WO2011160016A2 - Poches de liaison à e3 et identification et utilisation d'inhibiteurs de ligase e3 - Google Patents

Poches de liaison à e3 et identification et utilisation d'inhibiteurs de ligase e3 Download PDF

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WO2011160016A2
WO2011160016A2 PCT/US2011/040879 US2011040879W WO2011160016A2 WO 2011160016 A2 WO2011160016 A2 WO 2011160016A2 US 2011040879 W US2011040879 W US 2011040879W WO 2011160016 A2 WO2011160016 A2 WO 2011160016A2
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crin
agent
cbl
mammal
ubiquitination
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WO2011160016A3 (fr
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Brent Stockwell
Andras Bauer
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The Trustees Of Columbia University In The City Of New York
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/60—In silico combinatorial chemistry
    • G16C20/64—Screening of libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
    • C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
    • C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B35/00—ICT specially adapted for in silico combinatorial libraries of nucleic acids, proteins or peptides
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16C—COMPUTATIONAL CHEMISTRY; CHEMOINFORMATICS; COMPUTATIONAL MATERIALS SCIENCE
    • G16C20/00—Chemoinformatics, i.e. ICT specially adapted for the handling of physicochemical or structural data of chemical particles, elements, compounds or mixtures
    • G16C20/60—In silico combinatorial chemistry
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • This invention is directed to, inter alia, an isolated protein fragment that includes a binding pocket or active site on an E3 ligase that modulates the E2-E3 interface, and to an agent that interacts with such a binding pocket.
  • the invention is further directed to methods for identifying an agent that may inhibit a RING-domain E3 ubiquitin ligase, methods for treating a condition in a mammal using such an agent, and methods for selectively inhibiting the ubiquitination function of an E3 ligase.
  • sequence listing text file "0304815pct.txt", file size of 41 .7 KB, created on June 15, 201 1 .
  • sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. ⁇ 1 .52(e)(5).
  • RING-domain E3 ubiquitin ligases are a large class of enzymes that serve as key regulators of many cellular processes, including cell proliferation (1 ), DNA damage repair (2), cell death (3), vascularization (4), and receptor tyrosine kinase (RTK) signaling (5).
  • RTK receptor tyrosine kinase
  • E3 ligases have been largely resistant to targeting with small molecules, especially in the ligase domain per se (6), and the few such inhibitors discovered tend to suffer from low potency and selectivity (7).
  • Low sequence homology of the RING and substrate binding domains, substrate diversity and the lack of a traditional active site make these proteins challenging drug targets (8-10).
  • Cbl adapter proteins function as negative regulators of signal transduction pathways (5, 14-17). Their activities were initially thought to derive solely from substrate binding (16), a model that was confounded by the discovery that Cbl family proteins also function as E3 ligases, ubiquitinating substrate proteins, and targeting them for internalization (5).
  • Cbl ligases have been implicated in downregulating activated growth factor receptors such as EGFR (18-20) and PDGFR (21 ) as well as downstream kinases such as Src and PI3K (22).
  • the E3 ligase c-Cbl itself has been implicated in insulin signaling (23), and has been implicated in diabetes and obesity.
  • One embodiment of the present invention is an isolated protein fragment.
  • This isolated protein fragment comprises a binding pocket or active site on an E3 ligase that modulates the E2-E3 interface, the pocket defined by the capacity of residue F63 of E2 to fit within the pocket.
  • Another embodiment of the present invention is an agent that interacts with the binding pocket on the E3 ligase disclosed above.
  • a further embodiment of the present invention is a method for identifying an agent that may inhibit a RING-domain E3 ubiquitin ligase.
  • the method comprises
  • step (b) conducting an in vitro auto-ubiquitination screen of the candidate E3 ligase inhibitor(s) identified in step (a), wherein an agent that inhibits E3 auto-ubiquitination is a candidate RING-domain E3 ubiquitin ligase inhibitor.
  • Yet another embodiment of the present invention is a method for selectively inhibiting the ubiquitination function of an E3 ligase. This method comprises contacting a cell with an agent that interferes with the ability of E2 to complex with an E3.
  • An additional embodiment of the present invention is a method for treating a condition in a mammal. This method comprises administering to the mammal an effective amount of an agent identified by any method disclosed in the present application.
  • Another embodiment of the present invention is a method for reducing food intake in a mammal. This method comprises administering to the mammal an effective amount of an agent identified by any method disclosed in the present application.
  • Yet another embodiment of the present invention is a method for reducing weight gain in a mammal. This method comprises administering to the mammal an effective amount of an agent identified by any method disclosed in the present application.
  • Figure 1 shows that CRIN-1 targets a conserved binding pocket at the c-Cbl-UbcH7 interface.
  • Figure 1A shows a conserved binding groove (blue), which was found using structural and sequence alignment of 41 RING domain structures from the PDB. The conserved binding groove is highlighted here by the W408 residue of the c-Cbl RING domain in yellow.
  • Figure 1 B shows that the region surrounding F63 of UbcH7 defines a potential small-molecule binding site on the c- Cbl RING domain. S407 and W408 present key interacting residues with UbcH7.
  • Figure 1 C shows the structure of CRIN-1 .
  • Figure 1 D shows the predicted binding pose of CRIN-1 to the c-Cbl RING domain.
  • FIG. 1 E shows an overlay of Heteronuclear Single Quantum Coherence (HSQC) spectra of the c-Cbl RING domain with (red) and without the addition of CRIN-1 (blue). Residues with significant chemical shift changes are marked by arrows.
  • Figure 1 F shows the chemical shift changes of individual residues in the c-Cbl RING domain upon CRIN-1 binding. The line notes the level of significance, as determined by the standard deviation in chemical shifts between the two spectra.
  • HSQC Heteronuclear Single Quantum Coherence
  • Figure 2 shows that CRIN-1 binds to and prevents auto-ubiquitination by the wild-type c-Cbl RING domain.
  • Figure 2A shows the results of a plate-based auto-ubiquitination assay comparing the activity of GST-wild-type and GST-S407A RING domain in the presence of CRIN-1 . Ubiquitination was detected by HRP- linked anti-GST antibody directed against ubiquitinated GST-Cbl-RING domain.
  • Figure 2B shows the results of a 32 P-labeled auto-ubiquitination assay of purified wild-type GST-c-Cbl-RING domain and UbcH7, with increasing concentrations of CRIN-1 . Polyubiquitin-smears disappeared with increasing concentrations of CRIN-1 .
  • Figure 2C shows the results of a 32 P-labeled auto-ubiquitination assay with purified S407A GST-c-Cbl-RING domain, with increasing concentrations of CRIN-1 and UbcH7. Mutation of S407 to alanine was predicted to abolish a hydrogen bond with CRIN-1 , destabilizing small molecule binding.
  • FIG. 2D shows that CRIN-1 binds wild-type c-Cbl (47-472) with nanomolar affinity. CRIN-1 binding to immobilized GST-c-Cbl was monitored by SPR to reveal a binding affinity of 54 nM.
  • Figure 2E shows that CRIN-1 stabilized the c-Cbl RING domain in thermal stability shift assays. Increasing concentrations of CRIN-1 increased the thermal stability of the wild-type c-Cbl RING domain, indicating binding.
  • Figure 2F shows that CRIN-1 did not affect the thermal stability of c-Cbl RING S407A, indicating a lack of binding.
  • Figure 2G shows that CRIN-1 did not inhibit the formation of auto-ubiquitinated poly-ubiquitin of the MDM-2 or BRCA1 RING domains.
