WO2022159687A1 - Essai phénotypique pour identifier des agents de dégradation de protéines - Google Patents

Essai phénotypique pour identifier des agents de dégradation de protéines Download PDF

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WO2022159687A1
WO2022159687A1 PCT/US2022/013294 US2022013294W WO2022159687A1 WO 2022159687 A1 WO2022159687 A1 WO 2022159687A1 US 2022013294 W US2022013294 W US 2022013294W WO 2022159687 A1 WO2022159687 A1 WO 2022159687A1
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cells
test compound
inhibitor
small molecule
neddylation
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Mikolaj SLABICKI
Benjamin Ebert
Eric S. FISCHER
Nathanael S. Gray
Radoslaw Nowak
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Dana-Farber Cancer Institute, Inc.
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/104—Aminoacyltransferases (2.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/36—Post-translational modifications [PTMs] in chemical analysis of biological material addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination

Definitions

  • molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation (Stanton et al., Science 359 eaao5902 (2016)).
  • proteolysis-targeting chimeric molecules PROTAC®s
  • molecular glue compounds are small molecules (also known as small molecule degraders) that induce an interaction between a substrate receptor of an E3 ubiquitin ligase and a target protein leading to proteolysis of the target.
  • IMiDs immune modulatory drugs
  • thalidomide a substrate receptor
  • CTL4 Cullin-RING ubiquitin ligase 4
  • these molecular glue degraders act substoichiometrically to catalyze the rapid depletion of previously inaccessible targets (Chopra et al., Drug Discov. Today. Technol. 37:5-13 (2019)).
  • the present invention is directed to a method for identifying a cullin dependent small molecule (e.g., monofunctional or multi-functional) degrader, comprising: incubating mammalian cells with a small molecule test compound in an amount sufficient to cause a change in phenotype in the cells, and a neddylation inhibitor at concentration nontoxic or of low toxicity to the cells; and measuring a difference in phenotype in the cells following incubation relative to a control that comprises the cells incubated with the test compound but not the neddylation inhibitor, wherein a difference in the phenotype in the cells relative to the control indicates that the test compound causes cullin dependent degradation of a target protein.
  • a cullin dependent small molecule e.g., monofunctional or multi-functional
  • the test compound is a natural product. In some embodiments, the test compound is a synthetic compound. In some embodiments, the test compound is a semi-synthetic compound.
  • the test compound is a small molecule kinase inhibitor. In some embodiments, the test compound is a small molecule tyrosine kinase inhibitor.
  • the test compound is a small molecule therapeutic agent.
  • the test compound is a small molecule anticancer therapeutic agent.
  • the small molecule inhibitor is CR8, CC-885, or indisulam.
  • the neddylation inhibitor is MLN4924.
  • the concentration of the test compound is between about 0.001 pM to about 100 pM.
  • the concentration of the neddylation inhibitor is less than 1 pM. [0013] In some embodiments, the concentration of the neddylation inhibitor is between about 10 nM to about 500 nM.
  • the concentration of the neddylation inhibitor is about 100 nM.
  • the cells are pretreated with the neddylation inhibitor prior to treatment with the test compound.
  • the mammalian cells are human cells. In some embodiments, the cells are primary cells. In some embodiments, the primary cells are isolated from a tissue or a biopsy sample.
  • the human cells are myeloid cells, lymphoid cells, neural cells, epithelial cells, endothelial cells, stem or progenitor cells, hepatocytes, myoblasts, osteoblasts, osteoclasts, lymphocytes, keratinocytes, melanocytes, mesothelial cells, germ cells, muscle cells, fibroblasts, transformed cells, non-transformed cells, or cancer cells.
  • the transformed cells are Cas9 stable cells.
  • the cells are SNGM, P31FU, OVK18, PFEIFFER, ES-2, OCIM1, K562, HEK293, or HEK293T cells.
  • the difference in phenotype in the cells is increased protein abundance, decreased protein abundance, increased protein activity, decreased protein activity, increased gene expression, decreased gene expression, changed cellular proliferation, changed protein localization, increased or decreased mRNA transcript abundance of predetermined genes, or cell viability.
