP94 PTM. The present disclosure therefore proceeded to investigate the contribution of each factor to the observed HMW species: complexation, conformation and PTM. GRP94 complexation contributes to its heterogeneity [0038] Because HER2, an abundant RTK in SKBr3 cells, co-localizes with GRP94 at the PM in these cells (Figure 2D and (Li et al., 2015; Patel et al., 2013), the present disclosure probed the cellular fractions of this cell line for HER2. HER2 detected by Western blotting on native gels was observed as a HMW species in the whole cell lysate (WCL), total membrane (TM), and PM fractions (Figure 2E). These HMW HER2 species, absent in MCF7 cells, correspond to the same fractions that contained the HMW GRP94 species. Short-term treatment of SKBr3 cells with PU- WS13 reduced the amount of HMW HER2 in the F2 pool (Figure 2D, immunofluorescence and Figure 2E, native PAGE) without majorly changing the overall levels of HER2 (Figure 2E, WCL by SDS-PAGE), paralleling the observations seen with GRP94. [0039] It was observed that HER2 at the PM, but not intracellular HER2, co-localized with early endosomes following short-term treatment with PU-WS13 (Figure 2F, EEA1 staining), indicating that GRP94 inhibition initiates clearance of PM-resident HER2, which involves internalization and endosomal sorting to lysosomes for degradation (Cortese et al., 2013; Raja et al., 2008). Conversely, PU- 29F affected intracellular HER2 pools. [0040] Finally, the present disclosure introduced a biotinylated PU-WS13 reagent, PU- WS13-B, which was found to preferentially isolate the GRP94 found in the F2, and not in the ER, Golgi, or cytosolic fractions (F1) (Figure 10B) and to enrich for the ~100 kDa GRP94 species (Figure 2G, black arrow). This probe pulled down HER2 along GRP94 (Figure 2G). Similar results were seen in EGFR+ BC cells, where EGFR, another RTK, was GRP94-bound (Figure 10C). The present disclosure found PU-WS13-B preferentially isolated GRP94 bound to HER2 over free GRP94 (Figure 10D). Conversely, the G4420 antibody, which recognizes amino acids 733-750 in the C-terminal domain of GRP94, was able to capture both the HER2-bound and the free GRP94 (Figure 10D). Conformation is key in HMW GRP94 formation [0041] The present disclosure next determined if GRP94’s conformational state contributes to formation of the HMW protein pool. The present disclosure probed the HMW pool with two anti-GRP94 antibodies, the conformation-specific 9G10 antibody, and the G4420 antibody, which is not known to discriminate between different conformational states of the chaperone. A dose-dependent immunocapture of GRP94 and HER2 by the G4420 antibody was associated with a dose-dependent decrease in the HMW GRP94 and HER2 pools noted on Native PAGE and a decrease in both GRP94 and HER2 levels in the supernatant noted on Western blot (Figure 3A). Conversely, the antibody 9G10 captured GRP94 but not HER2; nonetheless, both the GRP94 and HER2 HMW pools were diminished on native PAGE but only GRP94 was reduced in the supernatant following 9G10 immunocapture, consistent with a conformational change induced by this antibody which is associated with release of the bound cargo. [0042] As noted above, PU-WS13-B preferentially captured the PM-localized GRP94 which was bound to HER2. To see if this corresponds to a specific conformation of the chaperone, the present disclosure treated cells with PU-WS13 prior to immunocapture with the two GRP94 antibodies (Figure 3B). Increasing amounts of PU-WS13 or increased duration of PU-WS13 exposure dramatically reduced the amount of GRP94 captured by the 9G10 antibody, indicating that the inhibitor changes GRP94 to a conformation that is no longer recognized by the antibody. Conversely, PU-WS13 treatment increased the amount of both GRP94 captured by the G4420 antibody and HER2 pulled down by the immunocapture of PU-WS13-bound GRP94, indicating that the ligand promotes the chaperone conformation that associates with surface-resident HER2. [0043] While this shows that PU-WS13 induces a conformational change in GRP94, it also indicates that a specific conformation of GRP94 exists which enables the formation of the GRP94 and HER2 stable HMW protein pools (Figure 3C). Without wishing to be bound by any particular theory, it is believed that by adopting this conformation, GRP94 stabilizes the PM- bound HER2, an important mechanism for the oncogenic function of RTKs. Hyperglycosylation favors HMW GRP94 formation [0044] GRP94 is post-translationally modified by phosphorylation and glycosylation (Cala, 2000), and the present disclosure investigates whether these PTMs contribute to the formation or stability of the HMW GRP94 complexes. Treatment with Endoglycosidase H (Endo H) or Peptide:N-glycosidase F (PNGase F), but not with lambda-protein phosphatase, led to a change in the electrophoretic mobility of GRP94 (Figure 11A). The mobility shift due to glycosylation differed between cellular fractions. The F2 fraction exhibited a greater mobility shift compared to the ER F1 fraction. This indicates that both species are N-glycosylated but that more residues are glycosylated on the GRP94 protein localized at the PM compared to GRP94 in other locations, such as the ER. Because an N-linked carbohydrate chain has a mass of 1.5-3 kDa, the slower migrating GRP94 species that was detected in the F2 likely contains two or more N-glycan modified Asn residues. The present disclosure terms this F2- specific species as hyper-N- glycosylated GRP94 (hgGRP94). [0045] Since hgGRP94 was the predominant species in the F2 fraction, the present disclosure investigated the effect of removing N-glycosylation on the stability of the HMW GRP94 and HER2 complexes (Figure 4A). Following deglycosylation under native conditions, The present disclosure probed for GRP94 and HER2 HMW complexes on native gels, or captured the GRP94 complexes with immobilized PU-WS13-B, or with the two GRP94 antibodies G4420 or 9G10. Glycan removal significantly reduced the amounts of HMW GRP94 and HER2 species seen on native gels, indicating that glycosylation is important for the stability of these complexes (Figure 4B). The amount of the GRP94 and HER2 (or EGFR) cargo captured by PU-WS13-B and G4420 pulldown was also decreased by deglycosylation treatment (Figure 4C and 11B). The effect was more dramatic when the sugars were completely stripped from Asn (such as by PNGase F), with near complete disappearance of the HMW GRP94 species on native PAGE (Figure 11C), similar to what was observed upon PU- WS13 treatment. Complete deglycosylation also abolished GRP94’s binding to PU-WS13-B (Figure 11C). [0046] As described above, G4420 favors the PU-WS13 bound GRP94-HER2 complex, and indeed it was observed that significantly more GRP94 and HER2 were captured by the antibody in cells pre-treated with PU-WS13 (Figure 4D). N-glycan removal however equally diminished the amount of GRP94 and HER2 captured by G4420 (Figure 4D), and the amount of HMW HER2 observed on native gels (Figure 4E), substantiating further that the N-glycans are key for maintaining the HMW GRP94 and HER2 pools (Figure 4F and 11C). [0047] GRP94 contains six potential N-glycan acceptor sites and under normal conditions the protein is predominantly monoglycosylated at N217 (Cloutier and Coulombe, 2013; Schwarz and Aebi, 2011). The present disclosure describes glycosylation site mapping by mass spectrometry and identified N62, N217 and N502 as putative glycosylated Asn sites on the hgGRP94 variant. Through knock-out (KO) and mutagenesis of endogenous GRP94 via CRISPR/Cas9, isolated and expanded four homozygous clones – N62Q, N217A, N62Q/N217A and KO (Figure 5A). Using these clones, the present disclosure confirmed that more than one Asn was glycosylated on hgGRP94 (see Endo H treatment, Figure 5B). The present disclosure found that N62 was a key residue needed for the formation of the HMW GRP94 pool, as evidenced by native PAGE (Figure 5C and 12A), insensitivity to PU-WS13 (see GRP94 pools on native PAGE, Figure 5C and p-ERK and EGFR on SDS-PAGE, Figure 5D and 12B), a decrease in RTK signaling activity (see ERK downstream signaling, Figure 5E and 12B), diminished interaction with the G4220 antibody (Figure 5F) and a significant decrease in GRP94 and RTK localized at the PM (Figure 5G and S5C) in the N62Q-containing clones when compared to WT. GRP94 KO mimicked the effects observed with the N62Q-containing mutants (i.e. a limited EGFR pool translocated to the PM, no hyperactive ERK as noted by p-ERK levels and insensitivity of basal p-ERK levels to PU- WS13). Conversely, N217 mutagenesis had little effect on these GRP94 and RTK functions. [0048] Collectively, these data indicate that GRP94’s ability to form long-lived, stable complexes with RTKs at the PM (as opposed to the dynamic interactions needed for RTK folding by GRP94 in the ER) (Eletto et al., 2010) is dependent on a specific hyperglycosylation pattern, with N62 being a key residue for the observed switch of GRP94 from a folding, ER chaperone, to an oncogenic protein that stabilizes and activates RTKs at the PM. HMW GRP94 – an oncogenic gain-of-function [0049] Because HMW GRP94 is observed in cells that have high PM expression and oncogenic downstream signaling of RTKs, the present disclosure investigates if the accumulation of GRP94 at the PM was sufficient to initiate such oncogenic signaling. To address this question, the present disclosure utilized a construct that directs myc tagged GRP94 to the PM by deletion of the KDEL sequence and incorporation of a transmembrane domain from platelet-derived growth factor receptor (Zheng et al., 2001). The present disclosure used a Meth A fibrosarcoma cell line that was stably transfected with this construct, TM96, and compared the properties of this cell line to those of WT Meth A cells. [0050] Fractionation of cell extracts expressing TM96 showed that the TM96 GRP94 construct was found only in the F2 fraction, but not the ER (F1) or cytoplasmic (C) fractions (Figure 6A). TM96 expressed GRP94 participated in the formation of stable HMW GRP94 complexes as evidenced by the characteristic electrophoretic migration pattern on native gels (Figure 6A), its glycosylation status suggestive of hyperglycosylation (Figure 13A) and its sensitivity to PU-WS13 (Figure 13B). Its introduction was sufficient to increase the PM expression of proteins that require GRP94 for cell surface presentation, such as LRP6 (Liu et al., 2013) (Figure 6B) and to increase the neoplastic nature of these cells (Figure 6A, 6B, see p-p65 and p- ERK and Figure 6C, see anchorage-independent growth). TM96 expressed GRP94 also augmented the formation of intracellular stable HMW complexes incorporating HSP90 (Figure 6A, HSP90 native PAGE), also referred to as HSP90-incorporating epichaperomes, which act as molecular scaffolding platforms that augment the activity of cytosolic protein pathways, including signaling pathways (Joshi et al., 2018; Kourtis et al., 2018; Rodina et al., 2016). PU-WS13 treatment was sufficient to reverse these effects, as evidenced by inhibition of the activated but not baseline signaling (Figure 6B) and the loss of the HMW GRP94 pool located at the PM (Figure 12B). Importantly, the increase in the HMW GRP94 species upon TM96 transfection significantly increased the vulnerability of cells to PU-WS13 treatment (Figure 6D). [0051] Together with the above findings in breast cancer, these results indicate that an increase in HMW GRP94 at the PM is sufficient to alter the properties of PM proteins, resulting downstream in the rewiring of the cytosolic protein networks for increased signaling output (Figure 6E). Importantly, this gain-of-function brought about through the formation of HMW GRP94 species also increased the cells’ dependence on this oncogenic mechanism, as indicated by increased vulnerability to its inhibition. HMW GRP94 is an actionable target in cancer [0052] Because PU-WS13 exhibits preference for the GRP94 pool incorporated into stable HMW complexes located at the PM of cancer cells, it can be used to address the targetability and safety of inhibiting this unusual GRP94 variant in cancer. Treatment is a balance between target engagement and therapeutic index, and the present disclosure evaluated whether target suppression can be safely achieved by PU-WS13 in vivo. To understand target engagement during the study, the present disclosure measured tumor and tissue pharmacokinetics (PK) and pharmacodynamics (PD) after either a single dose of PU-WS13 administered intraperitoneally (i.p.) or at the end of a long- term treatment (see Figure 14A for study design). Both AU565 HER2+ and MDA-MB-468 EGFR+ breast tumors were established orthotopically. [0053] It was observed that a dose of 75 mg/kg PU-WS13 significantly engaged the target as indicated by significant RTK downregulation (Figures 7A, 7B) and supported by the PU-WS13 concentration recorded in these tumors (Figures 7C). HSP70 levels, which are a marker of HSP90 inhibition (Yuno et al., 2018), remained unchanged at this dose, indicating that inhibition of this close paralog during treatment by PU-WS13 did not occur (Figure 14B, 14C). The present disclosure therefore investigated the efficacy of 75 mg/kg and 125 mg/kg PU-WS13 given three times weekly (M-W-F), every other day (qod) or daily (qd) with weekends off (Figure 7D). The present disclosure provides significant, and dose- and schedule-dependent effects of PU-WS13 (Figures 7E-7G), with complete tumor growth suppression observed under the daily treatment paradigm. Similar results were also noted when tumors were established subcutaneously (Figures 7H, 7I). [0054] PU-WS13 was well tolerated. Even for the long treatment regimens that delivered 37 to 62 doses of PU-WS13 to mice over 87 days, no treatment-related toxicities were observed: mice retained a normal weight throughout treatment (Figure 7J). The present disclosure conducted complete necropsies and analyzed hematology and serum chemistry panels on vehicle-treated mice and on mice receiving the 125mg/kg dose five times per week for 87 days (Figure 7K and Figure 15). All hematological and clinical chemistry findings were within normal parameters, and histopathology conducted on major organs showed no toxic changes induced by PU-WS13. [0055] The present disclosure also evaluated GI tract LRP6 levels after PU-WS13 administration (Figure 7L). Housekeeping GRP94 is essential for folding and regulating physiologic functions of the Wnt receptor LRP6 (Rachidi et al., 2015), and it is expected that compounds such as PU-WS13 selectively targeting the tumor-specific HMW GRP94 variant will act on tumor functions while leaving housekeeping GRP94 functions unaltered at similar or higher concentrations as those seen in the tumor. Because most small molecules, including PU-WS13, are largely cleared via the GI tract, it is a body site most exposed to such agents over the time they spend in the body. Indeed, the concentration of PU-WS13 detected in the stomach and large intestine, and the exposure to agent over 24 hr, was much higher in these organs than in the tumor (Figure 7C); nonetheless no decrease in LRP6 levels were detected by western blot. [0056] The present disclosure identifies a GRP94 variant in cancer, whereby by altering N- glycosylation, a new protein conformationally, dynamically and functionally distinct from the GRP94 of normal cells is created. In place of a chaperone that is confined to the ER and makes transient interactions with, and folds, client proteins, a specific increase in N-glycosylation promotes a conformational state that allows for stable interactions with oncoproteins at the PM. In this context hyperglycosylation is a modality used by GRP94 to alter its conformational fitness and stabilize a state most permissive for stable interactions. Through this stabilization, these proteins’ functions are enhanced, and cellular protein pathways are aberrantly remodeled - N-glycosylation thus transforms a chaperone, GRP94, from a folding to a scaffolding protein that remodels protein connectivity, with an end result of proteome-wide dysfunction. Therefore, the N-glycosylation pattern of GRP94 the present disclosure identifies is a specific modification exploited by cancer cells to alter the customary role of a chaperone. [0057] The aberrantly N-glycosylated GRP94 variant is present only in some tumors, is independent of total GRP94 levels, and is absent or scarce in non-transformed cells. The present disclosure found that the functions of one class of oncoproteins, RTKs, are modified by this GRP94 variant, and only in cancer cells driven by RTK overexpression. RTK overexpression is a form of proteome stress, and under these conditions GRP94 N-glycosylation at specific sites is key both to enhance the presence of these proteins at the plasma membrane by forming stable complexes with the RTKs, as well as maintain RTKs in a state that enables aberrant downstream signaling and a rewiring of cytosolic protein pathways. [0058] N-linked glycosylation is among the most ubiquitous protein modifications in eukaryotes. It is implicated in a myriad of housekeeping functions, including