  • EDTA inactived-MDM2 RING and the ligase inactive mutant BRCA1 I26A served as negative controls.
  • Figure 3 shows that CRIN-1 inhibited auto-ubiquitination of c-Cbl and selectively enhanced insulin response.
  • Figure 3A shows that a 2-hour CRIN-1 treatment of C33a cells increased levels of c-Cbl protein but not Cbl-b.
  • Figure 3B shows that transfection of c-Cbl S407A-luc in 293T cells confers resistance to CRIN- 1 .
  • Cells were transiently transfected with either wild-type or S407A c-Cbl-luciferase and subsequently treated with CRIN-1 .
  • FIG. 3C shows that CRIN-1 inhibits ubiquitination of c-Cbl in 3T3-L1 adipocytes.
  • Adipoctyes were transfected with HA-tagged ubiquitin and subsequently treated with CRIN-1 for 2 hours, lysed, and immunoprecipitated with anti-HA resin. Lysates and precipitated protein were blotted and stained for c-Cbl, revealing an abrogation of ubiquitination in samples treated with relevant concentrations of CRIN-1 .
  • Figure 3D shows that a 2-hour treatment of 3T3-L1 adipocytes with CRIN-1 increased levels of the insulin receptor (IR- ⁇ ).
  • Figure 3E shows that CRIN-1 co-treatment with insulin prolonged insulin response, as measured by p-Akt levels in serum-starved 3T3-L1 adipocytes, but has no effect on baseline insulin signaling.
  • Figure 3F shows that a 2 hour CRIN-1 treatment of DU-145 cells did not affect levels of the EGF receptor.
  • Figure 3G shows that CRIN-1 co-treatment of serum-starved 3T3-I1 adipocytes with IGF-1 , EGF or PDGF did not enhance or prolong growth factor receptor signaling.
  • Figure 4 shows that CRIN-1 induced and stimulated GLUT-1 -mediated glucose uptake and GLUT-4 membrane localization.
  • Figure 4A shows 3 H-labeled 2- deoxy-glucose uptake assay with De Vivo disease patient fibroblasts. Cells were serum-starved overnight, then incubated with 10 ⁇ CRIN-1 for 16 hours. The fibroblasts were then incubated with 3 H-labeled 2-deoxy-glucose, and the amount of glucose uptake determined by radiography.
  • Figure 4B shows the quantification of the cell-surface bound GLUT-4 fraction.
  • Differentiated GFP-Myc-GLUT-4 expressing 3T3-L1 adipocytes were serum-starved overnight, during the last 2 hours of starvation, CRIN-1 was added to half of the cells at a final concentration of 10 ⁇ .
  • the cells were stimulated with 0, 8 or 80 nM insulin for 10 minutes at 37°C, then externalized myc tag was stained using an AlexaFluor546-conjugated secondary antibody. Images were taken using a 20x objective to quantify fluorescence intensities due to myc and GFP, and the relative ratio of the fluorescence signals is plotted.
  • Figure 4C shows representative images taken using a 63x objective. The same acquisition conditions were used for all images, and these are displayed using the same dynamic range. Scale bar, 10 ⁇ .
  • Figure 5 shows that CRIN-1 improved motor performance and insulin sensitivity in mice in a CNS-dependent manner.
  • Figure 5A shows that CRIN-1 treatment of high fat diet fed mice improved glucose tolerance. Mice were treated with 50 mg/kg of CRIN-1 administered i.p. for the seven days of the high-fat diet. Glucose tolerance was measured after a 16 hour fasting period the day after the last injection.
  • Figure 5B shows that CRIN-1 treatment lowers basal levels of insulin and improves insulin response. Mice were treated with 50 mg/kg of CRIN-1 administered i.p. for the seven days of the high-fat diet. Insulin levels were measured after a 16 hour fasting period the day after the last injection.
  • Figure 5C shows that CRIN-1 decreases caloric intake in mice.
  • Figure 5D shows that CRIN-1 treatment improves rotorod performance of mice. Mice were treated with daily doses of 50 mg/kg CRIN-1 daily from birth until 2 months of age. Subsequently they were placed on the rotorod to measure initial motor coordination ability.
  • FIG. 6 shows that a plate-based auto-ubiquitination assay specifically detects c-Cbl-RING auto-ubiquitination.
  • the auto-ubiquitination assay disclosed in Example 1 below was conducted under different conditions and using different protein constructs to control for nonspecific signal sources.
  • the assay was conducted with the GST-c-Cbl-RING construct as well as the deletion mutant (Cbl-d), with no E3 ligase added (UbcH7), with non-biotinylated ubiquitin (Cbl and Cbl-d nonlabeled), with GST alone as well as without adding ATP or E1 enzyme.
  • EDTA a strong chelator of zinc was used as a control to disrupt the folding and function of the c-Cbl-RING domain.
  • Figure 7 shows the results of a plate-based auto-ubiquitination assay using UbcH5b, measuring the activity of GST-wild-type RING domains in the presence of CRIN-1 .
  • Ubiquitination was detected by HRP-linked anti-GST antibody directed against ubiquitinated GST- Cbl-RING domain.
  • Figure 8 shows that CRIN-1 specificity for c-Cbl is dependent on S407 but not E412.
  • the results of a 32 P labeled auto-ubiquitination assay with purified E412D GST-c-Cbl-RING domain and UbcH7, with increasing concentrations of CRIN-1 are shown.
  • Figure 10 shows that thermal denaturation of UbcH7 showed no effect by CRIN-1 . Fluorescence was normalized to minimum and maximum signal indicating completely folded and unfolded states.
  • Figure 1 1 shows an immunoblot of phosphorylated insulin receptor- ⁇ in 3T3-L1 cells following CRIN-1 or insulin treatment. Differentiated 3T3-L1 cells were treated with CRIN-1 for 2 hours, then immunoprecipitated with anti-phosphotyrosine antibody to show CRIN-1 increases the amount of activated insulin receptor.
  • Figure 12 shows immunoblots of the proteins as indicated.
  • DU-145 cells were serum-starved overnight, then treated with 80 ng/ml of EGF or 10 ⁇ CRIN-1 for the indicated time.
  • Figure 13 shows representative cells fixed and immunostained for c- Cbl and GLUT-1 .
  • Differentiated 3T3-L1 adipocytes were serum-starved overnight, then treated with 10 ⁇ CRIN-1 for 2 hours or 80 nM insulin for 5 minutes.
  • Figure 15 shows the predicted binding mode of CRIN-1 to the c-Cbl RING-domain. CRIN-1 is shown to establish a hydrogen-bond with residue S407, and forming a ⁇ -cationic interaction with W408.
  • Figure 16 shows the results of Surface Plasmon Resonance (SPR) analysis of CRIN-1 GST-Cbl (47-472) binding.
  • SPR Surface Plasmon Resonance
  • Figure 17 shows that CRIN-1 treatment caused a marked decrease in the amount of HA-ubiquitin-tagged c-Cbl.
  • the experimental condition of the results shown in Figure 17 are as follows: DU-145 cells were transfected with HA-tagged ubiquitin, then treated with CRIN-1 for 2 hours. Cells were then lysed, and ubiquitin was immunoprecipiated using anti-HA antibodies. The precipitated proteins were blotted for c-Cbl in the DU-145 cell line.
  • Figure 18 shows that CRIN-1 treatment caused a marked decrease in the amount of HA-ubiquitin-tagged insulin receptor ⁇ -subunit.