  • the difference in phenotype in the cells is measured by flow cytometry, fluorescence-activated cell sorting (FACS), mass cytometry, and/or magnetic sorting, protein localization, cellular morphology, gene expression by RNA sequencing (RNAseq), Luminex® multiplex bead assay, quantitative polymerase chain reaction (qPCR), next generation sequencing (NGS), or immunoblotting.
  • RNA sequencing RNA sequencing
  • Luminex® multiplex bead assay is the L1000 assay.
  • the invention is directed to high throughput screening (HTS) methods for identifying cullin dependent small molecule degraders.
  • HTS high throughput screening
  • the method of the present invention identifies small molecule (e.g, monofunctional and multiple-functional) degraders by observing a change phenotype in cells, e.g, cell viability or cell death, that is rescuable by co-treatment with the neddylation inhibitor.
  • the rescue identifies compounds that cause protein degradation mediated by E3 ligase machinery which produces a toxic effect on the cell.
  • the presence of the neddylation inhibitor prevents the degradation, thus rescuing the cell.
  • This approach is generalizable to any phenotype, and presents a new way of identifying small molecule degrader compounds.
  • FIG. 1A-FIG. IB are a set of graphs showing inhibition of neddylation as a readout for functional degradation (Slabicki et al., Nature DOI: 10.1038/s41586-020-2374-x (2020)).
  • FIG. 1 A shows that neddylation inhibitor MLN4924 is not toxic to cells ⁇ 1 pM.
  • FIG. IB shows that CR8 cell killing is rescued by low dose neddylation inhibitor MLN4924.
  • FIG. 2A-FIG. 2H are a set of graphs showing that MLN4924 rescues CR8 (cyclin- dependent kinase (CDK) inhibitor) induced toxicity in SNGM (FIG. 2A), P31FUJ (FIG. 2B), OVK18 (FIG. 2C), PFEIFFER (FIG.2D), ES-2 (FIG. 2E), OCIM1 (FIG. 2F), K562 (FIG. 2G), and HEK293T (FIG. 2H) cell lines.
  • CDK cyclin- dependent kinase
  • FIG. 3 A-FIG. 3H are a set of graphs showing that MLN4924 rescues CC885 (cereblon (CRBN) modulator) induced toxicity in SNGM (FIG. 3A), P31FUJ (FIG. 3B), OVK18 (FIG. 3C), PFEIFFER (FIG. 3D), ES-2 (FIG. 3E), OCIM1 (FIG. 3F), K562 (FIG. 3G), and HEK293T (FIG. 3H) cell lines.
  • CC885 cereblon (CRBN) modulator
  • FIG. 4A-FIG. 4H are a set of graphs showing that MLN4924 rescues indisulam (CDK) induced toxicity in SNGM (FIG. 4A), P31FUJ (FIG. 4B), OVK18 (FIG. 4C), PFEIFFER (FIG. 4D), ES-2 (FIG. 4E), OCIM1 (FIG. 4F), K562 (FIG. 4G), and HEK293T (FIG. 4H) cell lines.
  • CDK indisulam
  • transitional term “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps.
  • the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim.
  • the transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention.
  • determining generally refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of can include determining the amount of something present, as well as determining whether it is present or absent.
  • small molecule refers to an organic molecule or compound that is monofunctional and that ranges in size from about 50 to about 10,000 daltons, usually from about 50 to about 5,000 daltons and more usually from about 100 to about 1000 daltons.
  • small molecule degrader also known as “molecular glue,” refers to a small molecule that induces or stabilizes interactions between proteins.
  • Known small molecule degraders e.g., thalidomide analogs, bind to the substrate receptors of E3 ubiquitin ligases and recruit target proteins for their ubiquitination and subsequent degradation by proteasomal degradation. Small molecule degraders also are referred to herein as cullin dependent degraders.
  • concentration of low toxicity refers to a concentration that kills less than about 50% of untreated control cells.
  • a low toxicity concentration of neddylation inhibitor MLN4924 is between about 10 nM to about 999 nM, or between about 10 nM to about 500 nM.
  • a low toxicity concentration of neddylation inhibitor MLN4924 is about 200 nM.
  • natural product refers to a small molecule organic compound produced by an organism, including a primary and secondary metabolite.
  • synthetic compound refers to a small molecule organic compound made by chemical synthesis, especially to imitate a natural product.
  • si-synthetic compound refers to a small molecule compound made by synthesis from a naturally occurring compound.