modification of a protein’s folding capacity, stability, and oligomerization and aggregation status, ER quality control and protein trafficking, host cell-surface interactions, and modulation of enzyme activity (Lee et al., 2015). Changes in glycosylation are observed in cancer where they affect the interaction and subsequently activation capacity of RTKs (Mereiter et al., 2019). Conversely, there is no report of N-glycosylation increasing the oncogenic properties of a protein, indirectly, by modulating its complexation. [0059] Without wishing to be bound by any particular theory, it is believed that N- glycosylation does not induce significant changes in a protein’s structure, but decreases protein conformational dynamics, likely leading to an increase in protein stability (Lee et al., 2015). In this view, N-glycans act like molecular glues, holding together residues around the glycosylation sites through favorable interactions made with nearby protein residues, thus resulting in the stabilization of a specific protein conformation or disfavoring others (Sola and Griebenow, 2006). Stabilization of a specific conformation favoring the formation of complexes of enhanced stability was recently shown for HSP90, the cytosolic paralog of GRP94 (Inda et al., 2020; Kishinevsky et al., 2018; Kourtis et al., 2018; Rodina et al., 2016). There, an oncogenic stress such as MYC hyperactivation or a neuronal damaging stress such as tau overexpression redistributes the cytosolic pool of molecular chaperones and helpers into complexes of enhanced stability. These stable assemblies, termed epichaperomes, function as multi-component scaffolds to provide a framework on which the cell’s complement of proteins can work more efficiently or differently than they would without chaperome participation. A similar role for the hgGRP94 variant in the context of disease is suggested by data provided in the present disclosure. The heterogeneous HMW GRP94 species observed on native gels likely consists of numerous other proteins besides the RTKs thus far identified. The identity of these other components, as well as their role in working with GRP94 to regulate the oncogenic HER2 and EGFR functions, is not yet known. [0060] Because both HSP90 and GRP94 are stable as dimers even at high concentrations, a conformational change is possibly required to drive stress-induced complexation alteration. This may lead to the unmasking of a site that is absent in the dimer, giving rise to a new protein–protein interaction platform and a new quaternary structure that reverts the chaperone from a folding to a scaffolding protein. While for HSP90 the identity of such a conformational stabilizer remains to be elucidated, for GRP94 the present disclosure suggests it to be, at least in part, regulated by N- glycosylation. Apparently, in most cases, glycosylation does not induce permanent secondary structural formations, but rather prompts local conformational changes close to the glycosylation site. Often these result in a β turn, infrequently a β strand and rarely an α helix (Mitra et al., 2006). Whether such a conformational switch changes the direct interaction of GRP94 with HER2 or whether it mediates the creation of a stable multimeric platform with co-chaperones and other factors that mediate HER2 stabilization, similarly to that seen for HSP90, remains to be seen. [0061] The present disclosure shows that inhibition of the hgGRP94 variant with compounds such as PU-WS13 is feasible in cancer cells, human primary tumor specimens, and xenografted tumors in mice. Although GRP94 is abundant in most cells of the mammalian body, it is clear that the housekeeping variant and the N-glycosylated variant targeted in cancers are different, rendering compounds such as PU- WS13 selective for the cancer form. By introducing these PU-type chemical probes, the present disclosure also demonstrates that the N-glycosylated GRP94 variant, and the specific aberrant proteins and cellular processes enabled by this variant, are targetable in disease. Thus, inhibitors of the N- glycosylated GRP94 variant are an example of a ‘targeted protein degradation-based therapeutic’ that act specifically on dysfunctions, and protein networks, enabled by this variant, thus sparing the normal folding functions of GRP94. [0062] While the present disclosure specifically exemplifies the expression and significance of the hgGRP94 variant in BC, several lines of evidence suggest that it is implicated in other cancers as well. RTK overexpression (ex. EGFR, HER2, MET and others) is observed in a variety of cancer cells and in cells of a tumor supportive microenvironment (Butti et al., 2018; Contessa et al., 2008; Contessa et al., 2010; Siddals et al., 2011; Tan et al., 2018; Turrini et al., 2017). Overall, several tumors are characterized by an aberrant expression of RTKs (FGFR1 in lung and breast cancer (Dutt et al., 2011; Reis-Filho et al., 2006), FGFR3 in breast and bladder cancer (Fischbach et al., 2015; Helsten et al., 2016), ERBB4 in breast and gastric cancer (Kim et al., 2016; Shi et al., 2012), FLT3 in colon cancer (Moreira et al., 2015), KIT in melanoma and GIST (Carvajal et al., 2011; Tabone et al., 2005), and PDGFRA in GBM (Nobusawa et al., 2011)). RTK amplifications also allow tumor cells to escape therapeutic treatment (MET and HER2 amplification can be detected in EGFR-mutant lung cancers that become resistant to EGFR TKI therapy (Yu et al., 2013)). Because EGFR overexpression is often a side effect of radiation therapy (Cuneo et al., 2015), targeting the GRP94 variant with PU-WS13 may also radiosensitize tumors. [0063] It is noteworthy that inhibition of the GRP94 variant is more toxic to EGFR+ tumors than the direct inhibition of EGFR by kinase inhibitors or anti-EGFR antibodies. Approximately half of all triple- negative BCs (TNBCs) and inflammatory BCs overexpress EGFR. Nonetheless, clinical trials of EGFR inhibitors have reported little or no benefit (Masuda et al., 2012). It is believed that the ineffectiveness of these treatments is due to crosstalk between EGFR and c-Met or other RTKs, because strategies that knocked down EGFR, either by siRNA or by mixtures of antibodies that induced robust degradation of EGFR, led to reduced viability of TNBC cells (Ferraro et al., 2013; Mueller et al., 2012). As per the present findings, GRP94 inhibition by PU- WS13 also induces robust EGFR degradation and apoptosis in TNBC cells, and this effect can provide a therapeutic advantage over tyrosine kinase inhibitors. [0064] In conclusion, the present disclosure reports that increasing the interaction strength between GRP94 and RTKs and other receptors at the plasma membrane, which the present disclosure found to be regulated by a specific N-glycosylation pattern, is a mechanism used by a chaperone to enhance the stabilization and interaction of certain proteins. Without wishing to be bound by any particular theory, it is believed that these findings identify a biochemical mechanism whereby stress remodels a chaperone from a folding to a scaffolding protein creating a state of chaperone- mediated protein connectivity dysfunction. Here aberrant N-glycosylation of a fraction of the cellular pool of the chaperone GRP94 remodels GRP94 location and conformation, and in turn, its interaction strength and interaction partners, with the outcome being aberrantly remodeled protein pathways and a pathologic cellular phenotype. The present disclosure describes that the HMW form of GRP94 is an example of a ‘protein assembly mutation’ (Nussinov et al., 2019), a proteome malfunction defined by defective protein-protein interaction that portends pathologic activity. This variant is a target for cancers and other diseases. Compounds and Definitions [0065] Compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in “Organic Chemistry”, Thomas Sorrell, University Science Books, Sausalito: 1999, and “March’s Advanced Organic Chemistry”, 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference. [0066] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric (e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomic, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, some structures depicted here show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and/or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure. [0067] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C-enriched carbon are within the scope of this disclosure. [0068] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value (except where such number would be less than 0% or exceed 100% of a possible value). [0069] Aliphatic: The term “aliphatic” refers to a straight-chain (i.e., unbranched) or branched, optionally substituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation but which is not aromatic (also referred to herein as “carbocyclic” or “cycloaliphatic”), that has a single point of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., C1-4). In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynyl groups and hybrids thereof. In some embodiments, “aliphatic” refers to a straight- chain (i.e., unbranched) or branched, optionally substituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation that has a single point of attachment to the rest of the molecule. [0070] Alkyl: The term “alkyl”, used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched hydrocarbon group having (unless otherwise specified) 1-12, 1-10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12, C1-10, C1-8, C1-6, C1-4, C1-3, or C1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl. [0071] Carbocyclyl: The terms “carbocyclyl,” “carbocycle,” and “carbocyclic ring” as used herein, refer to saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 14 members, wherein the aliphatic ring system is optionally substituted as described herein. Carbocyclic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, norbornyl, adamantyl, and cyclooctadienyl. In some embodiments, “carbocyclyl” (or “cycloaliphatic”) refers to an optionally substituted monocyclic C3-C8 hydrocarbon, or an optionally substituted C7-C10 bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule. The term “cycloalkyl” refers to an optionally substituted saturated ring system of about 3 to about 10 ring carbon atoms. In some embodiments, cycloalkyl groups have 3–6 carbons. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. The term “cycloalkenyl” refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl. [0072] Alkenyl: The term “alkenyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched hydrocarbon chain having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. [0073] Alkynyl: The term “alkynyl”, used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl. [0074] Aryl: The term “aryl” refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, “aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, “aryl” groups are hydrocarbons. [0075] Heteroaryl: The terms “heteroaryl” and “heteroar–”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10- membered bicyclic heteroaryl); having 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Exemplary heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2- a]pyridinyl, thienopyrimidinyl, triazolopyridinyl, and benzoisoxazolyl. The terms “heteroaryl” and “heteroar–”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H–quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3–b]–1,4– oxazin–3(4H)–one, and benzoisoxazolyl. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted. [0076] Heteroatom: The term “heteroatom” as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. [0077] Heterocycle: As used herein, the terms “heterocycle”, “heterocyclyl”, and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic or 7- to 10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0–3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro- 2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+ (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3- dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). [0078] Partially Unsaturated: As used herein, the term “partially unsaturated”, when referring to a ring moiety, means a ring moiety that includes at least one double or triple bond between ring atoms. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aromatic (e.g., aryl or heteroaryl) moieties, as herein defined. [0079] Patient or subject: As used herein, the term “patient” or “subject” refers to any organism to which a provided composition is or may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients or subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some embodiments, a patient or subject is a human. In some embodiments, a patient or a subject is suffering from or susceptible to one or more disorders or conditions. In some embodiments, a patient or subject displays one or more symptoms of a disorder or condition. In some embodiments, a patient or subject has been diagnosed with one or more disorders or conditions. In some embodiments, a patient or a subject is receiving or has received certain therapy to diagnose and/or to treat a disease, disorder, or condition. [0080] Substituted or optionally substituted: As described herein, compounds of this disclosure may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. “Substituted” applies to one or more hydrogens that are either explicit or implicit from the structure (e.g.,
Figure imgf000029_0001
refers to at least
Figure imgf000029_0002
). Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable,” as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above. [0081] Treat: As used herein, the term “treat” (also “treatment” or “treating”) refers to any administration of a therapy that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. In some embodiments, such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. [0082] The present disclosure provides compounds that are inhibitors of N-glycosylated Grp94. Such compounds are represented schematically in Formula (I):
Figure imgf000030_0001
or a pharmaceutically acceptable salt thereof, wherein: Z2 is –N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; X1 is –H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; X2 is –H, halogen, or unsubstituted or substituted -(C1-C6)aliphatic; X3 and X5 are independently -halogen, unsubstituted or substituted -(C1-C12)aliphatic, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 3- to 10-membered heterocyclic group having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or unsubstituted or substituted 3- to 10-membered cycloalkyl group; or X2 and X3 are taken together with their intervening atoms to form a 5- to 8- membered partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R1 is –(CR1aR1b)3-NR2R3; each occurrence of R1a is independently hydrogen or methyl; each occurrence of R1b is independently hydrogen or methyl; R2 and R3 are independently hydrogen or unsubstituted or substituted -(C1-C8)aliphatic; each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, - CN, -OR, -SR, -N(R)2, -NO2, -C(O)R’, -C(O)OR, -C(O)N(R)2, -OC(O)R’, -OC(O)N(R)2, - OC(O)OR, -OSO2R, -OSO2N(R)2, -N(R)C(O)R’, -N(R)SO2R’, -SO2R’, -SO2N(R)2, - SO3R’, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -CN, - OR, -SR, -N(R)2, -NO2, -C(O)R’, -C(O)OR, -C(O)N(R)2, -OC(O)R’, -OC(O)N(R)2, - OC(O)OR, -OSO2R, -OSO2N(R)2, -N(R)C(O)R’, -N(R)SO2R’, -SO2R’, -SO2N(R)2, - SO3R’, or oxo; and each R’ is independently unsubstituted C1-6 aliphatic or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. [0083] In some embodiments, Z2 is –N-. In some embodiments, Z2 is –CR10-. In some embodiments, Z2 is –CH-. [0084] In some embodiments, X1 is –H. In some embodiments, X1 is halogen. In some embodiments, X1 is F. In some embodiments, X1 is Cl. In some embodiments, X1 is Br. In some embodiments, X1 is I. In some embodiments, X1 is F or Cl. [0085] In some embodiments, X2 is -H. In some embodiments, X2 is halogen. [0086] In some embodiments, X2 is –H, halogen, or unsubstituted or substituted -(C1- C6)aliphatic; and X3 and X5 are independently -halogen, unsubstituted or substituted -(C1- C12)aliphatic, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 3- to 10- membered heterocyclic group having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or unsubstituted or substituted 3- to 10-membered cycloalkyl group. [0087] In some embodiments, both X3 and X5 are halogen. In some embodiments, the halogen is Cl. In some embodiments, the halogen is F. In some embodiments, the halogen is I. In some embodiments, the halogen is Br. In some embodiments, X3 and X5 are identical halogen. In some embodiments, neither X3 nor X5 are hydrogen. In some embodiments, one of X3 and X5 is halogen. In some embodiments, one of X3 and X5 is hydrogen. [0088] In some embodiments, X3 and X5 are both Cl. In some embodiments, X3 and X5 are Cl and Br. In some embodiments, X3 and X5 and Cl and I. [0089] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted -(C1-C6)aliphatic. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted -(C1-C6)aliphatic. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted -(C1-C6)aliphatic. [0090] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted -(C1-C6)alkyl. In some embodiments, the -(C1-C6)alkyl group is substituted with one or more halogen, -OH, -CN, -NH2, or unsubstituted C1-6 aliphatic. In some embodiments, the -(C1-C6)alkyl group is substituted with -OH. [0091] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted -(C2-C6)alkenyl. In some embodiments, the -(C2-C6)alkenyl group is substituted with halogen, -OH, -CN, -NH2, or unsubstituted C1-6 aliphatic. In some embodiments, the -(C2-C6)alkenyl group is substituted with -OH. [0092] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted -(C2-C6)alkynyl. In some embodiments, the -(C2-C6)alkynyl group is substituted with halogen, -OH, -CN, -NH2, or unsubstituted C1-6 aliphatic. In some embodiments, the -(C2-C6)alkynyl group is substituted with -OH. [0093] In some embodiments, X3 and X5 are independently selected from halogen or the following groups:
Figure imgf000033_0001
[0094] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted phenyl. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted phenyl. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted phenyl. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted phenyl, wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, -CF3, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, -CF3, or -NH2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted phenyl, wherein the substituent is –NO2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted phenyl, wherein the substituent is –OMe. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted phenyl, wherein the substituent is –CF3. [0095] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted 5- to 6- membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. [0096] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted
Figure imgf000034_0001
. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted
Figure imgf000034_0002
. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000034_0003
wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. In some embodiments, a substituent is –NO2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000034_0004
wherein the substituent is –-OMe. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000034_0005
wherein the substituent is –CF3. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000034_0006
, wherein the substituent is –Me. [0097] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted
Figure imgf000034_0007
. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted
Figure imgf000034_0008
In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000034_0009
wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0001
, wherein the substituent is –NO2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0002
wherein the substituent is -OMe. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0003
wherein the substituent is –CF3. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0004
wherein the substituent is –Me. [0098] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted
Figure imgf000035_0005
. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted
Figure imgf000035_0006
In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0007
wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0008
, wherein the substituent is –NO2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0009
wherein the substituent is -OMe. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000035_0010
, wherein the substituent is –CF3. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000036_0001
or , wherein the substituent is –Me. [0099] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted
Figure imgf000036_0002
. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted
Figure imgf000036_0003
. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000036_0004
wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000036_0005
or
Figure imgf000036_0006
, wherein the substituent is –NO2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000036_0007
, wherein the substituent is -OMe. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000036_0008
wherein the su 3 5
Figure imgf000036_0009
bstituent is –CF3. In some embodiments, one of X and X is halogen, and the other of X3 and X5 is substituted
Figure imgf000036_0010
, wherein the substituent is –Me. [0100] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted
Figure imgf000037_0001
or or . In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or or . In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000037_0002
or or , wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000037_0003
or , wherein the substituent is –NO2. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000037_0004
, wherein the substituent is -OMe. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted
Figure imgf000037_0005
wherein the substituent is –CF3. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted or or , wherein the substituent is –Me. [0101] In some embodiments, X3 and X5 are independently selected from halogen or the following groups:
Figure imgf000038_0001
[0102] In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted or substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is unsubstituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, one of X3 and X5 is halogen, and the other of X3 and X5 is substituted 8- to 10- membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, wherein each substituent is independently halogen, -N(R)2, -OR, -CN, -NO2, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2. [0103] In some embodiments,
Figure imgf000038_0002
[0104] In some embodiments, one of R2 and R3 is H. In some embodiments, both R2 and R3 are H. In some embodiments, neither of R2 and R3 is H. [0105] In some embodiments, one of R2 and R3 is H and the other is unsubstituted or substituted -(C1-C8)aliphatic. In some embodiments, one of R2 and R3 is H and the other is unsubstituted or substituted -(C1-C8)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C1-C8)alkyl. In some embodiments, one of R2 and R3 is H and the other is methyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C2)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C3)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C4)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C5)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C6)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C7)alkyl. In some embodiments, one of R2 and R3 is H and the other is unsubstituted -(C8)alkyl. In some embodiments, one of R2 and R3 is H and the other is substituted -(C1-C8)alkyl, wherein each substituent is independently halogen, -OH, -CN, -NH2, or unsubstituted C1-6 aliphatic. [0106] In some embodiments, one of R2 and R3 is H and the other is unsubstituted or substituted -(C2-C8)alkenyl. [0107] In some embodiments, one of R2 and R3 is H and the other is unsubstituted or substituted -(C2-C8)alkynyl. [0108] In some embodiments, R2 and R3 are independently selected from the following groups: [
Figure imgf000039_0001
, [0110] In some embodiments, compounds of Formula (I) have the structure of Formula (Ia):
Figure imgf000039_0002
or a pharmaceutically acceptable salt thereof, wherein each of X1, X2, X3, X5, Z2, and R3 is as defined and described herein, both singly and in combination. [0111] In some embodiments, compounds of Formula (I) have the structure of Formula (Ib):
Figure imgf000040_0001
Ib or a pharmaceutically acceptable salt thereof, wherein each of X1, X3, X5, Z2, and R1 is as defined and described herein, both singly and in combination. [0112] In some embodiments, compounds of Formula (I) have the structure of Formula (Ic- i) or (Ic-ii):
Figure imgf000040_0002
Ic-ii or a pharmaceutically acceptable salt thereof, wherein each of X1, X3, Z2, and R1 is as defined and described herein, both singly and in combination. [0113] In some embodiments, compounds of Formula (I) have the structure of Formula (Id):
Figure imgf000041_0001
or a pharmaceutically acceptable salt thereof, wherein each of X1, X3, X5, Z2, and R3 is as defined and described herein, both singly and in combination. [0114] In some embodiments, compounds of Formula (I) have the structure of Formula (Ie- i) or (Ie-ii):
Figure imgf000041_0002
Ie-i
Figure imgf000042_0002
Ie-ii or a pharmaceutically acceptable salt thereof, wherein each of X1, X3, Z2, and R3 is as defined and described herein, both singly and in combination. [0115] In some embodiments, the present disclosure provides compounds selected from Table 1:
Figure imgf000042_0001
I-3 I-4
Figure imgf000043_0001
I-9 I-10
Figure imgf000044_0001
I-15 I-16
Figure imgf000045_0001
I-21 I-22
Figure imgf000046_0001
Figure imgf000047_0001
I-33 I-34
Figure imgf000048_0001
Figure imgf000049_0001
I-45 (SO-IV-26A) I-46 (SO-III-103A)
Figure imgf000050_0001
I-51 I-52
Figure imgf000051_0001
Figure imgf000052_0001
I-63 I-64
Figure imgf000053_0001
I-69 I-70
Figure imgf000054_0001
I-75 I-76
Figure imgf000055_0001
I-81 I-82
Figure imgf000056_0001
I-87 I-88
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
I-101 or a pharmaceutically acceptable salt thereof. [0116] In some embodiments, the present disclosure provides a method of treating cancer, inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis, or diabetes, comprising the step of administering to a subject suffering therefrom an effective amount of a compound having the structure of Formula I, Ia, Ib, Ic-i, Ic-ii, Id, Ie-i, or Ie-ii, or a pharmaceutically acceptable salt thereof. Compositions [0117] The present disclosure also provides compositions comprising a compound provided herein with one or more other components. In some embodiments, provided compositions comprise and/or deliver a compound described herein (e.g., compounds of Formulae I, Ia, Ib, Ic-i, Ic-ii, Id, Ie-i, and Ie-ii ). [0118] In some embodiments, a provided composition is a pharmaceutical composition that comprises and/or delivers a compound provided herein (e.g., compounds of Formulae I, Ia, Ib, Ic-i, Ic-ii, Id, Ie-i, and Ie-ii ) and further comprises a pharmaceutically acceptable carrier. Pharmaceutical compositions typically contain an active agent (e.g., a compound described herein) in an amount effective to achieve a desired therapeutic effect while avoiding or minimizing adverse side effects. In some embodiments, provided pharmaceutical compositions comprise a compound described herein and one or more fillers, disintegrants, lubricants, glidants, anti-adherents, and/or anti-statics, etc. Provided pharmaceutical compositions can be in a variety of forms including oral dosage forms, topical creams, topical patches, iontophoresis forms, suppository, nasal spray and/or inhaler, eye drops, intraocular injection forms, depot forms, as well as injectable and infusible solutions. Methods of preparing pharmaceutical compositions are well known in the art. [0119] In some embodiments, provided compounds are formulated in a unit dosage form for ease of administration and uniformity of dosage. The expression “unit dosage form” as used herein refers to a physically discrete unit of an active agent (e.g., a compound described herein) for administration to a subject. Typically, each such unit contains a predetermined quantity of active agent. In some embodiments, a unit dosage form contains an entire single dose of the agent. In some embodiments, more than one unit dosage form is administered to achieve a total single dose. In some embodiments, administration of multiple unit dosage forms is required, or expected to be required, in order to achieve an intended effect. A unit dosage form may be, for example, a liquid pharmaceutical composition containing a predetermined quantity of one or more active agents, a solid pharmaceutical composition (e.g., a tablet, a capsule, or the like) containing a predetermined amount of one or more active agents, a sustained release formulation containing a predetermined quantity of one or more active agents, or a drug delivery device containing a predetermined amount of one or more active agents, etc. [0120] Provided compositions may be administered using any amount and any route of administration effective for treating or lessening the severity of any disease or disorder described herein. Uses [0121] The present disclosure provides uses for compounds and compositions described herein. In some embodiments, provided compounds and compositions are useful in medicine (e.g., as therapy). In some embodiments, provided compounds and compositions are useful in research as, for example, analytical tools and/or control compounds in biological assays. [0122] In some embodiments, the present disclosure provides methods of administering provided compounds or compositions to a subject in need thereof. In some embodiments, the present disclosure provides methods of administering provided compounds or compositions to a subject suffering from or susceptible to a disease, disorder, or condition associated with N- glycosylated Grp94. [0123] In some embodiments, provided compounds are useful as N-glycosylated Grp94 inhibitors. In some embodiments, the present disclosure provides methods of inhibiting N- glycosylated Grp94 in a subject comprising administering a provided compound or composition. In some embodiments, the present disclosure provides methods of inhibiting N-glycosylated Grp94 in a biological sample comprising contacting the sample with a provided compound or composition. [0124] In some embodiments, the present disclosure provides methods of treating cancer, comprising administering a provided compound or composition to a subject in need thereof. In some embodiments, the present disclosure provides methods of treating proliferative diseases, comprising administering a provided compound or composition to a subject in need thereof. In some embodiments, the cancer is colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, leukemias, myelomas, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, or endometrial cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is non-small-cell lung cancer. [0125] In some embodiments, the present disclosure provides methods of treating a hematological malignancy, comprising administering a provided compound or composition to a subject in need thereof. In some embodiments, a hematological malignancy is leukemia (e.g., chronic lymphocytic leukemia, acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, or acute monocytic leukemia). In some embodiments, a hematological malignancy is lymphoma (e.g., Burkitt’s lymphoma, Hodgkin’s lymphoma, or non-Hodgkin’s lymphoma). In some embodiments, a hematological malignancy is myeloma (e.g., multiple myeloma). In some embodiments, a hematological malignancy is myeloproliferative neoplasm (e.g., polycythemia vera, essential thrombocytopenia, or myelofibrosis). In some embodiments, a hematological malignancy is myelodysplastic syndrome. [0126] In some embodiments, the present disclosure provides methods of treating an inflammatory disease, disorder, or condition (e.g., acute respiratory syndrome, hyperinflammation, and/or cytokine storm syndrome (including those associated with COVID-19) or atopic dermatitis), neurodegenerative diseases, rheumatoid arthritis, or diabetes comprising administering a provided compound or composition to a subject in need thereof. [0127] In some embodiments, a provided compound or composition is administered as part of a combination therapy. As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic or prophylactic regimens (e.g., two or more therapeutic or prophylactic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition. [0128] For example, in some embodiments, a provided compound or composition is administered to a subject who is receiving or has received one or more additional therapies (e.g., an anti-cancer therapy and/or therapy to address one or more side effects of such anti-cancer therapy, or otherwise to provide palliative care). Exemplary additional therapies include ERBB2 inhibitors, EGFR inhibitors, CDK4 inhibitors, CRAF inhibitors, BRAF inhibitors, AKT inhibitors, MET inhibitors, BCR-ABL inhibitors, JAK inhibitors, HIF-1α inhibitors, and p53 inhibitors. [0129] The present disclosure contemplates, among other things, the following numbered embodiments: 1. A method of treating cancer comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N-glycosylated Grp94. 2. A method of treating cancer characterized by the presence of N-glycosylated Grp94 comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N-glycosylated Grp94. 3. A method of treating a disease associated with N-glycosylated Grp94, comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N- glycosylated Grp94. 4. The method of embodiment 1, 2, or 3, wherein the compound has the structure of Formula (I):
Figure imgf000063_0002
or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000063_0001