  • the experimental condition of the results shown in Figure 18 are as follows: Differentiated 3T3-L1 adipocytes were transfected with HA-tagged ubiquitin, then treated with CRIN-1 for 2 hours. Cells were then lysed, and ubiquitin was immunoprecipiated using anti-HA antibodies. The precipitated proteins were blotted for c-Cbl and insulin receptor ⁇ - subunit in the 3T3-L1 adipocytes.
  • Figure 19 shows the results of a 3 H-labeled 2-deoxy-glucose uptake assay with Devivo-disease patient fibroblast cells.
  • Cells were serum-starved overnight then incubated with 10 ⁇ CRIN-1 , 100 ⁇ thioctic acid (TA) or their combination.
  • the fibroblasts were then incubated with 3 H-labeled 2-deoxy-glucose, and the amount of glucose uptake determined by radiography.
  • Figure 20A shows that CRIN-1 induces GLUT-4 membrane localization in GFP-Myc-GLUT-4 expressing 3T3-L1 fibroblasts.
  • the experimental conditions of the results shown in Figure 20A are as follows: Differentiated GFP-Myc-GLUT-4 expressing 3T3-L1 adipocytes were serum-starved overnight, then treated with 10 ⁇ CRIN-1 for 2 hours or with 80 nM insulin for 5 minutes. The cells were washed with PBS then stained with mouse anti-Myc and anti-mouse AlexaFluor-594 antibodies. Cells were then fixed in ethanol, and visualized.
  • Figure 20B shows the results of a microplate-based determination of the cell-surface bound GLUT-4 fraction. Cells were treated and stained as described above, then fixed for 1 hour in 50% ethanol. Cells were then washed and evaluated for GFP/594nm fluorescence ratios using a microplate-reader.
  • Figure 21 shows that CRIN-1 activated insulin signaling in mouse brain and fat tissue.
  • Figure 22 shows the docking site on the c-Cbl RING-domain.
  • the binding site was constrained as any residue within 6A of residue F63 of UbcH7, including residues S407 and W408.
  • Figure 23 shows the structure of CRIN-2 (des-methylamino CRIN-1 ).
  • Figure 24 shows thermal denaturation of the c-Cbl RING-domain with CRIN-2.
  • Figure 25 shows the results of a plate-based auto-ubiquitination assay according to the present invention with wild-type RING-domain.
  • IC 5 o w t 230 nM.
  • Figure 26 shows that 2 hour treatment of C33a cells with CRIN-1 or CRIN-2 increased c-Cbl levels selectively.
  • One embodiment of the present invention is an isolated protein fragment.
  • This protein fragment comprises a binding pocket or active site on an E3 Iigase that modulates the E2-E3 interface, the pocket defined by the capacity of residue F63 of E2 to fit within the pocket.
  • peptide means a linked sequence of amino acids, which may be natural, synthetic, or a modification or combination of natural and synthetic.
  • E3 As used herein, "E3”, “RING-domain E3 ubiquitin Iigase”, “E3 Iigase”, and “E3 ubiquitin Iigase” are used interchangeably. These terms mean a protein that, in combination with an E2 ubiquitin-conjugating enzyme, causes the attachment of one or more ubiquitins to a lysine on a target protein.
  • E3 ubiquitin ligases include, for example, c-Cbl (the human version of which is listed as SEQ ID NO:1 ), Cbl-b, MDM2, AMFR, ARIH1 , BARD1 , BRCA1 , BRD2, CCNB1 , CNOT4, DORFIN, GERP, E3A, Anaphase-promoting complex (APC), UBR5 (EDD1 ), SOCS/BC- box/eloBC/CUL5/RING, LNXp80, CBX4, HACE1 , HECTD1 , HECTD2, HECTD3, HECW1 , HECW2, HERC1 , HERC2, HERC3, HERC4, HUWE1 , IBRDC2, ITCH, KAP1 , KF1 , LOC, LRSAM1 , MARCH-I, MARCH2, MARCH4, MARCH5, MARCH6, MARCH8, MARCH9, Mel18, MIDI ,
  • the E2-E3 interface means the points of interaction between an E3 ligase and an E2.
  • An ⁇ 2 means a ubiquitin- conjugating enzyme, which is an enzyme that is able to accept an activated ubiquitin on a cysteine residue via a thioester bond and binds ubiquitin ligases or E3 ligases via a structurally conserved binding region.
  • Ubiquitin-conjugating enzymes include, without limitation, UbcH7 (the human version of which is listed as SEQ ID NO:2).
  • module means to mediate.
  • Residue F63 of E2 means the phenylalanine residue at position 63 of human UbcH7 as shown in SEQ ID NO:2 as well as the residues comparable to the phenylalanine residue 63 of human UbcH7 in other E2s, including UbcH7's isoforms, homologs, and orthologs, which residue fits within a binding pocket of an E3.
  • isoform means an alternative form of a protein resulting from differential transcription of the relevant gene either from an alternative promoter or an alternate splicing site.
  • Homolog means a gene related to a second gene by descent from a common ancestral DNA sequence.
  • Ortholog means a gene in a different species that evolved from a common ancestral gene by speciation.
  • Whether two residues are comparable or not may be determined by sequence alignment.
  • “Alignment” refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i.e., nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences.
  • An alignment of phylogenetically-related sequences may be used to identify conserved domains and relatedness within these domains.
  • An alignment may suitably be determined by means of computer programs known in the art such as MACVECTOR software (1999) (Accelrys, Inc., San Diego, Calif.) or ClustalX.COPYRGT. (Larkin et al., 2007).
  • Two or more sequences may be "optimally aligned" with a similarity scoring method using a defined amino acid substitution matrix such as the BLOSUM62 scoring matrix.
  • a preferred method uses a gap existence penalty and gap extension penalty that arrives at the highest possible score for a given pair of sequences. See, for example, Dayhoff et al. (1978) and Henikoff and Henikoff (1992).
  • the BLOSUM62 matrix is often used as a default scoring substitution matrix in sequence alignment protocols such as Gapped BLAST 2.0.
  • the gap existence penalty is imposed for the introduction of a single amino acid gap in one of the aligned sequences, and the gap extension penalty is imposed for each additional empty amino acid position inserted into an already opened gap.
  • the alignment is defined by the amino acids positions of each sequence at which the alignment begins and ends, and optionally by the insertion of a gap or multiple gaps in one or both sequences, so as to arrive at the highest possible score.
  • Optimal alignment may be accomplished manually or with a computer-based alignment algorithm, such as gapped BLAST 2.0 (Altschul et al, (1997). See U.S. Patent Application US20070004912.
  • the isolated protein fragment further comprises any residue on the E3 within 6 angstroms of residue F63 of E2, when E2 and E3 are complexed.
  • “complexed” means bound.
  • the isolated protein fragment is further defined by the presence of amino acid residues S407 and W408 of c-Cbl such as, e.g., SEQ ID NO:1 , or homologous residues thereof in other E3 ligases that enable complexing of an E2 with its corresponding E3.
  • homologous residues of c-Cbl means comparable residues in other E3s, including c-Cbl's isoforms, homologs, and orthologs, which facilitate complexing of an E2 with its corresponding E3.
  • a particular E3 "corresponds" to a particular E2 if that E3 is able to complex with that E2 and carry out its ubiquitination activity.
  • Another embodiment of the present invention is an agent that interacts with the binding pocket or active site on the E3 ligase disclosed above.
  • This agent may be selected from the group consisting of a small molecule, a biologic, and combinations thereof.