  • the present invention is directed to a method for identifying a cullin dependent small molecule (e.g., monofunctional or multi-functional) degrader, comprising: incubating mammalian cells with a small molecule test compound in an amount sufficient to cause a change in phenotype in the cells, and a neddylation inhibitor at a concentration that is nontoxic or of low toxicity to the cells; and measuring a difference in phenotype in the cells following incubation relative to a control that comprises the cells incubated with the test compound but not the neddylation inhibitor, wherein a difference in the phenotype in the cells relative to the control indicates that the test compound causes cullin dependent degradation of a target protein.
  • the control could be performed simultaneously or not, and with the same amount of the small molecule and for the same amount of incubation time.
  • the test compound is a natural product. In some embodiments, the test compound is a synthetic compound. In some embodiments, the test compound is a semi-synthetic compound.
  • the test compound is a small molecule kinase inhibitor. In some embodiments, the test compound is a small molecule tyrosine kinase inhibitor.
  • Tyrosine kinase inhibitors are a class of chemotherapy medications that inhibit, or block, one or more of the enzyme tyrosine kinases. Over 25 TKIs have been approved by the U.S. Food and Drug Administration for use in humans (Roskoski, Pharmacol Res. 144'.19-50 (2019)).
  • TKIs include Ruxolitinib, Tofacitinib, Lapatinib, Vandetanib, Sorafenib, Sunitinib, Axitinib, Nintedanib, Regorafenib, Pazopanib, Lenvatinib, Crizotinib, Ceritinib, Cabozantinib, DWF, Afatinib, Ibrutinib, B43, KU004, Foretinib, KRCA- 0008, PF-06439015, PF-06463922, Canertinib, GSA-10, GW2974, GW583340, WZ4002, CP- 380736, D2667, Mubritinib, PD153035, PD168393, Pelitinib, PF-06459988, PF-06672131, PF-6422899, PKI-166, Reveromycin A, Tyrphostin 1, Tyr
  • the test compound is a member of a library or collection of compounds or agents of interest (e.g, TKIs).
  • the library of compounds may be composed of a plurality of chemical compounds and agents that may be assembled from a single or multiple sources.
  • a library may include chemical compounds and agents such as chemically synthesized substances, products of biotechnological processes, naturally occurring substances or products resulting from combinatorial chemistry techniques or combinations thereof.
  • the library includes a plurality of clinical or preclinical small molecule drugs.
  • the test compound is a small molecule therapeutic agent.
  • the test compound is a small molecule anticancer therapeutic agent.
  • test compound is CR8, CC-885, or indisulam.
  • CR8 also known as (7?)-CR8 and (2R)-2-[[9-(Methylethyl)-6-[[[4-(2- pyridinyl)phenyl]methyl]amino]-9H-purin-2-yl]amino]-l-butanol, has the following structure:
  • CC-885 also known as N-(3-chloro-4-methylphenyl)-N'-[[2-(2,6-dioxo-3- piperidinyl)-2,3-dihydro-l-oxo-lH-isoindol-5-yl]methyl]-urea, has the following structure:
  • Indisulam also known as E7070 and N-(3-chloro-lH-indol-7-yl)benzene-l,4- disulfonamide, has the following structure: (Indisulam).
  • the amount of the test compound is sufficient to cause a measurable change in phenotype in the cells.
  • the concentration of the test compound is between about 0.001 pM to about 100 pM. In some embodiments, the concentration of the test compound is between about 0.001 pM to about 30 pM. In some embodiments, the concentration of the test compound is between about 0.03 pM to about 30 pM. In some embodiments, the concentration of the test compound is between about 0.1 pM to about 3 pM. In some embodiments, the concentration of the test compound is between about 0.001 pM to about 0.1 pM.
  • Neddylation is a post-translational protein modification that conjugates ubiquitin-like protein neuronal precursor cell-expressed developmentally down-regulated protein 8 (NEDD8). Similar to ubiquitination, neddylation is mediated by a cascade of three NEDD8 specific enzymes, an El activating enzyme, an E2 conjugating enzyme and one of the several E3 ligases. By altering its substrates’ conformation, stability, subcellular localization or binding affinity to DNA or proteins, neddylation regulates diverse cellular processes including the ubiquitin-proteasome system-mediated protein degradation, protein transcription, cell signaling, etc. (Kandala et al., Am J Cardiovasc Dis. 4(4): 140-158 (2014)).