(b) each of Z1 and Z3 is independently -CH- or -N-; (c) Z2 is -N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; (d) each of Z4, Z5, Z6, Z7 and Z8 is independently -C- or -N-, with the provisos that at least one of Z4, Z6 and Z7 is -C- and no three consecutive Z4 through Z8 are N; (e) X1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (f) each of X2, X3, X4 , X5, and X6 is independently -H, -halogen, -SR, -N(R)2, -OR, -CN, - NO2, -CN, -C(O)R, -C(O)2R, -S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6- membered)heterocyclic aromatic or heterocyclic non-aromatic group; with the provisos that at least one of X2, X4 and X5 is -H and that X2 is absent when Z4 is -N-, X3 is absent when Z5 is -N-, X4 is absent when Z6 is -N- and X5 is absent when Z7 is -N-; (g) R1 is -(C1-C6)aliphatic-N+-(R2)(R3)(R4), -(C1-C6)aliphatic-N-R3R4, -(C1-C6)aliphatic- C(=O)N-R3R4, -(C1-C6)aliphatic-R3R4, -(C1-C6)aliphatic-R2R3R4, -(C1-C6)aliphatic-N-CR2R3R4, - (C1-C6)aliphatic-C(halogen)3, -(C1-C6)aliphatic-alkenyl, -(C1-C6)aliphatic-alkynyl, -(C1- C6)aliphatic-(C3-C8)cycloalkyl, -(C1-C6)aliphatic-(C3-C8)heterocyclo, -(C1-C6)aliphatic-phenyl, - (C1-C6)aliphatic-(5 or 6-membered)heteroaryl, -(C1-C6)aliphatic-cyano, where the cycloalkyl, heterocyclo, heteroaryl, or phenyl is unsubstituted or substituted, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (h) R2 and R3 are independently hydrogen, -N(R)2, -CH2CH(OH)R4, -CH(OH)CH2R4, - CH2SO2NHR4, - CH2NHSO2R4, or unsubstituted or substituted -(C1-C6)aliphatic, or R3 and R4 form an unsubstituted or substituted 3- to 7-membered heterocyclic ring when taken together with the nitrogen to which they are attached; (i) R4 is hydrogen, halogen, or unsubstituted or substituted –(C1-C6)aliphatic; (j) each RY is independently R, -OR, or halogen; (k) Z3 can be cyclized with X2 to form a cyclic aryl, heteroaryl, alkyl or heteroalkyl ring; and (l) each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, - N(R)2, -OR, -CN, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2. 5. The method of embodiment 4, wherein:
Figure imgf000064_0001
(b) each of Z1 and Z3 is independently -CH- or -N-; (c) Z2 is -CH-, -N-, or –CR10-, wherein R10 is -(C1-C6)alkyl; (d) each of Z4, Z5, Z6, Z7 and Z8 is independently -C- or -N-, with the provisos that at least one of Z4, Z6 and Z7 is -C- and no three consecutive Z4 through Z8 are N; (e) X1 is -H, -halogen, -NH2, -CN, -(C1-C6)alkyl, -O(C1-C6)alkyl, -CH2OH, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -OC(halogen)3, -OCH(halogen)2, or -OCH2(halogen); (f) each of X2, X3, X4 , and X5 is independently -H, -halogen, -NH2, -CN, -(C1-C6)alkyl, -O(C1-C6)alkyl, -CH2OH, -C(halogen)3, -CH(halogen)2, -CH2(halogen), - OC(halogen)3, -OCH(halogen)2, -OCH2(halogen), or an unsubstituted or substituted (5- or 6-membered)aryl, heterocyclic aromatic, or non-aromatic group selected from pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl, phenyl, benzyl, thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl, with the provisos that at least one of X2, X4 and X5 is -H and that X2 is absent when Z4 is -N-, X3 is absent when Z5 is -N-, X4 is absent when Z6 is -N- and X5 is absent when Z7 is -N-; (g) X6 is -H when Z8 is -C- or absent when Z8 is -N-; (h) R1 is -(CH2)m-N+-(R2)(R3)(R4), -(CH2)m-N-R3R4, -(CH2)m-C(=O)N-R3R4, -(CH2)m-R3R4, -(CH2)m-C(halogen)3, -(CH2)m-alkenyl, -(CH2)m-alkenyl-CH3, -(CH2)m-alkynyl, -(CH2)m-alkynyl- CH3, -(CH2)m-(C3-C8)cycloalkyl, -(CH2)m-(C3-C8)heterocycloalkyl, -(CH2)m-phenyl, -(CH2)m-(5 or 6-membered)heteroaryl, -(CH2)m-cyano, where m is 1, 2, 3, 4 or 5 and where the cycloalkyl, heterocycle or phenyl is unsubstituted or substituted with one or more X1 groups, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (i) R2 and R3 are independently hydrogen, methyl, ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, isobutyl, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -CH2C(halogen)3, -CHCH(halogen)2, CHCH2(halogen), -CH2OH, -CH2CH2OH, -CH2C(CH3)2OH, -CH2CH(CH3)OH, -C(CH3)2CH2OH, -CH(CH3)CH2OH, -CH(CH3)CH(OH)R4, -CH2CH(OH)R4, -CH2SO2NHR4, -CH2NH SO2R4 or R3 and R4 form an unsubstituted or substituted aziridine, azetidine, pyrrolidine or piperidine ring when taken together with the nitrogen to which they are attached; (j) R4 is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or -C(halogen)3; and (k) Z3 can be cyclized with X2 to form a cyclic aryl, heteroaryl, alkyl or heteroalkyl ring. 6. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000065_0001
or a pharmaceutically acceptable salt thereof. 7. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000066_0001
or a pharmaceutically acceptable salt thereof. 8. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000066_0004
or a pharmaceutically acceptable salt thereof. 9. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000066_0002
or a pharmaceutically acceptable salt thereof. 10. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000066_0003
or a pharmaceutically acceptable salt thereof. 11. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000067_0001
or a pharmaceutically acceptable salt thereof. 12. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000067_0002
or a pharmaceutically acceptable salt thereof. 13. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000067_0004
or a pharmaceutically acceptable salt thereof. 14. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000067_0003
or a pharmaceutically acceptable salt thereof. 15. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000068_0001
or a pharmaceutically acceptable salt thereof. 16. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000068_0002
or a pharmaceutically acceptable salt thereof. 17. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000068_0003
or a pharmaceutically acceptable salt thereof. 18. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000068_0004
or a pharmaceutically acceptable salt thereof. 19. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000069_0004
or a pharmaceutically acceptable salt thereof. 20. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000069_0001
or a pharmaceutically acceptable salt thereof. 21. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000069_0002
or a pharmaceutically acceptable salt thereof. 22. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000069_0003
or a pharmaceutically acceptable salt thereof. 23. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000070_0001
or a pharmaceutically acceptable salt thereof. 24. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000070_0002
or a pharmaceutically acceptable salt thereof. 25. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000070_0003
or a pharmaceutically acceptable salt thereof. 26. The method of embodiment 5, wherein the compound has the following formula:
Figure imgf000070_0004
or a pharmaceutically acceptable salt thereof. 27. The method of embodiment 4, wherein the compound has the structure of Formula (II):
Figure imgf000071_0001
or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000071_0002
(b) each of Z1 and Z3 is independently -CH- or -N-; (c) Z2 is -N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; (d) X1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (e) each of X3 and X5 is independently -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, - CN, -C(O)R, -C(O)2R, -S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6- membered)heterocyclic aromatic or heterocyclic non-aromatic group; (f) R1 is -(C1-C6)aliphatic-N+-(R2)(R3)(R4), -(C1-C6)aliphatic-N-R3R4, -(C1-C6)aliphatic- C(=O)N-R3R4, -(C1-C6)aliphatic-R3R4, -(C1-C6)aliphatic-R2R3R4, -(C1-C6)aliphatic-N-CR2R3R4, - (C1-C6)aliphatic-C(halogen)3, -(C1-C6)aliphatic-alkenyl, -(C1-C6)aliphatic-alkynyl, -(C1- C6)aliphatic-(C3-C8)cycloalkyl, -(C1-C6)aliphatic-(C3-C8)heterocycloalkyl, -(C1-C6)aliphatic- phenyl, -(C1-C6)aliphatic-(5 or 6-membered)heteroaryl, -(C1-C6)aliphatic-cyano, wherein the cycloalkyl, heterocycloalkyl, phenyl, or heteroaryl is unsubstituted or substituted, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (g) R2 and R3 are independently hydrogen, -N(R)2, -CH2CH(OH)R4, -CH(OH)CH2R4, - CH2SO2NHR4, - CH2NHSO2R4, or unsubstituted or substituted -(C1-C6)aliphatic, or R3 and R4 form an unsubstituted or substituted 3- to 7-membered heterocyclic ring when taken together with the nitrogen to which they are attached; (h) each RY is independently R, -OR, or halogen; (i) R4 is hydrogen, halogen, or unsubstituted or substituted –(C1-C6)aliphatic; and (j) each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, -N(R)2, -OR, -CN, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2. 28. The method of embodiment 27, wherein:
Figure imgf000072_0001
(b) each of Z1 and Z3 is independently -CH- or -N-; (c) Z2 is -CH-, -N-, or –CR10-, wherein R10 is -(C1-C6)alkyl; (d) X1 is -H, -halogen, -NH2, -CN, -(C1-C6)alkyl, -O(C1-C6)alkyl, -CH2OH, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -OC(halogen)3, -OCH(halogen)2, or -OCH2(halogen); (e) each of X3 and X5 is independently -H, -halogen, -NH2, -CN, -(C1-C6)alkyl, -O(C1- C6)alkyl, -CH2OH, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -OC(halogen)3, -OCH(halogen)2, -OCH2(halogen), or a (5- or 6-membered)aryl, heterocyclic aromatic, or non-aromatic group selected from pyridyl, furyl, thiophenyl, pyrrolyl, oxazolyl, imidazolyl, phenyl, benzyl, thiazolidinyl, thiadiazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, triazinyl, morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperazinyl, 2,3-dihydrofuranyl, dihydropyridinyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, or tetrahydrothiopyranyl; (f) R1 is -(CH2)m-N+-(R2)(R3)(R4), -(CH2)m-N-R3R4, -(CH2)m-C(=O)N-R3R4, -(CH2)m-R3R4, -(CH2)m-C(halogen)3, -(CH2)m-alkenyl, -(CH2)m-alkenyl-CH3, -(CH2)m-alkynyl, -(CH2)m-alkynyl- CH3, -(CH2)m-(C3-C8)cycloalkyl, -(CH2)m-(C3-C8)heterocycloalkyl, -(CH2)m-phenyl, -(CH2)m-(5 or 6-membered)heteroaryl, -(CH2)m-cyano, where m is 1, 2, 3, 4 or 5 and where the cycloalkyl, heterocycle or phenyl is unsubstituted or substituted with one or more X1 groups, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (g) R2 and R3 are independently hydrogen, methyl, ethyl, ethenyl, ethynyl, propyl, butyl, pentyl, hexyl, isopropyl, t-butyl, isobutyl, -C(halogen)3, -CH(halogen)2, -CH2(halogen), -CH2C(halogen)3, -CHCH(halogen)2, CHCH2(halogen), -CH2OH, -CH2CH2OH, -CH2C(CH3)2OH, -CH2CH(CH3)OH, -C(CH3)2CH2OH, -CH(CH3)CH2OH, -CH(CH3)CH(OH)R4, -CH2CH(OH)R4, -CH2SO2NHR4, -CH2NHSO2R4, or R3 and R4 form an unsubstituted or substituted aziridine, azetidine, pyrrolidine or piperidine ring when taken together with the nitrogen to which they are attached; and (h) R4 is hydrogen, methyl, ethyl, isopropyl, t-butyl, isobutyl, or -C(halogen)3. 29. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000073_0001
or a pharmaceutically acceptable salt thereof. 30. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000073_0002
or a pharmaceutically acceptable salt thereof. 31. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000073_0003
or a pharmaceutically acceptable salt thereof. 32. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000074_0001
or a pharmaceutically acceptable salt thereof. 33. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000074_0002
or a pharmaceutically acceptable salt thereof. 34. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000074_0003
or a pharmaceutically acceptable salt thereof. 35. The method of embodiment 28, wherein the compound has the following formula:
Figure imgf000074_0004
or a pharmaceutically acceptable salt thereof. 36. The method of embodiment 1, 2, or 3, wherein the compound has the structure of Formula (III):
Figure imgf000075_0001
or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000075_0002
(b) each of Z1 and Z3 is independently -CH- or -N-; (c) Z2 is -N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; (d) each of Z6, Z7 and Z8 is independently -C- or -N-, with the proviso that at least one of Z6- Z8 is -C-; (e) X1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (f) each of X4 , X5, and X6 is independently -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, - CN, -C(O)R, -C(O)2R, -S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6- membered)heterocyclic aromatic or heterocyclic non-aromatic group; with the provisos that X4 is absent when Z6 is a nitrogen, X5 is absent when Z7 is a nitrogen and X6 is absent when Z8 is a nitrogen; (g) R7 is -(C1-C6)aliphatic-N+-(R2)(R3)(R4), -(C1-C6)aliphatic-N-R3R4, -(C1-C6)aliphatic- C(=O)N-R3R4, -(C1-C6)aliphatic-R3R4, -(C1-C6)aliphatic-R2R3R4, -(C1-C6)aliphatic-N-CR2R3R4, - (C1-C6)aliphatic-C(halogen)3, -(C1-C6)aliphatic-alkenyl, -(C1-C6)aliphatic-alkynyl, -(C1- C6)aliphatic-(C3-C8)cycloalkyl, -(C1-C6)aliphatic-(C3-C8)heterocycloalkyl, -(C1-C6)aliphatic- phenyl, -(C1-C6)aliphatic-(5 or 6-membered)heteroaryl, -(C1-C6)aliphatic-cyano, wherein the cycloalkyl, heterocycloalkyl, phenyl, or heteroaryl is unsubstituted or substituted, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (h) Q is fused benzo, fused (5- or 6-membered)heteroaryl, a fused 4 to 7-membered cycloalkyl ring or a fused 4- to 7-membered non-aromatic heterocyclic ring; (i) R2 and R3 are independently hydrogen, -N(R)2, -CH2CH(OH)R4, -CH(OH)CH2R4, - CH2SO2NHR4, -CH2NHSO2R4, or unsubstituted or substituted -(C1-C6)aliphatic, or R3 and R4 form an unsubstituted or substituted 3- to 7-membered heterocyclic ring when taken together with the nitrogen to which they are attached; (j) R4 is hydrogen, halogen, or unsubstituted or substituted –(C1-C6)aliphatic; (k) each R8 is independently -H, -halogen, -N(R)2, -OR, -CN, or a unsubstituted or substituted selected from –CH2-phenyl or -(C1-C6)aliphatic; (l) each RY is independently R, -OR, or halogen; (m) a is an integer selected from 0, 1 and 2; and (n) each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, -N(R)2, -OR, -CN, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2. 37. The method of embodiment 1, 2, or 3, wherein the compound has the structure of Formula (IV):
Figure imgf000076_0001
or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000077_0001
(b) each of Z1, Z3, Z9, Z10, Z11 and Z12 is independently -CH- or -N-; (c) Z2 is -N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; (d) each of X8 and X9 is independently -CH-, -S-, -N-, or -O-; (e) X1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (f) R7 is -(C1-C6)aliphatic-N+-(R2)(R3)(R4), -(C1-C6)aliphatic-N-R3R4, -(C1-C6)aliphatic- C(=O)N-R3R4, -(C1-C6)aliphatic-R3R4, -(C1-C6)aliphatic-R2R3R4, -(C1-C6)aliphatic-N-CR2R3R4, - (C1-C6)aliphatic-C(halogen)3, -(C1-C6)aliphatic-alkenyl, -(C1-C6)aliphatic-alkynyl, -(C1- C6)aliphatic-(C3-C8)cycloalkyl, -(C1-C6)aliphatic-(C3-C8)heterocycloalkyl, -(C1-C6)aliphatic- phenyl, -(C1-C6)aliphatic-(5 or 6-membered)heteroaryl, -(C1-C6)aliphatic-cyano, wherein the cycloalkyl, heterocycloalkyl, phenyl, or heteroaryl is unsubstituted or substituted, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (g) R2 and R3 are independently hydrogen, -N(R)2, -CH2CH(OH)R4, -CH(OH)CH2R4, - CH2SO2NHR4, -CH2NHSO2R4, or unsubstituted or substituted -(C1-C6)aliphatic, or R3 and R4 form an unsubstituted or substituted 3- to 7-membered heterocyclic ring when taken together with the nitrogen to which they are attached; (h) R4 is hydrogen, halogen, or unsubstituted or substituted –(C1-C6)aliphatic; (i) each R8 is independently -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (j) R9 is –H, (C1-C6)aliphatic-cycloalkyl, -(C1-C6)aliphatic-heterocycloalkyl, -(C1- C6)aliphatic-aryl, -(C1-C6)aliphatic-heteroaryl, or -(C1-C6)aliphatic-cyano, wherein each cycloalkyl, heterocycloalkyl, aryl, or heteroaryl is unsubstituted or substituted, with the proviso that R9 is absent when X9 is –S- or –O-; (k) each RY is independently R, -OR, or halogen; (l) a is an integer selected from 0, 1 and 2; and (m) each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2; and wherein each substituted group is substituted with one or more groups selected from halogen, -N(R)2, -OR, -CN, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2. 38. The method of embodiment 1, 2, or 3, wherein the compound has the structure of Formula (V):
Figure imgf000078_0001
or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000078_0002