  • the agent inhibits E3 ubiquitination.
  • small molecule includes any chemical or other moiety, other than biologies, that can act to affect biological processes, particularly to inhibit a RING-domain E3 ubiquitin ligase.
  • Small molecules can include any number of therapeutic agents presently known and used, or that can be synthesized in a library of such molecules for the purpose of screening for biological function(s).
  • Small molecules are distinguished from macromolecules by size.
  • the small molecules of the present invention usually have a molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1 ,000 Da, most preferably less than about 500 Da.
  • Small molecules include without limitation organic compounds, peptidomimetics and conjugates thereof.
  • organic compound refers to any carbon-based compound other than macromolecules such as nucleic acids and polypeptides.
  • organic compounds may contain calcium, chlorine, fluorine, copper, hydrogen, iron, potassium, nitrogen, oxygen, sulfur and other elements.
  • An organic compound may be in an aromatic or aliphatic form.
  • Non-limiting examples of organic compounds include acetones, alcohols, anilines, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, amino acids, nucleosides, nucleotides, lipids, retinoids, steroids, proteoglycans, ketones, aldehydes, saturated, unsaturated and polyunsaturated fats, oils and waxes, alkenes, esters, ethers, thiols, sulfides, cyclic compounds, heterocyclic compounds, imidizoles, and phenols.
  • An organic compound as used herein also includes nitrated organic compounds and halogenated (e.g., chlorinated) organic compounds.
  • Representative, non-limiting examples of small molecules according to the present invention include CRIN-1 and CRIN-2 disclosed in more detail below.
  • Preferred small molecules are relatively easier and less expensively manufactured, formulated or otherwise prepared. Preferred small molecules are stable under a variety of storage conditions. Preferred small molecules may be placed in tight association with macromolecules to form molecules that are biologically active and that have improved pharmaceutical properties. Improved pharmaceutical properties include changes in circulation time, distribution, metabolism, modification, excretion, secretion, elimination, and stability that are favorable to the desired biological activity. Improved pharmaceutical properties include changes in the toxicological and efficacy characteristics of the chemical entity.
  • a polypeptide mimetic is a molecule that mimics the biological activity of a polypeptide, but that is not peptidic in chemical nature. While, in certain embodiments, a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids), the term peptidomimetic may include molecules that are not completely peptidic in character, such as pseudo-peptides, semi-peptides, and peptoids.
  • biological means products derived from living sources as opposed to a chemical process.
  • Non-limiting examples of a “biologic” include proteins, nucleic acids, and partially purified products from tissues.
  • protein includes antibodies, antibody mimetics, domain antibodies, lipocalins, and targeted proteases.
  • the term also includes vaccines containing a peptide or peptide fragment intended to raise antibodies against the peptide or peptide fragment.
  • Antibody as used herein includes an antibody of classes IgG, IgM, IgA, IgD, or IgE, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, and bifunctional antibodies.
  • the antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof, which exhibits sufficient binding specificity to a desired epitope or a sequence derived therefrom.
  • the antibody may also be a chimeric antibody.
  • the antibody may be derivatized by the attachment of one or more chemical, peptide, or polypeptide moieties known in the art.
  • the antibody may be conjugated with a chemical moiety.
  • the antibody may be a human or humanized antibody.
  • antibody-like molecules are also within the scope of the present invention.
  • Such antibody-like molecules include, e.g., receptor traps (such as entanercept), antibody mimetics (such as adnectins, fibronectin based "addressable” therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.), domain antibodies (the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez-Retamozo et al., Cancer Res. 2004 Apr 15;64(8):2853-7)).
  • receptor traps such as entanercept
  • antibody mimetics such as adnectins, fibronectin based "addressable” therapeutic binding molecules from, e.g., Compound Therapeutics, Inc.
  • domain antibodies the smallest functional fragment of a naturally occurring single-domain antibody (such as, e.g., nanobodies; see, e.g., Cortez
  • Suitable antibody mimetics generally can be used as surrogates for the antibodies and antibody fragments described herein. Such antibody mimetics may be associated with advantageous properties (e.g., they may be water soluble, resistant to proteolysis, and/or be nonimmunogenic). For example, peptides comprising a synthetic beta-loop structure that mimics the second complementarity- determining region (CDR) of monoclonal antibodies have been proposed and generated. See, e.g., Saragovi et al., Science. Aug. 16, 1991 ;253(5021 ):792-5.
  • CDR complementarity- determining region
  • Peptide antibody mimetics also have been generated by use of peptide mapping to determine "active" antigen recognition residues, molecular modeling, and a molecular dynamics trajectory analysis, so as to design a peptide mimic containing antigen contact residues from multiple CDRs. See, e.g., Cassett et al., Biochem Biophys Res Commun. Jul. 18, 2003;307(1 ):198-205. Additional discussion of related principles, methods, etc., that may be applicable in the context of this invention are provided in, e.g., Fassina, Immunomethods. October 1994;5(2):121 -9.
  • peptide includes targeted proteases, which are capable of, e.g., substrate-targeted inhibition of post-translational modification such as disclosed in, e.g., U.S. Patent Application Publication No. 20060275823.
  • Nucleic acids refer to molecules composed of chains of monomeric nucleotides, adenine, cytosine, guanine, thymine, uracil, or any artificially constructed molecules to mimic such a chain. Nucleic acids include without limitation, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), glycol nucleic acid (GNA) and threose nucleic acid (TNA), as well as antisense nucleic acids and siRNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PNA peptide nucleic acid
  • LNA morpholino and locked nucleic acid
  • GAA glycol nucleic acid
  • TAA threose nucleic acid
  • to "inhibit E3 ubiquitination” means to reduce the activity of a E3 ubiquitin ligase, whether such activity is auto-ubiquitination or ubiquitination of a target protein.
  • these agents are specific, with little to no off target effects.
  • the agent is selected from the group consisting of CRIN-1 , CRIN-2, an analog of CRIN-1 or CRIN-2, a pharmaceutical salt of CRIN-1 , a pharmaceutical salt of CRIN-2, and pharmaceutical salts of the analogs.
  • an "analog of CRIN-1 or CRIN-2" means a compound having structural and functional similarities with CRIN-1 or with CRIN-2.
  • the present invention includes all possible isomers of a particular compound, such as, e.g., CRIN-1 or CRIN-2, whether shown herein or not.
  • a further embodiment of the present invention is a method for identifying an agent that may inhibit a RING-domain E3 ubiquitin ligase. This method comprises:
  • step (b) conducting an in vitro auto-ubiquitination screen of the candidate E3 ligase inhibitor(s) identified in step (a), wherein an agent that inhibits E3 auto-ubiquitination is a candidate RING-domain E3 ubiquitin ligase inhibitor.
  • an "in silico screen” means a filtering of large databases or libraries of possible agents through the use of computational approaches based on discrimination functions that permit the selection of agents to be tested for biological activity.
  • An exemplary in silico screen according to the present invention uses an in silico compound library, such as those generated using OMEGA1 .8. Screening may be performed using FRED2.0 as disclosed herein. Docking poses may be scored by PLP (35). Other approaches to in silico screens are known in the art. See, e.g., Plewcznyski et al., Chem. Biol. Drug. Res., 69(4):269-79 (2007), Lu et al., J. Med.
  • an auto-ubiquitination screen means an assay for testing whether an agent can inhibit the formation of poly-ubquititinated chains on a RING-domain E3 ubiquitin ligase.
  • a representative in vitro auto-ubiquitination screen according to the present invention is disclosed in more detail in Example 1 below.