  • the best- characterized substrates of neddylation are cullin family members that function as indispensable components of multiunit cullin-RING E3 ubiquitin ligases (CRLs), whose activity requires cullin neddylation (Zhou et al., Molecular cancer 75:77(2019)).
  • CTLs cullin-RING E3 ubiquitin ligases
  • the small molecule MLN4924 Panedistat
  • NAE adenosine sulfamate analog
  • Neddylation inhibitors effectively block cullin neddylation and inactivate CRLs. This in turn leads to accumulation of various CRL substrates, thus triggering multiple cellular responses, including cell cycle arrest, apoptosis, senescence and autophagy in a cell-type dependent manner (Zhou et al., Molecular cancer 18:77(2019)).
  • the neddylation inhibitor is MLN4924, also known as ((lS,2S,4R)-4-(4-((lS)-2,3-Dihydro-lH-inden-l-ylamino)-7H-pyrrolo(2,3-d)pyrimidin-7-yl)- 2-hydroxycyclopentyl)methyl sulphamate, has the following structure:
  • the neddylation inhibitor is a derivative or analog of MLN492.
  • Analogs of MLN492 that may be useful as neddylation inhibitors are described in U.S. Patent 7,951,810 B2 and U.S. Patent Application Publication 2018/0086785 Al.
  • TAS4464 Another exemplary NAE or neddylation inhibitor, which may be used in the present invention, is TAS4464, which is also known as 4-amino-7-[(2R,3R,4S,5R)-3,4-dihydroxy-5- [(sulfamoylamino)methyl]oxolan-2-yl]-5-[2-(2-ethoxy-6-fluorophenyl)ethynyl]pyrrolo[2,3- d] pyrimidine and has the following structure: [0056]
  • the neddylation inhibitor is a derivative or analog of TAS4464. Analogs of TAS4464 that may be useful as neddylation inhibitors are described in U.S. Patent 9,963,456 B2.
  • the concentration of the neddylation inhibitor is below IpM. In some embodiments, the concentration of the neddylation inhibitor is between about 10 nM to about 999 nM.
  • the concentration of the neddylation inhibitor is between about 10 nM to about 500 nM.
  • the concentration of the neddylation inhibitor is about 100 nM.
  • the cells may be pretreated with different concentrations of the neddylation inhibitor, typically for about 10 minutes to about 24 hours prior to treatment with the test compound in dose response assays.
  • the cells are incubated in the presence of the test compound and the neddylation inhibitor for an amount of time sufficient to allow for a measurable change in phenotype to occur.
  • the cells are incubated with neddylation inhibitor and test compound for at least 3 to 5 days.
  • the cells are incubated with neddylation inhibitor and test compound for at least 3 days.
  • incubation time may be shortened to 10 minutes - 24 hours to investigate protein levels or other more general readout, e.g, cellular differentiation and/or gene expression by RNA sequencing (RNAseq) or quantitative polymerase chain reaction (qPCR).
  • Mammalian cells suitable for use in the present methods are those that can be maintained or propagated in vitro and that naturally display, or that may be genetically modified to display, a phenotype (such as one described herein) the alteration of which is desired to be monitored for modulation.
  • cells may display a phenotype whose inhibition within the assay is to be determined.
  • Mammalian cells which display the appropriate phenotypic characteristics (phenotype) may be identified or obtained by any convenient technique or source, including those known to the person of ordinary skill in the art.
  • Such cells may be stable cell lines, such as those obtainable from American Type Culture Collection (ATCC) or other cell repositories.
  • ATCC American Type Culture Collection
  • the mammalian cells employed may be primary cells derived from a tissue or organ of an individual organism.
  • the mammalian cells may be transiently transfected with genetic constructs, such as those involved in the (desired) phenotype, for example a reporter gene.
  • the mammalian cells may be infected with a virus or bacterium which leads to an alteration in the phenotype.
  • human cells murine cells, hamster cells, rate cells, primate cells, and cells from domestic mammals (such as ovine, bovine, equine, canine or feline cells).
  • domestic mammals such as ovine, bovine, equine, canine or feline cells.
  • cells for employment in the present methods include mammalian cells derived from a cancer or tumor, or those associated with some other disease or abnormality of a mammal.