(b) each of Z1 and Z3 is independently -CH- or -N-; (c) Z2 is -N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; (d) each of Z4, Z5, Z6, Z7 and Z8 is independently -C- or -N-, with the proviso that no three consecutive Z4 through Z8 are N; (e) X1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (f) each of X4, X5, and X6 is independently -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, - CN, -C(O)R, -C(O)2R, -S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6- membered)heterocyclic aromatic or heterocyclic non-aromatic group; with the provisos that at least one of X2, X4 and X5 is -H and that X2 is absent when Z4 is -N-, X3 is absent when Z5 is -N-, X4 is absent when Z6 is -N- and X5 is absent when Z7 is -N-; (g) each of X2 and X3 is independently selected from (1) -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, -CN, -C(O)R, -C(O)2R, - S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6-membered)heterocyclic aromatic or heterocyclic non-aromatic group; or (2) X2 and X3 taken together form a fused benzo or fused (5- or 6-membered) heteroaryl that may be substituted with one or more R8 groups; (h) R7 is -(C1-C6)aliphatic-N+-(R2)(R3)(R4), -(C1-C6)aliphatic-N-R3R4, -(C1-C6)aliphatic- C(=O)N-R3R4, -(C1-C6)aliphatic-R3R4, -(C1-C6)aliphatic-R2R3R4, -(C1-C6)aliphatic-N-CR2R3R4, - (C1-C6)aliphatic-C(halogen)3, -(C1-C6)aliphatic-alkenyl, -(C1-C6)aliphatic-alkynyl, -(C1- C6)aliphatic-(C3-C8)cycloalkyl, -(C1-C6)aliphatic-(C3-C8)heterocycloalkyl, -(C1-C6)aliphatic- phenyl, -(C1-C6)aliphatic-(5 or 6-membered)heteroaryl, -(C1-C6)aliphatic-cyano, wherein the cycloalkyl, heterocycloalkyl, phenyl, or heteroaryl is unsubstituted or substituted, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (i) R2 and R3 are independently hydrogen, -N(R)2, -CH2CH(OH)R4, -CH(OH)CH2R4, - CH2SO2NHR4, - CH2NHSO2R4, or unsubstituted or substituted -(C1-C6)aliphatic, or R3 and R4 form an unsubstituted or substituted 3- to 7-membered heterocyclic ring when taken together with the nitrogen to which they are attached; (j) R8 is -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, -CN, -C(O)R, -C(O)2R, - S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6-membered)heterocyclic aromatic or heterocyclic non-aromatic group; (k) R4 is hydrogen, halogen, or unsubstituted or substituted –(C1-C6)aliphatic; and (l) each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, -N(R)2, -OR, -CN, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2. 39. The method of embodiment 1, 2, or 3, wherein the compound is:
,
Figure imgf000080_0001
NH
Figure imgf000081_0001
N
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
,
Figure imgf000086_0001
Figure imgf000087_0001
N
Figure imgf000088_0001
Figure imgf000089_0001
or a pharmaceutically acceptable salt thereof. 40. The method of embodiment 39, wherein the compound is:
Figure imgf000089_0002
or a pharmaceutically acceptable salt thereof. 41. The method of embodiment 27, wherein the compound has the structure of Formula (II):
Figure imgf000089_0003
or a pharmaceutically acceptable salt thereof, wherein: (a) Y is -S-; (b) each of Z1 and Z3 is -N-; (c) Z2 is -N-; (d) X1 is -H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; (e) each of X3 and X5 is independently -H, -halogen, -SR, -N(R)2, -OR, -CN, -NO2, - CN, -C(O)R, -C(O)2R, -S(O)R, -S(O)2R, -C(O)N(R)2, -SO2N(R)2, -OC(O)R, -N(R)C(O)R, -N(R)SO2R, -OC(O)N(R)2, unsubstituted or substituted -(C1-C6)aliphatic, or an unsubstituted or substituted group selected from (5- or 6-membered)aryl, (5- or 6-membered)arylalkyl, and (5- or 6- membered)heterocyclic aromatic or heterocyclic non-aromatic group; (f) R1 is -(C1-C6)aliphatic-N+-(R2)(R3)(R4), -(C1-C6)aliphatic-N-R3R4, -(C1-C6)aliphatic- C(=O)N-R3R4, -(C1-C6)aliphatic-N-CR2R3R4, with the proviso that when all of R2-R4 are present the compound further comprises a pharmaceutically acceptable counter ion; (g) R2 and R3 are independently hydrogen, -N(R)2, -CH2CH(OH)R4, -CH(OH)CH2R4, - CH2SO2NHR4, - CH2NHSO2R4, or unsubstituted or substituted -(C1-C6)aliphatic, or R3 and R4 form an unsubstituted or substituted 3- to 7-membered heterocyclic ring when taken together with the nitrogen to which they are attached; (h) each RY is independently R, -OR, or halogen; (i) R4 is hydrogen, halogen, or unsubstituted or substituted –(C1-C6)aliphatic; and (j) each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, -N(R)2, -OR, -CN, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, or -NH2. 42. The method of any one of the preceding embodiments, wherein the compound is part of a pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient. 43. The method of any one of the preceding embodiments, wherein the N-glycosylated Grp94 comprises two or more N-glycan modified residues. 44. The method of any one of the preceding embodiments, wherein the N-glycosylated Grp94 comprises two or more N-glycan modified Asn residues. 45. The method of any one of the preceding embodiments, wherein the N-glycosylated Grp94 comprises glycosylated Asn at the N62 residue. 46. The method of any one of the preceding embodiments, wherein the N-glycosylated Grp94 alters the function of one or more aberrant oncogenic proteins. 47. The method of embodiment 46, wherein the N-glycosylated Grp94 is characterized in that, prior to administration of the compound, it forms high molecular weight complexes (e.g., above 242 kDa) with one or more aberrant oncogenic proteins. 48. The method of any one of the preceding embodiments, wherein the cancer is characterized by overexpression of a receptor tyrosine kinase (RTK). 49. The method of embodiment 48, wherein the aberrant oncogenic protein is a receptor tyrosine kinase (RTK). 50. The method of embodiment 49, wherein the RTK is HER2 or EGFR. 51. The method of any one of the preceding embodiments, wherein the compound is characterized in that it exhibits a greater binding affinity for N-glycosylated Grp94 compared to “housekeeping Grp94”, Hsp90α and/or Hsp90β. 52. The method of any one of embodiments 1, 2, and 4-51, wherein the cancer is colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, leukemias, myelomas, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, or endometrial cancer. 53. The method of embodiment 52, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, esophageal cancer, or non-small-cell lung cancer. 54. The method of embodiment 53, wherein the cancer is breast cancer. 55. The method of any one of embodiments 3-51, wherein the disease is selected from the group consisting of inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis, and diabetes. Examples [0130] Methods and examples described herein may be carried out according to the disclosure of Yan et al., 2020, Cell Reports 31, 107840, June 30, 2020, incorporated herein by reference. Compounds described herein may be prepared according to the procedures described in WO 2015/023976, incorporated herein by reference. It will be appreciated that, although the present disclosure describes the synthesis of certain compounds of the present disclosure, the following methods and other methods known to one of ordinary skill in the art can be applied to make and use all compounds and subclasses and species of each of these compounds, as described herein. [0131] General procedure 1:
Figure imgf000092_0001
Scheme 1. Reagents and conditions: (a) CS2, NaHCO3, H2O, EtOH, reflux, 4 days; (b) neocuproine hydrate, CuI, NaOtBu, 3,5-dichloroiodobenzene, DMF, 115°C, 24 h; (c) 1,3- dibromopropane, Cs2CO3, DMF, rt, 2 h; (d) amines, DMF, rt, 18-48 h. [0132] 9-(3-(tert-butylamino)propyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine (I-38, HJP-V-142):
Figure imgf000093_0001
[0133] To a solution of 4a (12 mg, 0.0277 mmol) in dry DMF (2 ml) was added tert- butylamine (58 µL, 0.554 mmol) and the reaction mixture was stirred at rt for 18 h. Then, the solvent was removed under reduced pressure and the crude product was purified by column chromatography (CH2Cl2:MeOH-NH3 (7N), 100:1 to 30:1) to afford 7.9 mg (67%) of I-38 (HJP- V-142).1H NMR (600 MHz, CDCl3) δ 8.36 (s, 1H), 7.28 (s, 3H), 5.82 (br s, 2H), 4.32 (t, J = 7.1 Hz, 2H), 2.52 (t, J = 6.7 Hz, 2H), 1.94 (p, J = 6.9 Hz, 2H), 1.05 (s, 9H); 13C NMR (150 MHz, CDCl3) δ 154.70, 153.38, 151.60, 143.65, 135.81, 134.66, 128.38, 128.11, 120.25, 50.42, 41.98, 39.03, 31.08, 28.91; MS m/z 425.36 [M + H]+. [0134] 8-((3,5-dichlorophenyl)thio)-9-(3-((2,2,2-trifluoroethyl)amino)propyl)-9H-purin-6- amine (I-43, HJP-VI-102):
Figure imgf000093_0002
[0135] To a solution of 4a (12 mg, 0.027 mmol) in dry DMF (3 ml) was added 2,2,2- trifluoroethylamine (108.8 µL, 1.385 mmol) and the reaction mixture was stirred at rt for 48 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 5.2 mg (42%) of I-43 (HJP-VI-102).1H NMR (600 MHz, CDCl3/MeOD) δ 8.25 (s, 1H), 7.42 – 7.38 (m, 2H), 7.37 – 7.34 (m, 1H merged with CDCl3), 4.33 (t, J = 7.0 Hz, 2H), 3.16 (q, J = 9.5 Hz, 2H), 2.70 (t, J = 6.3 Hz, 2H), 2.04 – 1.92 (m, 2H); MS m/z 450.88 [M + H]+. [0136] 8-((3-bromo-5-iodophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine (I-50, HJP-VI-105):
Figure imgf000094_0001
[0137] To a solution of 4b (635 mg, 1.466 mmol) in dry DMF (5 ml) was added iso- propylamine (5.43 mL, 55.8 mmol) and the reaction mixture was stirred at rt for 24 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 260 mg (42.6%) of I-50 (HJP-V-105).1H NMR (600 MHz, CDCl3/MeOD) δ 8.28 (s, 1H), 7.86 – 7.85 (m, 1H), 7.73 – 7.71 (m, 1H), 7.56 – 7.54 (m, 1H), 4.30 (t, J = 7.0 Hz, 2H), 2.74 – 2.66 (m, 1H), 2.53 (t, J = 6.8 Hz, 2H), 1.96 (p, J = 6.9 Hz, 2H), 1.04 (d, J = 6.3 Hz, 6H); 13C NMR (150 MHz, CDCl3/MeOD) δ 154.58, 153.08, 151.41, 144.52, 139.95, 138.12, 133.85, 133.11, 123.70, 119.75, 94.91, 48.67, 43.50, 41.68, 29.93, 22.56; MS m/z 546.86 [M + H]+. [0138] 8-((3,5-dichlorophenyl)thio)-9-(3-(neopentylamino)propyl)-9H-purin-6-amine (I- 41, HJP-V-143):
Figure imgf000094_0002
[0139] To a solution of 4a (12 mg, 0.027 mmol) in dry DMF (3 ml) was added neopentylamine (65 µL, 0.554 mmol) and the reaction mixture was stirred at rt for 24 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 12.2 mg (86%) of I-41 (HJP-V-143).1H NMR (600 MHz, CDCl3/MeOD) δ 8.27 (s, 1H), 7.36 (t, J = 1.8 Hz, 1H), 7.32 (d, J = 1.8 Hz, 2H), 4.31 (t, J = 7.0 Hz, 2H), 2.57 (t, J = 6.8 Hz, 2H), 2.28 (s, 2H), 1.99 (p, J = 6.9 Hz, 2H), 0.92 (s, 9H); 13C NMR (150 MHz, CDCl3/MeOD) δ 154.68, 153.15, 151.37, 144.38, 136.02, 133.75, 129.02, 128.92, 119.79, 62.17, 47.10, 41.88, 31.37, 29.55, 27.83. [0140] General procedure 2:
Figure imgf000095_0001
Scheme 2. Reagents and conditions: (a) Cs2CO3, DMF, rt, 3-24 h; (b) TFA, DCM, 24 h. [0141] 1-(3-(6-amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9-yl)propyl)guanidine (I-45, SO-IV-26A):
Figure imgf000095_0002
[0142] To 3a (20 mg, 0.064 mmol) in dry DMF (2 mL) was added Cs2CO3 (23 mg, 0.070 mmol) and di-boc-guanidinopropyl bromide (37 mg, 0.096 mmol) and the reaction mixture was stirred at rt for 24 h. After filtering off excess Cs2CO3, the filtrate was concentrated under reduced pressure and the resulting residue was partially purified by preparative TLC (CH2Cl2:MeOH:AcOH at 100:0:0 to 30:1:0.5) to give a residue (38%), which was dissolved in TFA/CH2Cl2 (0.4:1.6 mL) and stirred for 24 hr at rt. The reaction mixture was concentrated and the residue purified by prep TLC (CHCl3:MeOH-NH3 (7N), 10:1) to yield I-45 (SO-IV-26A, 62.7 mg, 13% for two steps).1H NMR (500 MHz, MeOD) δ 8.27 (s, 1H), 7.53 – 7.49 (m, 3H), 4.36 (t, J = 6.9 Hz, 2H), 3.21 (t, J = 6.7 Hz, 2H), 2.10 – 2.01 (m, 2H); MS m/z 410.97 [M + H]+. [0143] 9-(3-aminopropyl)-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine I-42, SO-IV- 22A):
Figure imgf000096_0001
[0144] To 3a (100 mg, 0.32 mmol) in dry DMF (3 mL) was added Cs2CO3 (115 mg, 0.35 mmol) and 3-(boc-amino)propyl bromide (305 mg, 1.28 mmol) and the reaction mixture was stirred at rt for 3 h. After filtering off excess Cs2CO3, the filtrate was concentrated under reduced pressure and the resulting residue was partially purified by preparative TLC (CH2Cl2:MeOH:AcOH at 100:0:0 to 30:1:0.5) to give a residue (28 mg), which was dissolved in TFA/CH2Cl2 (0.4:1.6 mL) and stirred for 24 hr at rt. The reaction mixture was concentrated and the residue purified by prep TLC (CHCl3:MeOH-NH3 (7N), 15:1) to yield I-42 (SO-IV-22A, 19 mg, 16% for two steps).1H NMR (500 MHz, CDCl3/MeOD) δ 8.26 (s, 1H), 7.39 – 7.36 (m, 1H), 7.36 – 7.32 (m, 2H), 4.32 (t, J = 6.9 Hz, 2H), 2.68 (t, J = 6.6 Hz, 2H), 1.95 (p, J = 6.8 Hz, 2H); 13C NMR (125 MHz, CDCl3/MeOD) δ 154.66, 152.98, 151.23, 144.36, 135.95, 133.22, 129.13, 128.97, 119.58, 40.90, 37.81, 32.24; MS m/z 369.00 [M + H]+. [0145] General procedure 3:
Figure imgf000097_0001
Scheme 3. Reagents and conditions: (a) neocuproine hydrate, CuI, NaOtBu, 3,5-disubstituted iodobenzene, DMF, 115°C, 24 h; (b) SbCl3, t-BuONO, DCE/DMSO, 80°C, 48 h (c) HF/pyridine, NaNO2, 0°C, 1 h; (d) 1,3-dibromopropane, Cs2CO3, DMF, rt, 2 h; (e) i- propylamine, DMF, rt, 8-24 h. [0146] 8-((3-chloro-5-iodophenyl)thio)-2-fluoro-9-(3-(isopropylamino)propyl)-9H-purin-6- amine (I-92, HJP-VI-110):
Figure imgf000097_0002
[0147] To a solution of 10a (23 mg, 0.00423 mmol) in dry DMF (2 ml) was added i- propylamine (173 µL, 2.1194 mmol) and the reaction mixture was stirred at rt for 8 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 15.2 mg (69%) of I-92 (HJP-V-110).1H NMR (600 MHz, CDCl3) δ 7.63 (t, J = 1.6 Hz, 1H), 7.61 (t, J = 1.5 Hz, 1H), 7.33 (t, J = 1.7 Hz, 1H), 6.34 (br s, 2H), 4.25 (t, J = 7.1 Hz, 2H), 2.75 – 2.68 (m, 1H), 2.56 (t, J = 6.8 Hz, 2H), 1.94 (p, J = 6.8 Hz, 2H), 1.04 (d, J = 6.3 Hz, 6H).13C NMR (150 MHz, CDCl3) δ 159.26 (d, J = 212.0 Hz), 156.48 (d, J = 20.3 Hz), 152.94 (d, J = 19.2 Hz), 143.62 (d, J = 2.7 Hz) 136.86, 136.54, 135.81, 134.61, 129.26, 118.38 (d, J = 3.7 Hz), 94.50, 48.83, 43.81, 42.08, 30.18, 22.83; MS m/z 520.94 [M + H]+. [0148] 2-chloro-8-((3,5-dichlorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6- amine (I-86, SO-IV-33A):
Figure imgf000098_0001
[0149] To a solution of 10b (4 mg, 0.0115 mmol) in dry DMF (1 ml) was added i- propylamine (20 µL) and the reaction mixture was stirred at rt for 24 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH- NH3 (7N), 20:1) to afford 3.1 mg (60%) of I-86 (SO-IV-33A).1H NMR (500 MHz, CDCl3) δ 7.33 – 7.31 (m, 1H), 7.30 – 7.28 (m, 2H), 5.75 (br s, 2H), 4.29 (t, J = 6.9 Hz, 2H), 2.78 – 2.70 (m, 1H), 2.56 (t, J = 6.6 Hz, 2H), 1.99 – 1.91 (m, 2H), 1.05 (d, J = 6.2 Hz, 6H); MS m/z 444.92 [M + H]+. [0150] 8-((3-chloro-5-(prop-1-en-2-yl)phenyl)thio)-2-fluoro-9-(3-(isopropylamino)propyl)- 9H-purin-6-amine (I-93, HJP-VI-118):
Figure imgf000098_0002
[0151] To a solution of I-92 (12 mg, 0.023 mmol) in DMF:H2O (1:ml: 0.1 ml) was added Pd(PPh3)2Cl2 (3.23 mg, 0.0046 mmol), 4,4,5,5-tetramethyl-2-(prop-1-en-2-yl)-1,3,2-dioxaborolane (7.73 mg, 0.046 mmol) and NaHCO3 (5.8 mg, 0.069 mmol) and the reaction mixture was heated at 90°C for 24 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 5.2 mg (52%) of I-93 (HJP-VI- 118).1H NMR (600 MHz, CDCl3) δ 7.39 (t, J = 1.6 Hz, 1H), 7.37 (t, J = 1.7 Hz, 1H), 7.27 – 7.25 (m, 1H), 5.98 (br s, 2H), 5.37 (s, 1H), 5.18 – 5.15 (m, 1H), 4.26 (t, J = 7.0 Hz, 2H), 2.73 (dp, J = 12.4, 6.2 Hz, 1H), 2.56 (t, J = 6.8 Hz, 2H), 2.09 (s, 3H), 1.99 – 1.91 (m, 2H), 1.05 (d, J = 6.3 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ 159.10 (d, J = 211.8 Hz), 156.18 (d, J = 20.3 Hz), 153.04 (d, J = 19.0 Hz), 144.99 (d, J = 2.7 Hz), 144.22, 141.03, 135.24, 132.48, 128.90, 125.89 (d, J = 7.5 Hz), 115.06, 53.44, 48.91, 43.73, 41.93, 22.68, 21.54; MS m/z 435.02 [M + H]+. [0152] General procedure 4:
Figure imgf000099_0001
Scheme 4. Reagents and conditions: (a) Pd(PPh3)4, 2-(tri-tert-butylstannyl)oxazole, LiCl, DMF, 90°C, 20h; (b) Pd(PPh3)4Cl2, boronate esters, NaHCO3, 90°C, 24 h. [0153] 8-((3-chloro-5-(oxazol-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6- amine (I-84, HJP-VI-49):
Figure imgf000099_0002
[0154] To a solution of I-49 (15 mg, 0.02985 mmol) in DMF was added Pd(PPh3)4 (3.5 mg, 0.003 mmol), 2-(tri-tert-butylstannyl)oxazole (42.78 mg, 0.1194 mmol) and LiCl (2.6 mg, 0.0597 mmol) and the reaction mixture was heated at 90°C for 20 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH- NH3 (7N), 20:1) to afford 7.6 mg (57%) of I-84 (HJP-VI-49).1H NMR (600 MHz, CDCl3/MeOD) δ 8.24 (s, 1H), 8.09 (s, 1H), 8.04 (s, 1H), 7.77 (s, 1H), 7.55 (s, 1H), 7.30 (s, 1H), 4.40 (t, J = 6.6 Hz, 2H), 3.04 (dt, J = 12.8, 6.3 Hz, 1H), 2.75 (t, J = 6.7 Hz, 2H), 2.26 – 2.18 (m, 2H), 1.26 (d, J = 6.4 Hz, 6H); 13C NMR (150 MHz, CDCl3/MeOD) δ 159.49, 154.72, 152.74, 151.36, 145.38, 139.77, 136.14, 132.91, 132.50, 130.09, 128.84, 127.55, 126.85, 119.58, 50.11, 42.28, 40.88, 27.84, 20.49. [0155] 8-((3-chloro-5-(5-methylthiophen-2-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)- 9H-purin-6-amine (I-77, HJP-VI-122):
Figure imgf000100_0001
[0156] To a solution of I-49 (15 mg, 0.0298 mmol) in DMF:H2O (1:ml: 0.1 ml) was added Pd(PPh3)4Cl2 (4.2 mg, 0.00597 mmol), 4,4,5,5-tetramethyl-2-(3-methylthiophen-2-yl)-1,3,2- dioxaborolane (13.4 mg, 0.0597 mmol) and NaHCO3 (7.5 mg, 0.089 mmol) and the reaction mixture was heated at 90°C for 24 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 5.7 mg (40%) of I-77 (HJP-VI-122).1H NMR (600 MHz, CDCl3) δ 8.34 (s, 1H), 7.38 (t, J = 1.7 Hz, 1H), 7.36 (t, J = 1.6 Hz, 1H), 7.34 (t, J = 1.8 Hz, 1H), 7.24 (d, J = 5.1 Hz, 1H), 6.90 (d, J = 5.1 Hz, 1H), 5.71 (s, 2H), 4.34 (t, J = 7.0 Hz, 2H), 2.72 (dt, J = 12.5, 6.3 Hz, 1H), 2.56 (t, J = 6.7 Hz, 2H), 2.26 (s, 3H), 2.02 – 1.97 (m, 3H), 1.04 (d, J = 6.3 Hz, 6H); 13C NMR (150 MHz, CDCl3) δ 154.56, 153.20, 151.65, 144.62, 137.60, 135.27, 134.85, 134.62, 133.24, 131.37, 128.62, 128.56, 128.25, 124.69, 120.13, 48.83, 43.66, 41.70, 30.08, 22.66, 14.96; MS m/z 472.99 [M + H]+. [0157] 8-((3-chloro-5-(thiophen-3-yl)phenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin- 6-amine (I-78, HJP-VI-119):
Figure imgf000100_0002