  • an agent that "inhibits E3 auto-ubiquitination” means an agent that, e.g., reduces formation of poly-ubquititinated chains on a E3 ubiquitin ligase.
  • the in vitro auto-ubiquitination screen is a high throughput screen.
  • a high throughput screen defines a process in which large numbers of agents are tested rapidly and in parallel for binding activity or biological activity against target molecules.
  • "large numbers of agents" may be, for example, more than 100 or more than 300 or more than 500 or more than 1 ,000 agents.
  • the process is an automated process.
  • a HTS is a known method of screening to those skilled in the art.
  • the agent is selected from the group consisting of a small molecule, a biologic, and combinations thereof.
  • step (a) further comprises identifying an agent that binds to or interacts with an E3 binding pocket that includes any residues on the E3 within 6 angstroms of residue F63 of E2, when E2 and E3 are complexed.
  • step (a) further includes identifying an agent that binds to or interacts with an E3 binding pocket that includes amino acid residues S407 and W408 of c-CBI, such as, e.g., SEQ ID NO:1 , or homologous residues thereof in other E3 ligases that enable complexing of an E2 with its corresponding E3.
  • the E2-E3 interface is a c- Cbl-UbcH7 interface.
  • the in silico screen comprises identifying an agent that interacts with residues S407 and W408 of the c-Cbl RING domain or homologous residues in other E3s.
  • the in silico screen comprises identifying an agent that interferes with the ability of UbcH7 to complex with the c-Cbl RING domain.
  • the in silico screen comprises identifying an agent that interferes with the ability of residue F63 of UbcH7 to form a complex with a small hydrophobic pocket on the c-Cbl RING domain flanked by S407 and W408.
  • the agent is selected from the group consisting of CRIN-1 , CRIN-2, an analog of CRIN-1 or CRIN-2, a pharmaceutical salt of CRIN-1 , a pharmaceutical salt of CRIN-2, and pharmaceutical salts of the analogs.
  • a further embodiment of the present invention is a method for selectively inhibiting the ubiquitination function of an E3 ligase. This method comprises contacting a cell with an agent that interferes with the ability of E2 to complex with an E3.
  • the agent interacts with a binding pocket disclosed herein and inhibits formation of the E2-E3 interface.
  • the agent is selected from the group consisting of a small molecule, a biologic, and combinations thereof.
  • the agent is selected from the group consisting of CRIN-1 ,
  • CRIN-2 an analog of CRIN-1 or CRIN-2, a pharmaceutical salt of CRIN-1 , a pharmaceutical salt of CRIN-2, and pharmaceutical salts of the analogs.
  • the contacting step comprises administering to a mammal, preferably a human, a pharmaceutical composition comprising the agent.
  • a mammal preferably a human
  • a pharmaceutical composition comprising the agent.
  • the human suffers from a condition selected from the group consisting of diabetes, including diabetes type II, DeVivo disease, obesity, obesity-related disorders, Alzheimer's disease, and cardiovascular disease.
  • the condition is diabetes type II.
  • An additional embodiment of the present invention is a method for treating a condition in a mammal, preferably a human, comprising administering to the mammal an effective amount of an agent identified by any method disclosed in the present invention.
  • an "effective amount” or “therapeutically effective amount” of an agent identified by any of the methods disclosed herein is an amount of such an agent that is sufficient to effect beneficial or desired results as described herein when administered to a patient, which is a mammal, preferably a human.
  • Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine.
  • a suitable dose of an agent identified by any of the methods disclosed herein will be that amount of the agent, which is the lowest dose effective to produce the desired effect with no or minimal side effects.
  • a suitable, non-limiting example of a dosage of an agent identified by any of the methods disclosed herein is from about 1 ng/kg to about 1000 mg/kg, such as from about 1 mg/kg to about 100 mg/kg, including from about 5 mg/kg to about 50 mg/kg.
  • Other representative dosages of such an agent include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1000 mg/kg.
  • the effective dose of such an agent maybe administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
  • the condition is selected from the group consisting of diabetes, including diabetes type II, DeVivo disease, obesity, obesity-related disorders, Alzheimer's disease, and cardiovascular disease.
  • the condition is diabetes type II.
  • the agent is selected from the group consisting of CRIN-1 , CRIN-2, an analog of CRIN-1 or CRIN-2, a pharmaceutical salt of CRIN-1 , a pharmaceutical salt of CRIN-2, and pharmaceutical salts of the analogs.
  • the agent is part of a pharmaceutical composition.
  • Another embodiment of the present invention is a method for reducing food intake in a mammal, preferably a human. This method comprises administering to the mammal an effective amount of an agent identified by any method disclosed in the present application.
  • the mammal has a condition selected from the group consisting of diabetes, including diabetes type II, obesity, and obesity-related disorders.
  • the condition is diabetes type II.
  • the agent is selected from the group consisting of CRIN-1 , CRIN-2, an analog of CRIN-1 or CRIN-2, a pharmaceutical salt of CRIN-1 , a pharmaceutical salt of CRIN-2, and pharmaceutical salts of the analogs.
  • the agent is part of a pharmaceutical composition.
  • Yet another embodiment of the present invention is a method for reducing weight gain in a mammal, preferably a human. This method comprises administering to the mammal an effective amount of an agent identified by any method disclosed in the present application.
  • the mammal has a condition selected from the group consisting of diabetes, including diabetes type II, obesity, and obesity-related disorders.
  • the condition is diabetes type II.
  • the agent is selected from the group consisting of CRIN-1 , CRIN-2, an analog of CRIN-1 or CRIN-2, a pharmaceutical salt of CRIN-1 , a pharmaceutical salt of CRIN-2, and pharmaceutical salts of the analogs.
  • the agent is part of a pharmaceutical composition.
  • a pharmaceutical composition of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, a pharmaceutical composition of the present invention may be administered in conjunction with other treatments.
  • a pharmaceutical composition of the present invention maybe encapsulated or otherwise protected against gastric or other secretions, if desired.
  • compositions of the invention comprise one or more active ingredients, e.g. one or more agents identified by the methods of the present invention, in admixture with one or more pharmaceutically- acceptable carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically- acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).
  • Pharmaceutically acceptable carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters, sodium
  • Each pharmaceutically acceptable carrier used in a pharmaceutical composition of the invention must be "acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • Carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.
  • compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in such pharmaceutical compositions.
  • ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate;
  • compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or non-aqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste.
  • These formulations may be prepared by methods known in the art, e.g., by means of conventional pan-coating, mixing, granulation or lyophilization processes.
  • Solid dosage forms for oral administration may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents.
  • Solid compositions of a similar type maybe employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine.
  • the tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter.
  • compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • the active ingredient can also be in microencapsulated form.
  • Liquid dosage forms for oral administration include pharmaceutically- acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain suitable inert diluents commonly used in the art.
  • the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions may contain suspending agents.
  • compositions for rectal or vaginal administration may be presented as a suppository, which maybe prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
  • Pharmaceutical compositions which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants.
  • the active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable carrier.
  • the ointments, pastes, creams and gels may contain excipients.
  • Powders and sprays may contain excipients and propellants.
  • compositions suitable for parenteral administrations comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.
  • Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.
  • a drug e.g., pharmaceutical formulation
  • the rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle.
  • injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use.
  • sterile liquid carrier for example water for injection
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.