  • the mammalian cells are primary cells.
  • the primary cells are isolated from a tissue or a biopsy sample.
  • the mammalian cells are human cells.
  • the human cells are myeloid cells, lymphoid cells, neural cells, epithelial cells, endothelial cells, stem or progenitor cells, hepatocytes, myoblasts, osteoblasts, osteoclasts, lymphocytes, keratinocytes, melanocytes, mesothelial cells, germ cells, muscle cells, fibroblasts, transformed cells, non-transformed cells, or cancer cells.
  • the transformed cells are Cas9 stable cells.
  • the cells are SNGM, P31FU, OVK18, PFEIFFER, ES-2, 0CIM1, K562, HEK293, or HEK293T cells.
  • the phenotype to be monitored for change may be one having been specifically engineered for a given cell type.
  • a mammalian cell type may be genetically modified to result in a phenotype that is convenient or suitable for detection, such as a fluorescent protein/marker (such as GFP; eGFP), a luminescent protein/marker (such as luciferase) or a cell-surface marker than is detectable, e.g., with a labelled antibody, or intracellular proteins stained with labeled antibodies.
  • the phenotype of the mammalian cell is any one of the following, namely: one associated with a cell signaling pathway, preferably an activated cell signaling pathway; and/or one selected from the list consisting of: luminescence, fluorescence, viability, senescence, differentiation, migration, invasion, chemotaxis, apoptosis, immunological anergy, surface marker expression, progress through the cell cycle, transcriptional activity, protein expression, glycosylation, resistance to infection, permeability and reporter-gene activity.
  • the phenotype is not death and/or reduced (or decreased) growth, such as a phenotype that is not rescue of a cell from cytokine dependence, is not rescue from apoptosis (including neutrophil apoptosis/cell-death), is not induction of colony formation, or is not a rescue screen.
  • a screen for such a phenotype may be designed to detect a trait or characteristic of the cell that is not a rescue from cell death or an increase in growth of the mammalian cell.
  • the phenotypic difference in the cells is increased protein abundance, decreased protein abundance, increased protein activity, decreased protein activity, increased gene expression, decreased gene expression, changed cellular proliferation, changed protein localization, or cell viability.
  • the phenotype difference is an increased or decreased mRNA transcript abundance of predetermined genes.
  • immunoassays are binding assays involving binding between antibodies and antigen.
  • Representative examples of immunoassays include enzyme linked immunosorbent assays (ELISAs), enzyme linked immunospot assay (ELISPOT), radioimmunoassays (RIA), radioimmune precipitation assays (RIP A), immunobead capture assays, Western blotting, dot blotting, gel-shift assays, Flow cytometry, immunohistochemistry, fluorescence microscopy, protein arrays, multiplexed bead arrays, magnetic capture, in vivo imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery/localization after photobleaching (FRAPZFLAP).
  • ELISAs enzyme linked immunosorbent assays
  • ELISPOT enzyme linked immunospot assay
  • RIA radioimmunoassays
  • RIP A radioimmune precipitation assays
  • immunobead capture assays Western blotting, dot blotting, gel-
  • the phenotypic difference in the cells is measured by flow cytometry, fluorescence-activated cell sorting (FACS), mass cytometry, and/or magnetic sorting, protein localization, cellular morphology, gene expression by RNA sequencing (RNAseq), Luminex® multiplex bead assay, quantitative polymerase chain reaction (qPCR), next generation sequencing (NGS), or immunoblotting.
  • RNA sequencing RNA sequencing
  • Luminex® multiplex bead assay e.g., the L1000 assay.
  • L1000 is a high-throughput gene expression assay that measures the mRNA transcript abundance of 978 "landmark" genes from human cells. Landmark genes were chosen to be expressed across lineage which would enable the prediction of expression of other genes not directly measured.
  • the "L” in LI 000 refers to the Landmark genes measured in the assay. Measurements are made using the 500 colors of Luminex® beads such that two transcripts are identified by a single bead color. Expression of 80 control transcripts, chosen for their invariant expression across cell states, as well as the 978 Landmark genes are also measured. LI 000 is highly reproducible, comparable to RNA sequencing, and suitable for computational inference of the expression levels of 81% of non-measured transcripts. Subramanian et al., Cell 777(6 1437-1452.el7 (2017).