[0158] To a solution of I-49 (15 mg, 0.0298 mmol) in DMF:H2O (1:ml: 0.1 ml) was added Pd(PPh3)4Cl2 (4.2 mg, 0.00597 mmol), 4,4,5,5-tetramethyl-2-(thiophen-3-yl)-1,3,2-dioxaborolane (13.4 mg, 0.0597 mmol) and NaHCO3 (7.5 mg, 0.089 mmol) and the reaction mixture was heated at 90°C for 24 h. Then, the solvent was removed under reduced pressure and the crude product was purified by prep TLC (CH2Cl2:MeOH-NH3 (7N), 20:1) to afford 7.1 mg (52%) of I-78 (HJP-VI- 122).1H NMR (600 MHz, CDCl3/MeOD) δ 8.24 (s, 1H), 7.63 (s, 1H), 7.60 (t, J = 1.4 Hz, 1H), 7.55 – 7.52 (m, 1H), 7.43 (dd, J = 5.0, 3.0 Hz, 1H), 7.37 (t, J = 1.6 Hz, 1H), 7.34 (d, J = 5.0 Hz, 1H), 4.34 (t, J = 6.9 Hz, 2H), 2.84 (dt, J = 12.1, 5.9 Hz, 1H), 2.62 (t, J = 6.8 Hz, 2H), 2.12 – 2.03 (m, 2H), 1.12 (d, J = 6.3 Hz, 6H); 13C NMR (150 MHz, CDCl3/MeOD) δ 154.61, 152.74, 151.36, 145.98, 139.48, 138.99, 135.87, 131.71, 129.89, 128.10, 127.23, 127.21, 125.93, 122.35, 119.52, 50.16, 43.03, 41.33, 28.94, 21.59; MS m/z 459.01 [M + H]+. GENERAL EXPERIMENTAL MODEL AND SUBJECT DETAILS Human Subjects [0159] De-identified pathology discarded specimens were obtained in accordance with the guidelines and approval of the Institutional Review Board #09-121 (breast cancer, PI: Dr. Modi) and #10-018 (esophagogastric cancer, PI: Dr. Janjigian) at Memorial Sloan Kettering Cancer Center (New York, NY). The patients provided signed informed consent prior to participation. Animals [0160] All animal studies were conducted in compliance with MSKCC’s Institutional Animal Care and Use Committee (IACUC) guidelines under an approved protocol. All mice were housed at MSKCC animal facility and Research Animal Resource Center (RARC) provided husbandry and clinical care. Athymic nude mice (Hsd:Athymic Nude-Foxn1 nu , female, 20-25 g, 6 weeks old; RRID:MGI:5652489) were obtained from Envigo and NSG mice (NOD.Cg-Prkdc scid
Figure imgf000101_0001
female, 20-25 g, 8 weeks old, IMSR Cat# JAX:005557, RRID:IMSR_JAX:005557) were obtained from the Jackson Laboratory. Human Cell Lines [0161] Cell lines were obtained from laboratories at MSKCC, or purchased from the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). Cells were cultured as per the providers’ recommended culture conditions. Cells were authenticated using short tandem repeat profiling and tested for mycoplasma before and after use. The breast cancer cell lines (MDA-MB-468 (HTB-132), HCC1806 (CRL- 2335), MDA-MB-231 (HTB-26), MDA-MB-415 (HTB-128), MCF-7 (HTB-22), BT474 (HTB- 20), BT20 (HTB-19), MDA- MB-361 (HTB-27), SKBr3 (HTB-30), MDA-MB-453 (HTB-131), T47D (HTB-133), AU565 (CRL-2351) and the non-transformed cell line HMEC (human primary mammary epithelial cells, PCS- 600-010) were obtained from the American Type Culture Collection (ATCC). The pancreatic cancer cell lines include: MiaPaCa2 (CRL-1420), Panc-1 (CRL-1469), BxPc-3 (CRL-1687), Capan-1 (HTB-79), SU.86.86 (CRL-1837), HPAF2 (CRL- 1997), ASPC-1 (CRL-1682), PL45 (CRL-2558), CFPAC (CRL-1918), Capan-2 (HTB-80) were purchased from ATCC; 931102 and 931019 are patient derived cell lines provided by Dr. Y. Janjigian, MSKCC. The lung cancer cell lines NCI-H3122, NCI-H299 were kindly provided by Dr. M. Moore, MSKCC; NCI-H1373 and NCI- H525 were obtained from Dr. N. Lecomte, MSKCC. The stomach cancer cell lines SNU-1 (CRL- 5971) and NCI-N87 (CRL-5822) were obtained from ATCC. The ovarian cancer cell lines PEO-1, PEO-4, OVCAR4, OV1847, A2780, IGROV-1 and OVCAR5 were kindly provided by Dr. D. Solit, MSKCC. The renal cancer lines SKRC38 and SKRC52 were provided by Dr. S. Larson, MSKCC. Neuroblastoma cells SY5Y (CRL-2266) was purchased from ATCC; LAN5 and SMS-KCNR were obtained from the Children’s Oncology Group (COG). Ewing’s sarcoma cells TC71 and A673 were kindly provided by Dr. S. Ambati, MSKCC. Lymphoma cell lines include: SU-DHL-6 (CRL- 2959), Toledo (CRL-2631), Farage (CRL-2630) and BC3 (CRL-2277) were purchased from ATCC; HBL-1, MD901 and U2932 were kindly provided by J. Angel Martinez-Climent, Centre for Applied Medical Research, Pamplona, Spain; Karpas422 (ACC-32), RCK8 (ACC-561) and SU- DHL-4 (ACC-495) were obtained from the DSMZ; OCI-LY1, OCI-LY3 and OCI-LY7 were obtained from the Ontario Cancer Institute; TMD8 was kindly provided by L. M. Staudt, NIH. Leukemia cell lines KASUMI-1 (CRL-2724), K562 (CCL-243) were purchased from ATCC; MOLM-13 (ACC-554) was obtained from DSMZ. Multiple Myeloma cell lines U266, PCNY1 and MM.1R were kindly provided from Dr. Z. Li, OSU. Murine Cell Lines [0162] Wild type and TM96 expressing MethA fibrosarcoma cells were kindly provided by Dr. Z. Li, OSU. The cells were established as previously reported (Zheng et al., 2001) and cultured in RPMI medium with 10% heat-inactivated FBS (VWR) and 1% Penicillin/Streptomycin. Reagents [0163] PU-WS13, PU-WS13-biotin, inactive-WS13-biotin, PU29F, PU-H71, HJP-149 and SO-33 were synthesized using a previously reported protocol (Patel et al., 2015; Patel et al., 2013; Rodina et al., 2016). Briefly, PU-WS13 was synthesized via CuI-catalyzed coupling of 8- mercaptoadenine with 3,5-dichloroiodobenzene at 110 °C resulting in 8-(3,5-dichloro- phenylsulfanyl)adenine in 72% yield, which was heated with 3-(tertbutoxycarbonyl-isopropyl- amino)-propyl tosylate in DMF at 80 °C under nitrogen protection for 30 min. The Boc- deprotection of resulting tert-butyl(3-(6- amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9- yl)propyl)(isopropyl)carbamate from the previous step with TFA gave crude material which upon purification by preparatory TLC with CH 2 Cl 2 :MeOH- NH3 (7N) at 20:1 yielded PU-WS13 (45% yield). PU-WS13-biotin was synthesized through alkylation of 8-(3,5-dichloro-phenyl sulfanyl)adenine at position N9 with N-(8- bromooctyl)phthalimide in the presence of Cs 2 CO 3 in DMF at room temperature to obtain 2-(8-(6- amino-8-((3,5-dichlorophenyl)thio)-9H-purin-9- yl)octyl)isoindoline-1,3-dione in 21% yield. The resulting product from the previous step was subjected to phathalimide-deprotection with hydrazine hydrate in a mixture of CH 2 Cl 2 and CH 3 OH to afford 9-(3-aminohexyl)-8-((3,5- dichlorophenyl)thio)-9H-purin-6-amine in 50% yield. In the final step, the free amine was reacted with NHS-active ester of biotin in DMF to result in a crude residue of PU-WS13-biotin. This residue was purified by preparatory TLC (CH 2 Cl 2 -MeOH- NH 3 (7N), 10:1) to give 34% yield of PU-WS13- biotin. The synthetic route to inactive-WS13- biotin comprises S-alkylation of 8-mercaptoadenine with 1-iodo-2-methoxyethane in aqueous KOH solution providing 87% yield of 8-((2- methoxyethyl)thio)-9H-purin-6-amine. Further reaction with N-(8-bromooctyl)phthalimide, followed by phathalimide-deprotection and coupling with NHS-active ester of biotin in DMF gave crude inactive-WS13-biotin. This resulting residue was purified by preparatory TLC (CH 2 Cl 2 :MeOH-NH 3 (7N), 10:1) to give in 72% yield the inactive- WS13-biotin. For the synthesis of HJP-149, C-S coupling was achieved by reacting 8- mercaptoadenine with 1-chloro-3,5- diiodobenzene in the presence CuI and neocuproine in DMF at 110 °C. The resulting product was then N9-alkylated with 1,3-dibromopropane affording 9-(3- bromopropyl)-8-((3-chloro-5- iodophenyl)thio)-9H-purin-6-amine in 25% yield. In the final step, the reaction of bromo derivative with excess of isopropylamine, followed by purification using preparative TLC (CH 2 Cl 2 :MeOH-NH 3 (7N), 20:1 or 15:1) afforded HJP-149 [8-((3-chloro-5- iodophenyl)thio)-9-(3- (isopropylamino)propyl)-9H-purin-6-amine] in 83% yield. Synthesis of SO- 33 includes cyclocondensation of 2,4,5,6-tetraaminopyrimidine sulphate with CS2 in ethanol to obtain 2,6- diamino-9H-purine-8-thiol in quantitative yields. Next, copper-catalyzed coupling of 2,6-diamino- 9H-purine-8-thiol with 3,5-dichloroiodobenzene resulted in 65% yield of 8-((3,5- dichlorophenyl)thio)-9H-purine-2,6-diamine. Transformation of C2-amino group of 8-((3,5- dichlorophenyl)thio)-9H-purine-2,6-diamine to Cl was achieved with SbCl3 and t-BuONO in 10:1 mixture of DCE and DMSO at 80 °C resulting in 2-chloro-8-((3,5-dichlorophenyl)thio)-9H-purin- 6- amine. The sequential reaction of 2-chloro-8-((3,5-dichlorophenyl)thio)-9H-purin-6-amine with 1,3-dibromopropane, followed by treating the bromo derivative from the previous step with excess isopropylamine resulted in crude SO-33. The crude residue was purified by preparatory TLC (CH 2 Cl 2 -MeOH-NH 3 (7N), 20:1) to give 60% yield of SO-33 [2-chloro-8-((3,5- dichlorophenyl)thio)-9-(3-(isopropylamino)propyl)-9H-purin-6-amine]. Synthesis of PU29F commenced with the coupling of 2,4,5,6-tetraaminopyrimidine with the acid fluoride of the 3,4,5- trimethoxyphenylacetic acid resulting in N-(2,4,6-triaminopyrimidin-5-yl)-2-(3,4,5- trimethoxyphenyl)acetamide. The acid fluoride was generated by treating phenylacetic acid derivative with cyanuric fluoride and pyridine in CH2Cl2. The acetamide derivative was cyclized to 8-(3,4,5-trimethoxybenzyl)-9H-purine-2,6-diamine by heating it in alcoholic NaOMe. Further transformation of the C2-amino group to fluorine was conducted by a modified Schiemann diazotization-fluorodediazoniation reaction in HF/pyridine and NaNO2 to yield 2-fluoro-8-(3,4,5- trimethoxybenzyl)-9H-purin-6-amine. The fluoro derivative was then N9-alkylated using the Mitsunobu reaction with 2-isopropoxyethan-1-ol in the presence of PPh3 and di-tert-butylazodi- carboxylate to give PU29F [2-Fluoro-9-(2-isopropoxy-ethyl)-8-(3,4,5-trimethoxy-ben-zyl)-9H- purin-6-ylamin] in 86% yield. For the synthesis of PU-H71, the cyclocondensation of 4,5,6- triaminopyrimidine sulfate with CS2 gave 8-mercaptoadenine in quantitative yield which was used directly without additional purification. CuI-catalyzed coupling of 8-mercaptoadenine with 5- iodobenzo[d][1,3]dioxole at 110 °C resulted in 8-(benzo[1,3]dioxol-5-ylsulfanyl)adenine in 58% yield. Next, iodination of 8-(benzo[1,3]dioxol-5-ylsulfanyl)adenine with NIS/TFA afforded 8-(6- iodo-benzo[1,3]dioxol-5-ylsulfanyl)adenine which was heated with 3-(tertbutoxycarbonyl- isopropyl-amino)-propyl tosylate and Cs 2 CO 3 in DMF at 80 °C under nitrogen atmosphere. The resulting tert-butyl(3-(6-amino-8-((6-iodobenzo[d][1,3]dioxol-5-yl)thio)-9H-purin-9- yl)propyl)(isopropyl)carbamate underwent Boc-deprotection with TFA at room temperature to give crude product which was further purified by preparatory thin-layer chromatography on silica gel (CHCl3/MeOH/NH4OH at 10:1:0.5) to afford PU-H71 in 98% yield. All final compounds were characterized using 1 H-NMR, 13 C-NMR and HRMS and their purity determined by HPLC to be higher than 98%. Taxol (S1150), Erlotinib (S1023) and Lapatinib (S2111) were purchased from Selleckchem. Cetuximab was received as leftover from the MSKCC Clinical Pharmacy. Lambda protein Phosphatase (Lambda PPase, P0753S), Endo H (P0703S) and PNGase F (P0709S) were purchased from NEB Inc. High capacity Streptavidin Agarose (20361) was purchased from ThermoFisher Scientific. Cell Fractionation and Immunoblotting [0164] Cells were either treated with DMSO (vehicle) or indicated compounds and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 0.5% NP40) supplemented with cocktail protease inhibitors (Roche) to produce whole-cell lysates. Lysates for cytosol and total membrane fractions were harvested and processed using ProteoExtract Subcellular Proteome Extraction Kit (Millipore Sigma) following the manufacturer's instructions. Plasma membrane proteins were prepared using the Minute Kit (Invent Biotechnologies Inc.) according to manufacturer’s instructions. Protein concentrations were determined using the BCA kit (Pierce). Ten to fifty micrograms of total protein were examined by immunoblotting with indicated antibodies. The following primary antibodies were used: HSP90 (SMC-107) from Stressmarq; HER2 (28-0004), myc (R950-25) from Invitrogen; Calnexin (610523) from BD Biosciences; HSP70 (SPA-810), GRP94 (SPA-850) from Enzo; GAPDH (ab8245), GRP78 (ab21685), HSP90α (ab2928) from Abcam; cleaved PARP (G7341) from Promega; EGFR (4267), LRP6 (2560), p-AKT (S473) (9271), AKT (4691), Caspase 7 (9494), p-ERK1/2 (T202/Y204) (4377), ERK1/2 (4695), p-STAT3 (9145), STAT3 (12640), p-p65 (S536) (3033), p65 (8242), Flotillin-1 (3253) from Cell Signaling Technology; GRP94 (G4420), α- Tubulin (T5168), β-actin (A1978) and Concanavalin A-HRP (L6397) from Sigma-Aldrich. The blots were washed with TBS/0.1% Tween 20 and incubated with appropriate HRP-conjugated secondary antibodies. Chemiluminescent signal was detected with Enhanced Chemiluminescence Detection System (GE Healthcare) according to the manufacturer’s instructions. Native Gel Electrophoresis [0165] Cells were lysed in the RIPA buffers (WCL), or fractionated (C, TM, F1, F2) and diluted with Felts buffer (20 mM Hepes pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl 2 , 20 mM Na 2 MoO 4 and 0.01 % NP40 with 0.1 mg/mL BGG). Twenty-five to one hundred μg of protein was loaded onto 5% native gel and resolved at 4°C. After running, gels were soaked in Tris-Glycine- SDS running buffer for 15 min prior to gel transfer for 75 min at 100 v, then immunoblotted as described above. Native Gel Electrophoresis under Denaturing Condition [0166] Cell lysates were mixed with 10 M urea (dissolved in Felts buffer) to reach the final concentrations of 2M, 4M, 6M and 8M. The mixtures were incubated for 15 min at room temperature and the volumes were adjusted by using Felts buffer. Immediately after the reaction, the samples were mixed with native loading buffer, and loaded onto native gel and resolved at 4 o C. The GRP94 and HER2 signals were then detected by immunoblotting as described above. Chemical bait Precipitation and Coimmunoprecipitation [0167] Protein extracts were prepared in the indicated buffers and diluted in Felts buffer. Samples were incubated with D-biotin (Control), Inactive-WS13-biotin (Control), PU-WS13-biotin (GRP94 bait) or GRP94 antibodies for 3 hr at 4°C, followed by incubation with High Capacity Streptavidin agarose beads (ThermoFisher Scientific) or Protein A/G agarose beads (Roche) for another 2 hr at 4°C. The beads were washed with cold Felts buffer three times and subjected to immunoblotting. Sequential Capture [0168] PU-WS13-biotin beads were prepared by incubating 20 μM PU-WS13-biotin (chemical bait) with High Capacity Streptavidin agarose beads (ThermoFisher Scientific) for 3 hr at 4 °C followed by washing with Felts buffer for three times. Antibody beads were prepared by incubating 9G10 or G4420 anti-GRP94 antibodies with protein A/G agarose beads (Roche) for 2 hr at 4 °C followed by washing with Felts buffer for three times. The pre-formed chemical bait or antibody bait was then added into the cell lysate and the mixture was incubated on a rotator for 3 hr at 4 °C. After separating the beads by centrifugation, the supernatant was collected and incubated with new pre-formed chemical bait or antibody bait. The sequential capture experiment was carried out by repeating the chemical precipitation (CP)/immunoprecipitation (IP) three times before the final IP with the indicated antibody bait. Captured cargos at each step were washed with Felts buffer three times before loading onto SDS-PAGE and subjecting to immunoblotting. siRNA knock-down of GRP94 [0169] Transient transfections were carried out using Lipofectamine RNAiMax reagent (ThermoFisher) according to the manufacturer's instructions. siGRP94 (Gene HSP90B1) and scramble siRNA were purchased from Qiagen. Cells were transfected with 5 nM or 20 nM siRNA. The knockdown efficiency and other cellular markers were evaluated at 72 hr post transfection by immunoblotting. Immunofluorescence [0170] Cells were seeded and grown onto Lab-Tek II chamber slides for 24 hr before the experiment. After washing two times with cold PBS, cells were fixed by treating at RT for 20 min with 2% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 in PBS containing 10% FBS for 10 min and blocked with 2% BSA for 1 hr. After washing four times with PBS, primary antibodies were added into the chambers, and incubated overnight at 4 °C. The cells were washed again with PBS, followed by incubation with the secondary antibody for 1 hr at RT. The cells were finally washed again with PBS for four times and mounted with ProLong Diamond Anti-fade Mountant with DAPI (Molecular Probes). Slides were cured for 2 hr at RT and stored at 4 o C overnight before imaging under microscope (×63 oil lens, Leica Upright Confocal SP5). Protein co- association was quantified using Coloc2 plugin in Fiji software to determine the mean Pearson’s Correlation Coefficient R value (PCC R value). Background was subtracted by using a Rolling- Ball Background Substraction, threshold regression was set as costes and the costes randomizations were set at 10. The following primary antibodies were used for the immunofluorescence: HER2 (Zymed; 28004; 1:50), HER2-FITC (BD; 340553; 1:200), GRP94 (Sigma; G4420; 1:100), EEA1 (Abcam; ab70521; 1:100) and Calnexin (Abcam; 22595; 1:100). Enzymatic Deglycosylation and Dephosphorylation [0171] Cell lysates were treated with Endo H or PNGase F according to the manufacturer’s instructions. After reacted at 37°C for 1h, the samples were mixed with protein loading buffer and subjected to immunoblotting. For the deglycosylation reactions under native condition (used for further pulldown assays), the lysates were firstly diluted in Felts buffer and then incubated with the enzymes without protein denaturing on ice overnight. The deglycosylated samples were further used for detatured or native gel electrophoresis, chemical or immuno precipitation experiments. To compare GRP94 protein’s sensitivity to different enzymes (i.e. Figure 11A), same amount of cell lysate