  • the suspension was lysed by sonication and the protein purified using glutathione sepharose beads (Amersham Biosciences, GE Healthcare Bio-Sciences Corp., Piscataway, NJ). The protein was eluted in suspension buffer containing 15 mM glutathione, then concentrated to 6 mg/ml.
  • Point mutants of the RING-domain were created by site-directed mutagenesis (Quikchange II, Stratagene, Agilent Technologies, Inc., Santa Clara, CA), then expressed and purified as above.
  • site-directed mutagenesis For thermal denaturation assays, 1 mg of protein was incubated with 8 units of PreScission (Amersham Biosciences) protease overnight at 4°C. The protein was then incubated for 30 minutes with glutathione sepharose to remove the cleaved GST-tag. The protein concentration of the cleaved RING domain was adjusted to about 0.25 mg/ml and the protein was dialysed into a buffer containing 150 mM NaCI, 100 mM HEPES pH 7.5.
  • Protein purification and sample preparation was done according to a protocol described previously (46). In short, 50 ml starter cultures were grown to an OD of 0.6 in minimal media and used to inoculate 1 L of minimal medium. The culture was then induced using 0.5 mM IPTG overnight at 20°C. Cells were harvested by centrifugation, and suspended in 20 ml_ of buffer containing 0.15 M NaCI, 50 mM Tris pH 7.6 and 5 mM DTT. Cells were lysed by sonication, then the protein was purified using glutathione sepharose beads. The RING domain was removed from the beads using thrombin cleavage overnight at 4°C. Protein was concentrated and measured.
  • the reaction mixture was transferred to streptavid in-coated 384-well plates (Reacti-bind, Pierce) and incubated for 1 hour at room temperature.
  • the plate was washed 3 times with 90 ⁇ /well of 10% goat serum in PBS with 0.1 % Tween-20, then incubated for one hour with wash buffer containing 1 :1000 HRP-linked goat anti-GST antibody (GST-HRP-13, Alpha Diagnostic Intl. Inc., San Antonio, TX).
  • the plate was washed again, then incubated with standard ECL for 5 minutes, and read on a Victor plate reader. Radiolabeled in vitro ubiquitination assay
  • PK-ubiquitin was a generous gift of Dr. Masha Poyurovsky.
  • DU-145 cells were cultured in MEM (Gibco 1 1370, Invitrogen Corp., Carlsbad, CA) containing 10% FBS, 2 mM glutamine and 1 mM pyruvate.
  • 3T3-L1 adipocytes and 3T3-L1 cells stably expressing GLUT4-7Myc-GFP were cultured as described previously by Yu et al. (41 ).
  • Cells were lysed with 100 ⁇ ⁇ buffer (50 mM HEPES, 40 mM NaCI, 2 mM EDTA, 0.5% Triton-X, 1 .5 mM sodium orthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium beta- glycerophosphate and protease inhibitor tablet (Roche, Nutley, NJ), (pH 7.4)). Samples were separated using SDS-polyacrylamide gel electrophoresis.
  • samples were then transferred to a polyvinylidene difluoride membrane, blocked for 1 hour at room temperature in Licor Odyssey Blocking Buffer and incubated with the necessary primary and secondary antibodies: anti-Cbl (Abeam ab 2235, Abeam pic, Cambridge, MA), anti-c-Cbl (BD transduction lab 610442, BD Biosciences, San Jose, CA), anti-EGFR (sc03, Santa Cruz Antibodies, Santa Cruz, CA), anti-Insulin Receptor ⁇ -subunit (L55B10, Cell Signaling Technology Inc, Denvers, MA), anti-phosphotyrosine (Upstate 06-427, Millipore, Billerica, MA), anti-p- Akt (Ser 473) (Cell Signaling 9271 S), anti-alpha-tubulin (T6199, Sigma, St.
  • anti-Cbl Abeam ab 2235, Abeam pic, Cambridge, MA
  • anti-c-Cbl BD transduction lab 610442, BD Biosciences,
  • 3T3-L1 adipocytes were grown to confluence, then grown for another two days in DMEM (Gibco) with 10% FBS, 2 mM gutamine, and 100 g/ml penicillin/streptomycin. Confluent cells were then incubated with the above media containing 10 g/ml of insulin, 1 mM isobutyl-methylxantine and 20 ⁇ prednisolone for 6 days. The media was changed every two days, then switched back to regular media for two days. Cells were then trypsinized and seeded, approximately 1 million cells per well in six-well dishes.
  • 3T3-L1 and DU-145 cells were transfected with HA-Ubiquitin (for HA- tagged pulldown) or were serum-starved overnight (phospho-tyrosine IP) prior to the experiments.
  • Cells were lysed in 150 ⁇ of buffer (20 mM Tris, 0.3M NaCI, 0.1 % NP- 40, 2mM EDTA, 1 mM PMSF) and incubated overnight with 30 ⁇ of Protein A beads conjugated with 4 ⁇ g of the appropriate antibody. Precipitated proteins were removed by boiling in 5 ⁇ _ 6x loading dye. Samples were separated and blotted as described above. Fluorescence microscopy
  • 3T3-L1 adipocytes expressing the GLUT4 reporter were prepared for imaging using a modified protocol described by Yu et al. (41 ). Externalized Myc epitope was stained with 9E10 antibodies and Texas Red conjugated secondary antibodies at 1 :100 dilution in PBS containing 5% GS at 4°C for 120 minutes. Cells were then washed in cold PBS, then mounted using fluoromount. Images were obtained using a FV500 confocal microscope (Olympus America Inc., Center Valley, PA) using a 60* (1 .4 NA) objective. Fluoview Tiff images were processed with ImageJ UCSD Plugins.
  • 3T3-L1 cells stably expressing a GLUT4-7Myc-GFP reporter protein were differentiated on coverslips. Cells were starved for 18 hours. During the last 2 hours of starvation, CRIN-1 was added to half of the cells at a final concentration of 10 ⁇ . The cells were stimulated with 0, 8 or 80 nM insulin for 10 minutes at 37°C, washed twice with ice-cold PBS, and fixed for 5 minutes at room temperature with 4% paraformaldehyde.
  • the cells were blocked with 5% normal goat serum for 30 minutes at room temperature before being incubated with mouse monoclonal antibody to myc (1 :200 dilution in 5% normal goat serum, 9E10, Abeam) for 1 hour at room temperature, followed by alexa 546 anti-mouse antibody (1 :500 dilution in 5% normal goat serum, Invitrogen) for 45 minutes at room temperature.
  • Cells were imaged using a Zeiss Axiovert microscope (Carl Zeiss Microimaging, LLC, Thornwood, NY) equipped with a cooled CCD camera with the Colibri illumination system driven by AxioVision imaging software (Carl Zeiss).
  • LED modules 470 nm and 540 nm were used for GFP and alexa 546 excitation, respectively, and FL Filter Set 62 HE BFP+GFP+HcRed was used for detection (Carl Zeiss). Images were acquired with either a Zeiss plan apochromat 63* l ⁇ A oil objective or Zeiss plan neofluar 20*/0.5 objective. Identical acquisition conditions were used for all cells. To quantify GLUT4 translocation, the ratio of cell-surface myc staining to total GFP intensity was used from images taken with a 20* objective. An average of values from 20 images (two independent experiments) for each condition was normalized to that of control, unstimulated cells to obtain the relative fold change.