  • the disclosed methods entail high throughput screening (HTS) for identifying cullin dependent small molecule degraders.
  • HTS high throughput screening
  • the term “high-throughput” relates to an assay system for the rapid testing of a plurality of compounds in a short time. Thus, the assaying time per tested compound is minimized.
  • the assay is preferably carried out in multiwell plates, e.g., 6-well plates, 12-well plates, 24-well plates, 96-well plates or 384-well plates, or 1536 plates, even more preferably in 96-well plates, 384-well or 1536 plates.
  • the term “high-throughput” encompasses screening activity in which human intervention is minimized, and automation is maximized.
  • high-throughput screening involves automated pipetting, mixing, and heating.
  • a high-throughput method is one in which for example hundreds of compounds can be screened per 24-hour period by a single individual operating a single suitable apparatus.
  • Automation refers to a method or any one or more steps thereof that is fully or partly controlled and/or carried out by one or more technical devices, preferably pipetting robots.
  • Methods for conducting a variety of different HTS assays, in particular HTS screening assays are known in the art. The skilled person knows how to conduct HTS methods by the methods used in the context of the present invention.
  • Example 1 Inhibition of neddylation as a readout for functional degradation.
  • HEK293T cells were treated with various concentration of MG132 (proteasome inhibitor), MLN7243 (El ubiquitin-activating enzyme inhibitor) or MLN4924 (cullin neddylation inhibitor) for three days.
  • Cell viability was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega®, G7572) on CLARIOstar® Plus Multi-mode Microplate Reader, MARS 3.4 (BMG LabTech).
  • Cell viabilities were calculated relative to DMSO controls. Standard four-parameter log-logistic curves are fitted with the ‘dr4pl’ R package. The results are shown in FIG. 1A.
  • FIG. 1A-FIG. IB show that titration of MLN4924 up to 1 pM did not have a significant effect on cell viability (FIG. 1 A), but a 100 nM dose of MLN4924 was able to rescue CR8 induced toxicity (FIG. IB). These results confirmed that addition of low dose of MLN4924 can achieve a functional effect of reduced toxicity, when the toxicity is caused by neddylation dependent mechanism, such as degradation of Cyclin K and CDK13 by CR8.
  • Ubiquitin proteasome system (UPS) compounds including the neddylation inhibitor MLN4924, are known to be toxic at high to mid concentrations (>1 pM). It was believed that low doses would not inhibit neddylation. Applicant surprisingly found that low dose MLN4924 (e.g, -100 nM) was nontoxic to cells and it potently blocked neddylation generating a sufficient window to observe phenotypic difference in the cells.
  • Example 2 MLN4924 rescue of CR8 (cyclin-dependent kinase (CDK) inhibitor) induced toxicity in multiple cell lines.
  • CDK cyclin-dependent kinase
  • HEK293T-Cas9 cells were resuspended at 0.15 x 10 6 per ml and plated on a 384-well plate with 50 pL per well and MLN4924, MLN7243 or MG132 with or without CR8 serially diluted with D300e Digital Dispenser (Tecan).
  • a panel of SNGM, P31FUJ, OVK18, PFEIFFER, ES-2, 0CIM1 or HEK293T cells was resuspended at -0.15 x 10 6 per ml and plated on a 384-well plate with 50 pL per well. Cells were pretreated with MLN4924 at 0, 0.05, 0.1 or 0.2 pM concentration and treated with CR8 in dose response. [0085] After three days of drug exposure, cell viability was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega®, G7572) on CLARIOstar® Plus Multi-mode Microplate Reader, MARS 3.4 (BMG LabTech). Cell viabilities were calculated relative to DMSO controls. The half-maximum effective concentration (ECso) values were derived from standard four-parameter log-logistic curves fitted with the ‘dr4pl’ R package.
  • Example 3 MLN4924 rescue of CC885 (cereblon (CRBN) modulator) induced toxicity in multiple cell lines.
  • CC885 cereblon (CRBN) modulator
  • HEK293T-Cas9 cells were resuspended at 0.15 x 10 6 per ml and plated on a 384-well plate with 50 pL per well and MLN4924, MLN7243 or MG132 with or without CC-885 serially diluted with D300e Digital Dispenser (Tecan).