was diluted into 1× enzyme reaction buffer (1× Protein MetalloPhosphatase (PMP) buffer for lambda PPase, 1× glyco buffer 3 for Endo H or 1× glyco buffer 2 for PNGase F. The control sample was diluted into PMP buffer. Enzymes were added into the reaction tubes and incubated for 1hr at 30 o C for PPase, at 37 o C for Endo H and PNGase F. Immediately after the reaction was finished, the samples were mixed with the protein loading buffer, heated at 95 o C for 5 min, stored on ice before loading into the gels. Assessment of Cell-Surface Proteins [0172] Cell surface protein isolation kit (Pierce) was used to purify the cell surface proteins according to the manufacturer’s instructions. Briefly, cell surface proteins were biotinylated by incubating the live cells with Sulfo-NHS-SS-biotin for 30 min at 4 °C. The reaction was quenched and cells were lysed. The biotinylated surface proteins were affinity-purified by NeutrAvidin Agarose beads, and then eluted with protein loading buffer and subjected to immunoblotting. The flow-through was used as intracellular protein fraction. CRISPR/Cas9 mediated Knock-Out and targeted Mutagenesis of endogenous GRP94 [0173] Design and construction of CRISPR sgRNAs: GRP94 CRISPR sgRNAs were designed using the online tools CRISPOR (http://crispor.tefor.net/) and CHOPCHOP (https://chopchop.cbu.uib.no/). All sgRNA sequences were further validated using the online Nucleotide BLAST tool (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) to target unique genomic sites. sgRNAs against hGRP94 used in this study are: sg1948, GAAGAAGCTATTCAGTTGGA; sg3859, CAACGATACCCAGCACATCT. The single- stranded oligos were synthesized by Intergrated DNA Technologies, cloned into PX458 (pSpCas9(BB)-2A- GFP, Addgene plasmid #48138, (Ran et al., 2013)) via BsaI, and the positive clones were validated by plasmid sequencing (Genewiz). [0174] Cell transfection and GFP sorting: ssODN repair template were synthesized by Genewiz and the sequences used for mutagenesis are: N62Q, gaactatgccatgcaatatttgcttacctaactgatttccttagagaggaggaggctattcagttggatggattaCAGgcatcacaaata agagaacttagagagaagtcggaaaagtttgccttcca; N217A, ctattccgccttccttgtagcagataaggttattgtcacttc aaaacacaacGCTgatacccagcacatctgggagtctgactccaatgaattttctgtaattgctgacccaagaggaaacac. Breast cancer cells, MDA-MB-468, were transfected using Neon nucleofection system (ThermoFisher) according to the manufacturer’s instructions. Briefly, 1×10 6 cells were mixed with 2 μg PX458-sgRNA plasmid, 4 μl ssODN repair template (10 μM) in 100μL Electroporation tip. Parameters were set at 1100 pulse voltage, 30 ms pulse width, 2 pulses.48 hr post-transfection, GFP positive cells were sorted and collected via Flow. The positive cells were re-seeded into the dishes and left to recover for additional 24 hr. [0175] Isolation of clonal cell lines by dilution: Cells were dissociated from the plates and then passed through a 35 μm cell strainer to prevent cell clumping. After counting the cell number, the cells were diluted to a final concentration of 0.5 cells per 100 μL and plated into 96 well plates. The plates were closely inspected every other day one week after the plating. Single cell clones were expanded and transferred into 12-well or 6-well plates for further validation. [0176] T7 endonuclease I assay: Genomic regions flanking the CRISPR sgRNA target sites were PCR amplified with Fusion Flash High-Fidelity PCR Master Mix (F548S, ThermoFIsher Scientifics) using gene-specific primers. PCR products were purified with MinElute PCR Purification Kit (Qiagen) and hybridized in PCR buffer (95°C, 5 min; 95-85°C at −2°C/s; 85- 25°C at −0.1°C/s; hold at 4°C). After treatment with T7 endonuclease I (2.5 U, NEB) at 37°C for 1 hr, the fragments were subjected to electrophoresis in a 2.5% agarose gel and visualized by staining with SyBR Safe DNA Gel stain (Invitrogen). [0177] Clone cell validations: When the single clone expanded to grow to a significant cell number, the genomic DNAs were extracted via the Phenol:Chloroform method and subjected for PCR and sequencing. The sequencing primers used in this study are: N62-F, ccattttaacccccaagaca; N62-R, atcaggccgtgaacctattt; N217-F, cactttcagaaaaggccataaaa; N217-R, caggaaaattaaggcccaga. Whole cell lysates were also validated by immunoblotting with GRP94 antibodies. Soft Agar Colony Formation Assay [0178] 6-well plates were coated with a bottom layer of 2 mL 1% low-melting-point agarose (Invitrogen) dissolved in the complete culture medium. Cell suspension in culture medium containing 0.4% low-melting-point agarose was then added on the top of the layer. Colony growth was quantified and images were taken after staining with 0.05% crystal violet. Cell Viability Assessment [0179] Annexin V staining. Cells were labelled with Annexin V-FITC and 7AAD after PU-WS13 treatment for 48 hr, as previously reported (Rodina et al., 2016). The samples were analyzed by flow cytometry (LSR-II, BD Biosciences). FlowJo software was used for data analysis (FlowJo LLC)). The necrotic cells were defined as Annexin V+/7AAD+, and the early apoptotic cells were defined as Annexin V + /7AAD-. [0180] ATP based cell viability assessment. Cell viability was assessed using CellTiter-Glo luminescent Cell Viability Assay (Promega) after a 72 hr PU-WS13 treatment. The method determines the number of viable cells in culture based on quantification of ATP amount, which signals the presence of metabolically active cells, and was performed as previously reported (Rodina et al., 2007). Example 1: Ex vivo Studies [0181] The fresh tissue slicing method maintains tissue integrity and architecture within an intact tumor- microenvironment-macroenvironment context throughout treatment, providing a more clinically- relevant means to assess the inhibitors’ effects. This is important because interactions among tumor and stromal cells are known to play a major role in cancer growth and progression and in the anti-tumor efficacy of agents. De-identified pathology discarded specimens were obtained in accordance with the guidelines and approval of the Institutional Review Board# 09-121 (PI: Dr. Modi). The primary breast cancer specimens or fresh esophagogastric PDX samples were processed as reported before (Corben et al., 2014). Briefly, the sample was delivered in a fresh state, harvested in a sterile environment under 30 minutes from the surgical procedure. Tumor tissue was chosen from the periphery of the index lesion to avoid potential frank central necrosis (cell death). The necrotic tissue may be grossly recognizable by any of the following criteria: loss of color or paleness of the tissue; loss of strength in which necrotic tissue is soft and friable; a distinct demarcation between the necrotic and viable tissue. Immediate after sampling is received, the sample was placed in wet ice and transported to the laboratory for ex vivo fresh tissue sectioning. Samples were then embedded into 5% Agarose gel and cut into 200 μm thick sections on a Leica VT 1000S vibratome. The live sections were transferred into 24-well tissue culture plates and treated for 24 hr or 48 hr with the indicated concentration of PU-WS13. Sections were then fixed in 4% formalin for 1hr at room temperature, and transferred into 70% ethanol. Following paraffin embedding, sectioning and mounting, the sections were stained with hematoxylin and eosin (H&E) and evaluated by the pathologists. Apoptosis and necrosis of the tumor cells was assessed by reviewing all the H&E slides of the case (controls and treated ones) blindly. The effect to any precursor lesions (if present) and any off-target effects to benign surrounding tissue was also evaluated. Example 2: In vivo Studies in Mice [0182] For the breast cancer model: Athymic nude mice (Hsd:Athymic Nude-Foxn1 nu , female, 20-25 g, 6 weeks old; RRID:MGI:5652489) were obtained from Envigo and were allowed to acclimatize at the MSKCC vivarium for 1 week prior to implanting tumors. Tumor xenografts were established subcutaneously into the dorsal flank or orthotopically into the 4 th mammary fat pad. Tumors were initiated by subcutaneous injection of 5 × 10 6 cells for MDA-MB-468 or orthotopic injection of 5 × 10 6 cells for AU565 in a 200 μL cell suspension of a 1:1 v/v mixture of PBS with reconstituted basement membrane (BD matrigel, Collaborative Biomedical Products Inc.). Tumor Volume was determined by measurement with Vernier calipers, calculated using the formula - length × width 2 × 0.5 and analyzed on indicated days as the median tumor volume ± SD Mice were randomized prior to treatments, and euthanized after similar PU-WS13 treatment periods and at a time before tumor reached a size that resulted in discomfort or difficulty in physiological functions in the individual treatment group, in accordance with the IUCAC protocol. For the esophagogastric cancer model: Esophagogastric PDX model was generated as previously described (Mattar et al., 2018). Briefly, patient specimens (~0.5 g) collected under the approved IRB protocol (10-018, PI: Dr. Janjigian) were minced, mixed with Matrigel (1:1 v/v) and implanted subcutaneously in 8 weeks old female NSG mice (NOD.Cg-Prkdc scid Il2rg tm1W
Figure imgf000112_0001
l /SzJ, the Jackson Laboratory, IMSR Cat# JAX:005557, RRID:IMSR_JAX:005557). When tumors reached 1-1.5 cm 3 , they were excised and transplanted in recipient mice, and models were considered successfully established following 3 rounds of transplantation. Example 3: Pharmacokinetic (PK) and Pharmacodynamic (PD) Studies [0183] PK study. Frozen tumors or tissues were dried and weighed prior to homogenization in acetonitrile/H2O (3:7). PU-WS13 was extracted in methylene chloride, and the organic layer was separated and dried under vacuum. Samples were reconstituted in mobile phase. Concentrations of PU-WS13 in tissue or plasma were determined by high-performance LC-MS/MS. PU-H71 was added as the internal standard. Compound analysis was performed on the 6410 LC-MS/MS system (Agilent Technologies) in multiple reaction monitoring mode using positive-ion electrospray ionization. For tissue samples, a Zorbax Eclipse XDB-C18 column (2.1 × 50 mm, 3.5 μm) was used for the LC separation, and the analyte was eluted under an isocratic condition (80% H 2 O + 0.1% HCOOH: 20% CH 3 CN) for 3 min at a flow rate of 0.4 mL/min. For plasma samples, a Zorbax Eclipse XDB-C18 column (4.6 x 50 mm, 5 μm) was used for the LC separation, and the analyte was eluted under a gradient condition (H 2 O+0.1% HCOOH:CH 3 CN, 95:5 to 70:30) at a flow rate of 0.35 mL/min. [0184] PD study. Tumors or tissues were homogenized in tissue lysis buffer (50 mM Tris- HCl pH 7.5, 50 mM KCl, 150 mM NaCl, 2 mM EDTA, 0.1% Sodium deoxycholate, 0.5% NP40, 0.5% Triton X-100, 0.5% SDS) using Bullet Blender Tissue Homogenizer (Next Advance Inc.). Protein concentrations were determined using the BCA kit (Pierce) according to the manufacturer’s instructions. Protein lysates (20-100 μg) were electrophoretically resolved by SDS/PAGE, transferred to nitrocellulose membrane, and probed with the indicated antibodies. Example 4: Efficacy Studies [0185] Efficacy studies: Mice bearing MDA-MB-468 or AU565 xenograft tumors reaching a volume of 100–150 mm 3 were treated i.p. with PU-WS13 (75mg/kg or 125mg/kg, dissolved in 60mM citrate buffer (pH 4.0) with 30% Captisol) or vehicle, on a 3-times or 5 times per week schedule, as indicated. Tumor volume (in mm 3 ) was determined by measurement with Vernier calipers, and was calculated as the product of its length × width 2 × 0.5. Mice were sacrificed after similar PU-WS13 treatment periods, and at a time before tumors reached a size that resulted in a discomfort or difficulty in physiological functions of mice in the individual treatment group, in accordance with our IUCAC protocol. All animals were observed daily for mortality from the time of animal receipt through the end of the study. Body weights for all animals were recorded no more than three times, but no fewer than once per week during the administration of the test article. All mice were observed for clinical symptoms at the time the animals were received and on all days in which the test article was administered. Example 5: Toxicology Studies [0186] Toxicology studies: The study assessed the safety and relevant toxicities of PU- WS13 administered by i.p. injection (125 mg/kg, five injections a week) over a chronic administration period (87 days). On the final day of the efficacy study of Example 4, mice were anesthetized with isoflurane and approximately 100 μL of whole blood was collected from the orbital plexus of each mouse into a labeled tube containing EDTA anticoagulant. Within 2 hr of blood collection, complete necropsies hematology and clinical chemistry were analyzed. A necropsy was performed on each animal. Gross examinations of each animal including internal organs were performed by a pathologist and any macroscopic lesions or other abnormal findings were recorded using standard terminology. For histopathology, tissues were collected and preserved in formalin. After at least 24 hr in fixative, the tissues were embedded in paraffin, sectioned and stained with hematoxylin and eosin or subjected to Immuno- staining. All tissues slides were examined by a pathologist. Lesions were recorded using morphologic diagnoses following standardized nomenclature. Example 6: Identification of N-Glycosylation sites using nano LC-Mass Spectrometry (LC- MS/MS) [0187] Samples were treated with EndoH as described. Initially, non-treated samples, as well as samples treated with EndoH were utilized to develop the mass spectrometric workflow used. Protein samples were separated by SDS-PAGE and processed using standard published protocols (Rodina et al., 2016) with the following modifications: Gel regions containing endoplasmin were generously excised and subjected to in-gel tryptic digestion with 200-300ng Trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) overnight, and after acidification with 10% formic acid (final concentration of 0.5-1% formic acid) resulting peptides were desalted using hand packed reversed phase Empore C18 Extraction Disks (3M, St. Paul, MN, USA) using a method described before (Rappsilber et al., 2007). Desalted peptides were concentrated to a very small droplet by vacuum centrifugation and reconstituted in 10 μL 0.1% formic acid in water. Approximately 90% of the peptide material was used for liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). A Q Exactive HF mass spectrometer was coupled directly to an EASY-nLC 1000 (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a self-packed 75 μm x 20-cm reverse phase column (360 mm OD, 75 mm ID, 10 mm ID tip Picotip emitter, New Objective, Woburn MA column packed with ReproSil-Pur C18, 3 μM beads, Dr. Maisch GmbH, Germany) for peptide separation. Peptides were eluted with a 3-40% acetonitrile gradient over 110 min at a flow rate of 250 nL/min. The mass spectrometer was operated in DDA mode with survey scans acquired at a resolution of 120,000 (at m/z 200) over a scan range of 300- 1750 m/z. Up to 15 most abundant precursors from the survey scan were selected with an isolation window of 1.6 Th for fragmentation by higher-energy collisional dissociation with normalized collision energy (NCE) of 27. The maximum injection time for the survey and MS/MS scans was 60 ms and the ion target value (AGC) for both scan modes was set to 3e6. [0188] Mass Spectrometry Data Processing: All mass spectra were first converted to mgf peak list format using Proteome Discoverer 1.4 (Thermo Fisher Scientific, Waltham, MA, USA) and the resulting mgf files searched against a human UniProt protein database using Mascot (Matrix Science, London,UK; version 2.5.0; www.matrixscience.com). Decoy protein sequences with reversed sequence were added to the database to allow for the calculation of false discovery rates (FDR). The search parameters were as follows: (i) up to two missed tryptic cleavages were allowed; (ii) precursor ion mass tolerance = 10 ppm; (iii) fragment ion mass tolerance = 0.3 Da; and (iv) variable protein modifications were allowed for N-linked glycosylation [N-Acetylhexosamine (HexNAc)], methionine oxidation, deamidation of asparagine and glutamines, and protein N- terminal acetylation. MudPit scoring was typically applied using significance threshold score p < 0.01. Decoy database search was always activated and, in general, for merged LS-MS/MS analysis of a gel lane with p < 0.01, false discovery rate averaged around 1%. Generated .RAW files further were analyzed with Thermo Scientific Xcalibur (version 4.1.31.9) software 4 (Thermo Fisher Scientific, Waltham, MA, USA). Layouts containing target precursors (modified by HexNAc) were created with mass accuracy set to 5ppm. The intensity of each specific precursor was extracted and noted. The present disclosure also noted the presence of the oxonium ion at 204.087 and GlcNAc fragment ions at m/z 126.055, 138.055, 168.065, and 186.075 in each MS/MS spectrum of modified peptides (Hagglund et al., 2004). [0189] Quantification and Statistical Analysis: Data were collected and statistical analyses performed using GraphPad Prism (version 6; GraphPad