  • 3T3-L1 adipocytes were differentiated and seeded with 1 million cells per well in a 6-well dish. Cells were serum starved, treated with compounds, and stained for externalized Myc epitope as above. Cells were then suspended by gentle pipetting in 1 ml of PBS containing 5% GS. The cell suspension was transferred onto a 96-well plate (Corning Inc., Corning, NY), fluorescence was read at 594 nm and 510 nm on a Victor III (Perkin Elmer, Santa Clara, CA) plate reader, and the ratio of internal/external GLUT4 was calculated. In vivo experiments in mice
  • mice Male C57BL/6J mice were purchased at 10 weeks of age (Jackson Lab, Boston, MA) and individually housed under controlled temperature (23°C) and lighting (12:12 hour light/dark cycle, lights on at 7:00 AM) with free access to water and food. After one week of acclimatization, regular chow (2018S, Harlan Teklad, Madison, Wl) was changed for a high-fat diet (55% calories from fat; TD 93075, Harlan Teklad, Madison, Wl) three days before the injections. The CRIN-1 compound was injected intraperitoneally once daily for seven days at a dose of 50 mg/kg-day (diluted in NaCI 0.9%).
  • Body composition was assessed using a Bruker Minispec analyzer (Bruker, The Woodlands, TX). Metabolic parameters and physical activity were measured during the first four days of injections using the Oxymax system from Columbus Instruments (Columbus, OH). Intraperitoneal glucose tolerance tests were performed the day after the last injection following an overnight fasting (16 hours). Plasma samples were obtained from the tail at 0, 15, 30, 45, 60, 90 and 120 minutes. Glucose was measured using a YSI 2700D glucose analyzer (YSI Inc, Yellow Springs, OH) and insulin was measured by RIA kit (Millipore, Billerica, MA).
  • C57BL/6J mice were treated with compound or vehicle (NaCI 0.9%) for 2 hours, then euthanized, and dissected.
  • Tissue samples were taken from the brain, liver and fat tissue, then flash frozen in liquid nitrogen. Tissue samples were thawed and homogenized manually, then resuspended in 3 ⁇ of buffer (50 mM HEPES pH 7.6, 1 mM EDTA, 0.1 % NP-40, 0.1 M LiCI, 0.7% Sodium deoxycholate) for every 1 mg of tissue.
  • the tissue lysate was then sonicated for 5 minutes in a sonicating waterbath (Branson 1510), then spun at 10,000 rpm for ten minutes. The supernatant were then filtered through MiraclothTM (EMD Biosciences) and spun again. 15 ⁇ aliquots of lysates were loaded on Tris-glycine gels and Western blotted as described above.
  • ubiquitination activity of RING E3 ligases is mediated by the RING domain
  • substrate specificity is typically determined by a distinct substrate- binding domain (32).
  • Inhibiting ubiquitination of target proteins can be accomplished by either targeting the substrate-binding domain, as was done with the MDM2-p53 interaction inhibitors named nutlins (7), or by targeting the RING domain directly in a generalized strategy that might be adaptable to many RING-domain E3 ligases. Because the aim was to develop inhibitors that can serve as scaffolds for targeting other E3 ligases, the inventors focused on the interface of c-Cbl's RING domain interface with that of its substrate carrier protein, UbcH7 (33).
  • AQCPHCRAPLQLRELVNCRWAEEVTQQLDTLQLCSL [0135] The x-ray crystallographic structure of the c-Cbl-RING-UbcH7 complex (33) allowed for in silico screening to discover compounds binding to the protein- protein interaction interface between the ubiquitin carrier (E2) and the c-Cbl RING domain. Using the modeling suite MOE 2006.08 (Chemical Computing Group, Montreal, Canada), the inventors defined the potential binding site on the c-Cbl RING domain, constrained as residues in proximity to residue F63 of UbcH7.
  • This residue plays a key role in the interface between E2 and E3 ligases in the available crystal structures in which UbcH7 is complexed with an E3 ligase (34).
  • F63 fits into a small hydrophobic pocket on the c-Cbl RING domain, flanked by Trp408, a residue crucial for UbcH7 binding (46), providing potential for hydrophobic stacking interactions for potential small molecule inhibitors.
  • S407 a residue unique to the c-Cbl RING-domain, might enable the formation of a hydrogen bond with small molecule inhibitors ( Figure 1 B and 22).
  • a docking site was selected to include any residue within 6A of F63 on UbcH7, a binding site that included the binding pocket defined by W408.
  • An in silico compound conformer library composed of two million commercially available compounds, including 47,000 already purchased compounds, was generated using OMEGA1 .8 (Openeye Scientific Software: Santa Fe, NM), which created 200 conformers (maximum) per compound. These conformers were then screened using FRED2.0 (Fast Rigid Exhaustive Docking, Openeye Scientific
  • these 720 compounds were tested in a high-throughput microplate- based auto-ubiquitination assay using purified GST-c-Cbl RING-domain protein
  • CRIN-1 selectively binds to and inhibits auto-ubiquitination by c-Cbl in vitro
  • CRIN-1 's ability to inhibit auto-ubiquitination of the c-Cbl RING-domain was tested in a plate-based ubiquitination assay used for the high-throughput screen ( Figure 6).
  • the ubiquitination pathway was reconstructed using purified proteins, and CRIN-1 significantly inhibited the c-Cbl-RING domain at nanomolar concentrations using either UbcH7 ( Figure 2A) or UbcH5b ( Figure 7) as ubiquitin carriers.
  • the same assay was performed using the S407A mutant of the c-Cbl RING domain.
  • a plate-based ubiquitination assay was also developed in 384-well format to facilitate rapid testing of analogs (see Example 1 ). After determining CRIN- 1 's potency against wild-type c-Cbl and the S407A c-Cbl mutant in this plate-based format ( Figure 1 e), it was found that CRIN-1 showed more than 60-fold selectivity for the wild-type RING-domain over the S407A mutant. To further investigate the isoform specificity of CRIN-1 , glutamate 412 was mutated to aspartate on the c-Cbl RING domain.
  • CRIN-1 CRIN-1 's activity on two other E3 ligases was also examined. CRIN-1 did not inhibit auto-ubiquitination of either BRCA1/BARD1 or the MDM-2 RING domain ( Figure 2G), suggesting a level of specificity against Cbl-RING domain.
  • CRIN-1 inhibits auto-ubiquitination by c-Cbl and selectively enhances insulin response in cell culture
  • 3T3-L1 adipocytes were transfected with HA-tagged ubiquitin, followed by CRIN-1 treatment.
  • the cells were lysed and HA-tagged proteins were immunoprecipitated with anti-HA antibodies.
  • c-Cbl was present in the untreated samples, indicating auto- ubiquitination, CRIN-1 treatment caused a marked decrease in the amount of HA- ubiquitin-tagged c-Cbl ( Figure 3C).
  • CRIN-1 failed to enhance signaling of any of these growth factors ( Figure 3G), indicating that ubiquitination by c-Cbl may not be critical to downregulation of the activated EGF, PDGF or IGF-1 receptors. This suggests that CRIN-1 specifically enhances insulin receptor and insulin signaling.
  • CRIN-1 treatment triggers relocalization of insulin-sensitive glucose
  • c-Cbl is required for insulin receptor down-regulation, and has been implicated in a variety of diseases and phenotypes associated with insulin action and glucose uptake (25, 39). Because localization of GLUT1 is known to be affected by levels of activated c-Cbl (48), CRIN-1 's effect on glucose uptake in mouse adipocytes was investigated. Serum-starved, differentiated 3T3-L1 adipocytes were treated with CRIN-1 , fixed, and stained by immunofluorescence for glucose transporter-1 (GLUT-1 ) and c-Cbl ( Figure 13). CRIN-1 treatment resulted in enrichment of GLUT-1 and c-Cbl in the plasma membrane, an effect similar to that induced by insulin treatment after five minutes ( Figure 13).