  • a panel of SNGM, P31FUJ, OVK18, PFEIFFER, ES-2, OCIM1 or HEK293T cells was resuspended at -0.15 x 10 6 per ml and plated on a 384-well plate with 50 pL per well. Cells were pretreated with MLN4924 at 0, 0.05, 0.1 or 0.2 pM concentration and treated with CC-885 in dose response.
  • FIG.3A-FIG. 3H show that MLN4924 rescues CC-885 induced toxicity in multiple cell lines.
  • Example 4 MLN4924 rescue of indisulam (CDK) induced toxicity in multiple cell lines.
  • HEK293T-Cas9 cells were resuspended at 0.15 x 10 6 per ml and plated on a 384-well plate with 50 pL per well and MLN4924, MLN7243 or MG132 with or without indisulam serially diluted with D300e Digital Dispenser (Tecan).
  • a panel of SNGM, P31FUJ, OVK18, PFEIFFER, ES-2, OCIM1 or HEK293T cells was resuspended at -0.15 x 10 6 per ml and plated on a 384-well plate with 50 pL per well. Cells were pretreated with MLN4924 at 0, 0.05, 0.1 or 0.2 pM concentration and treated with indisulam in dose response. [0095] After three days of drug exposure, cell viability was assessed using CellTiter-Glo® Luminescent Cell Viability Assay (Promega®, G7572) on CLARIOstar® Plus Multi-mode Microplate Reader, MARS 3.4 (BMG LabTech). Cell viabilities were calculated relative to DMSO controls. The half-maximum effective concentration (EC50) values were derived from standard four-parameter log-logistic curves fitted with the ‘dr4pl’ R package.
  • CR8 induces the degradation of Cyclin K via direct binding of CR8-CDK12-Cyclin K to damage specific DNA binding protein 1 (DDB1).
  • CC-885 is a CRBN based degrader of G1 to S phase transition 1 (GSPT1).
  • Indisulam is a DDB1 and CUL4 associated factor 15 (DCAF15) based degrader of splicing factor RNA binding motif protein 39 (RBM39). All of the three compounds caused dose dependent cell killing via proteasomal degradation.
  • FIG. 2A-FIG. 4H illustrate co-treatments of CC-885, CR8 and Indisulam with three doses of MLN4924 (50, 100, 200 nM).
  • Increased doses of neddylation inhibitor decreased CellTiter-Glo® levels for different cell lines.
  • Optimal concentration of MLN4924 may be characterized with increased toxicity.

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Abstract

L'invention concerne un procédé d'identification d'agents de dégradation de petites molécules dépendantes de la culline par criblage phénotypique.
PCT/US2022/013294 2021-01-22 2022-01-21 Essai phénotypique pour identifier des agents de dégradation de protéines WO2022159687A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180140578A1 (en) * 2016-11-23 2018-05-24 Board Of Regents Of The University Of Texas System Methods and compositions for the diagnosis and selective treatment of cancer
US20190374657A1 (en) * 2017-02-08 2019-12-12 Dana-Farber Cancer Institute, Inc. Tunable endogenous protein degradation with heterobifunctional compounds
US20200348285A1 (en) * 2017-11-09 2020-11-05 Dana-Farber Cancer Institute, Inc. Methods to prevent teratogenicity of imid like molecules and imid based degraders/protacs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180140578A1 (en) * 2016-11-23 2018-05-24 Board Of Regents Of The University Of Texas System Methods and compositions for the diagnosis and selective treatment of cancer
US20190374657A1 (en) * 2017-02-08 2019-12-12 Dana-Farber Cancer Institute, Inc. Tunable endogenous protein degradation with heterobifunctional compounds
US20200348285A1 (en) * 2017-11-09 2020-11-05 Dana-Farber Cancer Institute, Inc. Methods to prevent teratogenicity of imid like molecules and imid based degraders/protacs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SłABICKI MIKOłAJ; KOZICKA ZUZANNA; PETZOLD GEORG; LI YEN-DER; MANOJKUMAR MANISHA; BUNKER RICHARD D.; DONOVAN KATHERINE A: "The CDK inhibitor CR8 acts as a molecular glue degrader that depletes cyclin K", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 585, no. 7824, 3 June 2020 (2020-06-03), London, pages 293 - 297, XP037241512, ISSN: 0028-0836, DOI: 10.1038/s41586-020-2374-x *

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