Software) or R statistical package. In each group of data, estimate variation was taken into account and is indicated in each figure as SD or SEM. If a single panel is presented, data are representative of 2 or 3 biological or technical replicates, as indicated. P values for unpaired comparisons between two groups with comparable variance were calculated by two-tailed Student’s t-test. Pearson’s tests were used to identify correlations among variables. Significance for all statistical tests was shown in figures for not significant (NS), p < 0.05 (*), p < 0.01 (**), p <0.001 (***) and p < 0.0001 (****). No samples or animals were excluded from analysis, and sample size estimates were not used. Animals were randomly assigned to groups. Studies were not conducted blinded, with the exception of all patient specimen histological analyses. Example 7: Structure-Activity Relationship Study of Various Inhibitors of N-glycosylated Grp94 [0190] Fluorescence Polarization (FP) measurements: FP competition assays were performed on an Analyst GT instrument (Molecular Devices, Sunnyvale, CA) and carried out in black 96-well microplates (Corning, no.3650) in a total volume of 100 µL in each well. A stock of 10 µM cy3B-GM and PU-FITC3 was prepared in DMSO and diluted with Felts buffer (20 mM Hepes (K), pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4 and 0.01% NP40 with 0.1 mg/mL BGG). To each well was added the fluorescent dye–labeled FP ligand, protein and tested inhibitor (initial stock in DMSO) in a final volume of 100 µL Felts buffer. Compounds were added in duplicate or triplicate wells. For each assay, background wells (buffer only), tracer controls (free, FP probe only) and bound controls (FP probe in the presence of protein) were included on each assay plate. The assay plate was incubated on a shaker at 4 °C for 24 h, and the FP values (in mP) were measured. The fraction of FP probe bound to protein was correlated to the mP value and plotted against values of competitor concentrations. The inhibitor concentration at which 50% of bound FP probe was displaced was obtained by fitting the data. For cy3B-GM, an excitation filter at 530 nm and an emission filter at 580 nm were used with a dichroic mirror of 561 nm. For PU-FITC3, an excitation filter at 485 nm and an emission filter at 530 nm were used with a dichroic mirror of 505 nm. All of the experimental data were analyzed using SOFTmax Pro and plotted using Prism 9.0 (GraphPad Software Inc., San Diego, CA), and binding affinity values are given as relative binding affinity values (IC50FP, concentration at which 50% of FP probe was competed off by compound). [0191] Cell Viability Assessment by AlamarBlue assay: AlamarBlue® assay (Invitrogen, Carlsbad, CA, USA) was performed to evaluate anti‐proliferative activity of the drugs in MDA- MB-468 cells. Cells were plated in 96‐well plates (5 × 105 cells/well in 200 μL of medium). After 12 h, compound was added to each well at a particular concentration and incubated for 72 h. At the end of the incubation period, 20 μL of stock solution (0.312 mg/mL) of the AlamarBlue was added to each well. Absorbance was measured using the Analyst GT instrument (Molecular Devices, Sunnyvale, CA). The drug effect was quantified as the percentage of control absorbance at 540 nm and 585 nm. Optical density was determined for 3 replicates per treatment condition and cell proliferation in drug‐treated cells was normalized to their respective controls (IC50cyt, concentration at which 50% of inhibition was observed compared to control well). [0192] For IC50 values described herein: FP refers to the fluorescent polarization binding assay with purified Grp94 protein – equilibrium binding. cyt refers to alamar blue cytotoxicity assay in MDA-MB-468 breast cancer cells – N-glyc GRP94 dependent cancer cell. See Yan et al., Cell Reports 2020, 31, 107840, June 30, 2020, incorporated herein by reference. [0193] Results of FP and cyt Assays:
Figure imgf000117_0001
Figure imgf000118_0001
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Example 8: Competitive Binding to Recombinant Protein and Cytotoxicity in a N- Glycosylation+ Cancer
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
Figure imgf000127_0001
Example 9: Compound Profiles in Native Page and co-IP Studies [0194] SDS PAGE and Immunoblotting. Cells were either treated with DMSO (vehicle) or indicated compounds and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 0.5% NP40) supplemented with cocktail protease inhibitors (Roche) to produce whole-cell lysates. Protein concentrations were determined using the BCA kit (Pierce). The protein lysates (10–50 µg) were electrophoretically resolved by SDS-PAGE, transferred onto nitrocellulose membranes and probed with the indicated antibodies. The following primary antibodies were used: EGFR (4267) from Cell Signaling Technology; cleaved PARP (G7341) from Promega; HSP70 (SPA-810), GRP94 (SPA-850) from Enzo; GAPDH (ab8245) from Abcam; HER2 (28-0004) from Invitrogen. The membranes were washed with TBS/0.1% Tween-20 and incubated with appropriate HRP-conjugated secondary antibodies. Chemiluminescent signal was detected with Enhanced Chemiluminescence Detection System (GE Healthcare) according to the manufacturer’s instructions. [0195] Native Gel Electrophoresis (Native PAGE) and Coomassie stain. Cells were lysed in the RIPA buffers. Twenty-five to one hundred µg of protein was loaded onto 5% native gel and resolved at 4°C in Native Running Buffer. After the electrophoresis, gels were soaked in Tris-Glycine-SDS running buffer for 15 min prior to gel transfer for 75 min at 100 v, then immunoblotted as described above. For Coomassie stain, the gel was first washed in water for 5 min, repeat 3 times, stained with Bio-Safe Coomassie (G-250, BIO-RAD) for 1 hr with shaking and rinsed in water for 30 min. [0196] Co-immunoprecipitation (Co-IP). Cells were either treated with DMSO (vehicle) or compounds for indicated time and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% sodium deoxycholate and 0.5% NP40) supplemented with cocktail protease inhibitors (Roche) to produce cell lysates. The lysates were diluted in Felts buffer (20 mM Hepes pH 7.3, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 20 mM Na2MoO4 and 0.01 % NP40 with 0.1 mg/mL BGG). Samples were incubated with GRP94 antibodies (9G10 from Enzo; or G4420 from Sigma) for 3 hr at 4°C, followed by incubation with Protein A/G agarose beads (Roche) for another 2 hr at 4°C. The beads were washed with cold Felts buffer three times and subjected to immunoblotting. [0197] Comparison of WS12 (inactive), SO33A (active), and HJP110 (active). Native Gel Assay: MDA-MB-468, 5µM, 4h treatment, See Figure 16. [0198] Comparison of WS13 (active), SO33A (active), SO26A (active), and SO116A (inactive). Native Gel Assay: MDA-MB-468, 5 µM /10µM, 4h treatment. 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[0200] While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

Claims

CLAIMS What is claimed is: 1. A method of treating cancer, inflammatory diseases, neurodegenerative diseases, rheumatoid arthritis, or diabetes in a patient suffering therefrom, comprising the step of administering to said patient an effective amount of a compound that inhibits N-glycosylated Grp94.
2. A method of treating cancer in a patient suffering therefrom, comprising the step of administering to said patient an effective amount of a compound that inhibits N-glycosylated Grp94, wherein the patient has been identified as having a cancer characterized by N-glycosylated Grp94.
3. A method of treating cancer in a patient suffering therefrom, comprising the steps of: (i) determining that the patient’s cancer is characterized by N-glycosylated Grp94; and (ii) administering to said patient an effective amount of a compound that inhibits N-glycosylated Grp94.
4. The method of claim 1, wherein the method is for treating cancer.
5. A method of treating cancer characterized by the presence of N-glycosylated Grp94 comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N-glycosylated Grp94.
6. A method of treating a disease associated with N-glycosylated Grp94, comprising the step of administering to a subject suffering therefrom an effective amount of a compound that inhibits N- glycosylated Grp94. Page 137 of 164
7. The method of any one of the preceding claims, wherein the N-glycosylated Grp94 comprises glycosylated Asn at the N62, N217, and/or N502 residue.
8. The method of any one of the preceding claims, wherein the N-glycosylated Grp94 comprises glycosylated Asn at the N62 residue.
9. The method of any one of the preceding claims, wherein the N-glycosylated Grp94 alters the function of one or more aberrant oncogenic proteins.
10. The method of any one of the preceding claims, wherein the N-glycosylated Grp94 is characterized in that it forms high molecular weight complexes (e.g., above 242 kDa) with one or more aberrant oncogenic proteins.
11. The method of any one of the preceding claims, wherein the cancer is characterized by overexpression of a receptor tyrosine kinase (RTK).
12. The method of claim 11, wherein the aberrant oncogenic protein is a receptor tyrosine kinase (RTK).
13. The method of any one of the preceding claims, wherein the compound has a structure of Formula (I):
Figure imgf000139_0001
or a pharmaceutically acceptable salt thereof, wherein: Z2 is –N- or –CR10-, wherein R10 is H or unsubstituted or substituted -(C1-C6)aliphatic; X1 is –H, -halogen, -N(R)2, -OR, -CN, or unsubstituted or substituted -(C1-C6)aliphatic; X2 is –H, halogen, or unsubstituted or substituted -(C1-C6)aliphatic; X3 and X5 are independently -halogen, unsubstituted or substituted -(C1-C12)aliphatic, unsubstituted or substituted phenyl, unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, unsubstituted or substituted 3- to 10-membered heterocyclic group having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or unsubstituted or substituted 3- to 10-membered cycloalkyl group; or X2 and X3 are taken together with their intervening atoms to form a 5- to 8- membered partially unsaturated or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur; R1 is –(CR1aR1b)3-NR2R3; each occurrence of R1a is independently hydrogen or methyl; each occurrence of R1b is independently hydrogen or methyl; R2 and R3 are independently hydrogen or unsubstituted or substituted -(C1-C8)aliphatic; each R is independently hydrogen, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2; wherein each substituted group is substituted with one or more groups selected from halogen, - CN, -OR, -SR, -N(R)2, -NO2, -C(O)R’, -C(O)OR, -C(O)N(R)2, -OC(O)R’, -OC(O)N(R)2, - OC(O)OR, -OSO2R, -OSO2N(R)2, -N(R)C(O)R’, -N(R)SO2R’, -SO2R’, -SO2N(R)2, - SO3R’, oxo, unsubstituted C1-6 aliphatic, or C1-6 aliphatic substituted with halogen, -CN, - OR, -SR, -N(R)2, -NO2, -C(O)R’, -C(O)OR, -C(O)N(R)2, -OC(O)R’, -OC(O)N(R)2, - OC(O)OR, -OSO2R, -OSO2N(R)2, -N(R)C(O)R’, -N(R)SO2R’, -SO2R’, -SO2N(R)2, - SO3R’, or oxo; and each R’ is independently unsubstituted C1-6 aliphatic or C1-6 aliphatic substituted with halogen, -OH, -CN, -NO2, or -NH2.
14. The method of claim 13, wherein R1 is –(CH2)3-NR2R3.
15. The method of claim 13 or 14, wherein one of R2 and R3 is hydrogen, and the other is unsubstituted or substituted -(C1-C8)aliphatic.
16. The method of claim 15, wherein one of R2 and R3 is hydrogen, and the other is selected from the group consisting of:
Figure imgf000141_0001
17. The method of any one of claims 13-16, wherein X1 is halogen.
18. The method of any one of claims 13-16, wherein X1 is hydrogen.
19. The method any one of claims 13-18, wherein X2 is H.
20. The method of any one of claims 13-19, wherein both X3 and X5 are independently –halogen.
21. The method of any one of claims 13-19, wherein one of X3 and X5 is –halogen, and the other is unsubstituted or substituted -(C1-C12)aliphatic.
22. The method of claim 21, wherein one of X3 and X5 is –halogen, and the other is selected from the group consisting of:
Figure imgf000142_0001
23. The method of any one of claims 13-19, wherein one of X3 and X5 is –halogen, and the other is unsubstituted or substituted phenyl.
24. The method of claim 23, wherein one of X3 and X5 is –halogen, and the other is selected from the group consisting of:
Figure imgf000142_0002
25. The method of any one of claims 13-19, wherein one of X3 and X5 is –halogen, and the other is unsubstituted or substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
26. The method of claim 25, wherein one of X3 and X5 is –halogen, and the other is selected from the group consisting of:
Figure imgf000142_0003
Figure imgf000143_0001
27. The method of any one of claims 13-26, wherein Z2 is –N-.
28. The method of any one of claims 13-26, wherein Z2 is –CH-.
29. The method of any one of claims 13-27, wherein the compound has the structure of Formula (Ia):
Figure imgf000143_0002
Ia or pharmaceutically acceptable salt thereof.
30. The method of any one of claims 13-27, wherein the compound has the structure of Formula (Ib):
Figure imgf000143_0003
Ib or pharmaceutically acceptable salt thereof.
31. The method of any one of claims 13-27, wherein the compound has the structure of Formula (Ic-i) or (Ic-ii):
Figure imgf000144_0001
Ic-ii or pharmaceutically acceptable salt thereof.
32. The method of any one of claims 13-27, wherein the compound has the structure of Formula (Id):
Figure imgf000145_0001
Id or pharmaceutically acceptable salt thereof.
33. The method of any one of claims 13-27, wherein the compound has the structure of Formula (Ie-i) or (Ie-ii):
Figure imgf000145_0002
Ie-i
Figure imgf000146_0001
Ie-ii or pharmaceutically acceptable salt thereof.
34. The method of any one of the preceding claims, wherein the compound is:
Figure imgf000146_0002
Figure imgf000147_0001
Figure imgf000148_0001
Figure imgf000149_0001
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Figure imgf000153_0001
Figure imgf000154_0001
or a pharmaceutically acceptable salt thereof.
35. The method of any one of claims 1-27 and 29-33, wherein the compound is:
Figure imgf000154_0002
Figure imgf000155_0001
Figure imgf000156_0001
I I I
Figure imgf000157_0001
Figure imgf000157_0002
Figure imgf000158_0001
Figure imgf000159_0001
Figure imgf000160_0001
or a pharmaceutically acceptable salt thereof.
36. The method of any one of the preceding claims, wherein the cancer is colorectal cancer, pancreatic cancer, thyroid cancer, basal cell carcinoma, melanoma, renal cell carcinoma, bladder cancer, prostate cancer, a lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, neuroblastoma, gastrointestinal cancers including gastrointestinal stromal tumors, esophageal cancer, stomach cancer, liver cancer, gallbladder cancer, anal cancer, brain tumors including gliomas, lymphomas including follicular lymphoma and diffuse large B-cell lymphoma, leukemias, myelomas, myeloproliferative neoplasms and gynecologic cancers including ovarian, cervical, or endometrial cancer.
37. The method of claim 36, wherein the cancer is breast cancer, ovarian cancer, gastric cancer, esophageal cancer, or non-small-cell lung cancer.
38. The method of claim 37, wherein the cancer is breast cancer.
39. The method of any one of the preceding claims, wherein the patient is receiving or has received a chemotherapeutic agent selected from the group consisting of ERBB2 inhibitors, EGFR inhibitors, CDK4 inhibitors, CRAF inhibitors, BRAF inhibitors, AKT inhibitors, MET inhibitors, BCR-ABL inhibitors, JAK inhibitors, HIF-1α inhibitors, and p53 inhibitors, so that the patient receives combination therapy with both.
40. The method of any one of the preceding claims, wherein the compound is characterized in that its IC50 cyt in the cell viability assay described in Example 7 is less than about 10 µM.
41. The method of any one of the preceding claims, wherein the compound is characterized in that its IC50 cyt in the cell viability assay described in Example 7 is less than about 0.1 µM.
42. The method of any one of the preceding claims, wherein the compound is characterized in that its IC50 FP against Grp94 in the fluorescence polarization assay described in Example 7 is greater than about 0.1, about 0.15, about 0.2, about 0.25, or about 0.3 µM.
43. The method of any one of the preceding claims, wherein the compound is characterized in that its IC50 FP against Hsp90α in the fluorescence polarization assay described in Example 7 is less than about 300, about 200, about 100, or about 50 µM.
44. The method of any one of the preceding claims, wherein the compound is other than:
Figure imgf000161_0001
45. The method of any one of the preceding claims, wherein the compound is other than
Figure imgf000162_0001
46. The method of any one of the preceding claims, wherein the compound is other than
Figure imgf000162_0002
47. A compound having the structure:
Figure imgf000162_0003
Figure imgf000163_0001
Figure imgf000164_0001
or a pharmaceutically acceptable salt thereof. 48. A pharmaceutical composition comprising the compound of claim 47.
PCT/US2021/039230 2020-06-26 2021-06-25 Inhibition of n-glycosylated grp94 WO2021263194A1 (en)

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