  • De vivo disease 40 patient fibroblast cell lines were used in a glucose uptake assay.
  • De vivo disease or GLUT-1 deficiency, is a hereditary disease that results from deletion of one copy of GLUT-1 , resulting in decreased glucose uptake in the central nervous system of patients.
  • CRIN-1 treatment of wild-type and patient-derived fibroblasts resulted in an increase of 2-deoxy-glucose uptake ( Figure 4A), elevating the uptake of the patient cells almost to the level of untreated wild-type cells.
  • CRIN-1 also affected localization of GLUT-4, the predominant glucose transporter in fat and muscle cells, which is more insulin-responsive than GLUT1 .
  • 3T3-L1 cells stably expressing a GLUT4-7myc-GFP reporter protein were serum- starved, then treated with CRIN-1 to monitor the compound's effect on GLUT-4 distribution (41 ).
  • GLUT4 is present in the plasma membrane, the myc tag of the reporter is located in the extracellular space, allowing for staining of cell-surface- bound GLUT-4 in non-permeabilized cells (41 ).
  • CRIN-1 treatment improves glucose tolerance by suppressing appetite in mice
  • CRIN-1 As insulin signaling is known to suppress appetite in a CNS-dependent way (45), CRIN-1 's effect on signaling markers in tissue from treated mice was examined. CRIN-1 -treated mice displayed elevated levels of c-Cbl, insulin receptor and highly elevated levels of S473-phosphorylated Akt in both brain and fat tissue ( Figure 5E and Figure 14), with the effect being more than two-fold higher in brain tissue, indicating CRIN-1 's effect on the central nervous system. These experiments suggest that CRIN-1 may be acting to suppress appetite in mice through the insulin signaling pathway.
  • the inventors have identified a small molecule nanomolar-potency inhibitor of the ligase activity of c-Cbl by directly targeting the RING domain.
  • the inventors were able to target the UbcH7/UbcH5b binding site on the RING-domain and find high-affinity inhibitors of the E3 ligase c- Cbl. Because the binding pocket itself is a common feature of most RING-domains, but the residues lining the pocket show little conservation, this presents a generalizable approach to targeting RING-domain ligases.
  • E3 ligase inhibitors achieve their activity by targeting substrate interfaces, such as nutlin-3a's inhibition of the p53-MDM2 interface (7).
  • substrate interfaces such as nutlin-3a's inhibition of the p53-MDM2 interface (7).
  • the approach disclosed herein enables the targeting of a common feature in all RING-containing ligases, enabling more facile development of small molecule ligase inhibitors, similar to what has been done for kinase inhibitors.
  • CRIN-1 specifically binds to and inhibits the activity of c-Cbl's RING domain over that of the close homolog Cbl-b's RING domain.
  • c-Cbl plays a crucial role in the regulation of kinase signaling, as well as glucose uptake.
  • CRIN-1 prevents c-Cbl ubiquitination of itself in vitro and in cell culture and increases the levels of insulin receptor in a dose-dependent manner.
  • the inventors observed increased and prolonged activation of insulin receptor signaling and Akt phosphorylation in differentiated adipocytes in response to insulin co-treatment, an indication that c-Cbl inhibition does indeed result in increased downstream signaling in the insulin receptor.
  • the inventors have not found an indication of prolonged or enhanced activation in case of EGF receptor signaling, where CRIN-1 treatment did not change EGF receptor levels and caused very little stimulation-dependent Akt activation.
  • Impaired glucose uptake plays a role in many chronic medical conditions, including type II Diabetes and GLUT-1 deficiency, as well as Alzheimer's disease (42).
  • the inventors examined glucose uptake in a patient fibroblast model of GLUT-1 deficiency, as well as in GLUT4-reporter-expressing 3T3-L1 adipocytes, and demonstrated that treatment with CRIN-1 resulted in increased glucose transporter localization to the cell membrane, and that this re-localization led to increased glucose uptake.
  • the inventors also observed improved glucose tolerance in a mouse model of insulin resistance upon CRIN-1 treatment and determined that CRIN-1 acts on the central nervous system, reducing food intake, suppressing appetite and improving motor coordination.
  • E3 ligase c-Cbl is involved in the pathogenesis of many metabolic conditions, including obesity (43), making CRIN-1 a versatile and useful tool both in the research of, and potential treatment of, these conditions.
  • Woods SC Lotter EC, McKay LD, & Porte D, Jr. (1979) Chronic intracerebroventricular infusion of insulin reduces food intake and body weight of baboons. Nature 282(5738):503-505.

Abstract

La présente invention concerne, entre autres, un fragment de protéine isolé qui comprend une poche de liaison ou un site actif sur une ligase E3 qui module l'interface E2-E3, où la poche de liaison est définie par la capacité du résidu F63 de E2 à s'ajuster dans la poche. La présente invention concerne en outre un agent qui interagit avec une telle poche de liaison sur une ligase E3. La présente invention concerne en outre des procédés pour identifier un agent qui peut inhiber une ubiquitine ligase E3 de domaine RING, des procédés pour traiter une affection chez un mammifère en utilisant un tel agent, et des procédés pour inhiber sélectivement la fonction d'ubiquitination d'une ligase E3.
PCT/US2011/040879 2010-06-17 2011-06-17 Poches de liaison à e3 et identification et utilisation d'inhibiteurs de ligase e3 WO2011160016A2 (fr)

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WO2013106643A3 (fr) * 2012-01-12 2013-09-06 Yale University Composés et procédés pour la dégradation améliorée de protéines cibles et d'autres polypeptides par une e3 ubiquitine ligase
US9988376B2 (en) 2013-07-03 2018-06-05 Glaxosmithkline Intellectual Property Development Limited Benzothiophene derivatives as estrogen receptor inhibitors
US9993514B2 (en) 2013-07-03 2018-06-12 Glaxosmithkline Intellectual Property Development Limited Compounds
US10071164B2 (en) 2014-08-11 2018-09-11 Yale University Estrogen-related receptor alpha based protac compounds and associated methods of use
US9938264B2 (en) 2015-11-02 2018-04-10 Yale University Proteolysis targeting chimera compounds and methods of preparing and using same
WO2017144016A1 (fr) * 2016-02-26 2017-08-31 胡卓伟 Polypeptide, dérivés correspondants et application correspondante dans la préparation de médicaments présentant une résistance à la fibrose pulmonaire
WO2018050286A1 (fr) 2016-09-16 2018-03-22 Helmholtz Zentrum Muenchen - Deutsches Forschungszentrum Für Gesundheit Und Umwelt (Gmbh) Inhibiteurs de traf 6
WO2019180207A1 (fr) 2018-03-22 2019-09-26 Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Inhibiteurs de traf 6
CN110812472A (zh) * 2019-11-19 2020-02-21 福建医科大学 E3泛素连接酶stub1在抑制乙型肝炎病毒复制中的应用
CN110812472B (zh) * 2019-11-19 2022-10-21 福建医科大学 E3泛素连接酶stub1在抑制乙型肝炎病毒复制中的应用
WO2022132301A1 (fr) * 2020-12-17 2022-06-23 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Rnf167 et castor1 en tant que nouvelles cibles de mtor
WO2022266321A1 (fr) * 2021-06-17 2022-12-22 The Regents Of The University Of California Inhibiteurs de ligase e3 et leurs méthodes d'utilisation

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