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Title:
SCREENING METHODS TO IDENTIFY COMPOUNDS INHIBITING THE ACTIVITY OF E2 ENZYMES BY STABILIZATION OF NON-COVALENT UBIQUITIN-E2 COMPLEXES AND PHARMACEUTICAL APPLICATIONS RELATED TO E2 INHIBITORS
Document Type and Number:
WIPO Patent Application WO/2014/094138
Kind Code:
A1
Abstract:
The invention exploits stabilization of the non-covalent donor ubiquitin interaction with E2 enzymes, including Cdc34-ubiquitin interaction. The present invention concerns an isolated and purified E2-ubiquitin complex comprising a binding pocket into which inhibitors of E2 enzymatic activity fit. The invention also concerns various aspects related to that binding pocket including inhibitors, pharmaceutical compositions, screening methods for identifying inhibitors, and therapeutic methods for inhibiting enzymes involved in the cell ubiquitin-proteasome system (UPS).

Inventors:
TYERS MICHAEL (CA)
SICHERI FRANK (CA)
CECCARELLI DEREK FRANK JOSEPH (CA)
HUANG HAO (CA)
ORLICKY STEPHEN (CA)
VAN DER SLOOT ALBERT MARTINUS (CA)
ST-CYR DANIEL (CA)
MOORE SUSAN JENNIFER (CA)
Application Number:
PCT/CA2013/001079
Publication Date:
June 26, 2014
Filing Date:
December 20, 2013
Export Citation:
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Assignee:
UNIV MONTREAL (CA)
MOUNT SINAI HOSPITAL CORP (CA)
International Classes:
C12N9/00; A61K45/00; C07K14/47; C12N9/96; C12Q1/25; G01N33/573; G06F19/16
Other References:
CECCARELLI, D. F. ET AL.: "An allosteric inhibitor of the human Cdc34 ubiquitin-conjugating enzyme.", CELL, vol. 145, no. 7, 24 June 2011 (2011-06-24), pages 1075 - 1087
PAGE, R. C. ET AL.: "Structural insights into the conformation and oligomerization of E2-Ubiquitin conjugates.", BIOCHEMISTRY, vol. 51, no. 20, 22 May 2012 (2012-05-22), pages 4175 - 4187
DOU, H. ET AL.: "Structure ofBIRC7-E2 ubiquitin conjugate reveals the mechanism of ubiquitin transfer by a RING dimer.", NAT. STRUCT. MOL. BIOL., vol. 19, no. 9, September 2012 (2012-09-01), pages 876 - 883
PULVINO, M. ET AL.: "Inhibition of proliferation and survival of diffuse large B- cell lymphoma cells by a small-molecule inhibitor of the ubiquitin-conjugating enzyme Ubc13-Uev1A.", BLOOD., vol. 120, no. 8, 23 August 2012 (2012-08-23), pages 1668 - 1677
HUANG, H. ET AL.: "E2 enzyme inhibition by stabilization of a low-affinity interface with ubiquitin.", NATURE CHEMICAL BIOLOGY., vol. 10, no. 2, February 2014 (2014-02-01), pages 156 - 163
Attorney, Agent or Firm:
FASKEN MARTINEAU DUMOULIN LLP (Stock Exchange TowerP.O. Box 242,Suite 370, Montreal Québec H4Z 1E9, CA)
Download PDF:
Claims:
CLAIMS:

1. An isolated and purified E2-ubiquitin complex comprising a binding pocket, wherein ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and wherein said binding pocket comprises Gly47Ub, Lys48Ub, Glu51Ub and Gln49Ub. 2. The isolated and purified E2-ubiquitin complex of claim 1 , wherein the E2- ubiquitin complex is a Cdc34-ubiquitin complex, wherein said Cdc34 comprises SEQ ID NO : 1 , and wherein said Cdc34-ubiquitin complex comprises one or more of the following:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162. 3. An isolated and purified stabilized non-covalent ubiquitin-Cdc34 complex, comprising Cdc34 of SEQ ID NO : 1 and ubiquitin of SEQ ID NO: 2, said complex further comprising a compound that fits within a stabilized binding pocket of said ubiquitin-Cdc34 complex and wherein said stabilized binding pocket comprises:

1) electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc34 side chain and side chains of Gln49Ub, Arg42U , and Arg72U , and (iii) the Ser129Cdc34 side chain and the side chains of Gln49Ub and Arg72U ; and

2) promotion of hydrophobic contacts between a ridge formed by Leu8U , Ile44u , and Val70U , and/or a complementary groove formed by Thr122 Cdc34, Leu 25 Cdc34, Ser126 Cdc34, Ile128 Cdc34 and Ser129 Cdc34.

4. A method of inhibiting Cdc34 catalytic activity, comprising fitting a compound into a binding pocket of a Cdc34-ubiquitin complex, wherein said Cdc34 comprises SEQ ID NO : 1 , wherein said ubiquitin comprises SEQ ID NO: 2, and wherein said binding pocket comprises:

- Gly47Ub, Lys48Ub, Glu51U and Gln49U ; and

one or more of the following:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C terminal end of helix a3 and α3-α4 linker; - a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58,

Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52,

Pro134, Tyr148, Trp151 , Tyr161 , and Thr162. 5. A method of inhibiting Cdc34 catalytic activity, comprising contacting Cdc34 comprising SEQ ID NO : 1 and ubiquitin comprising SEQ ID NO: 2 with a compound that stabilizes non-covalent ubiquitin-Cdc34 complex, wherein said compound fits within a stabilized binding pocket of said ubiquitin-Cdc34 complex and wherein said stabilized binding pocket comprises:

1 ) electrostatic and H-bond interactions selected from the group consisting of:

(i) Asn132Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc34 side chain and side chains of Gln49Ub, Arg42Ub, and Arg72Ub, and (iii) the Ser129Cdc34 side chain and the side chains of Gln49U and Arg72Ub ; and

2) promotion of hydrophobic contacts between a ridge formed by Leu8Ub, Ile44ub, and Val70U , and/or a complementary groove formed by Thr122 Cdc34, Leu125 Cdc34, Ser126 Cdc34, lle128 Cdc34 and Ser129 Cdc34.

6. The method of claim 4 or 5, wherein said compound makes direct contact with the following amino acids: Gly47Ub, Lys48U , Glu51 U and Gln49U .

7. The method of any one of claims 4 to 6, for treating a cancer, a neurological disorder, an immunological disorder, an infectious disease, diabetes, hemochromatosis, cystic fibrosis, mental retardation, and/or infertility.

8. The method of claim 7, wherein the cancer is selected from the group consisting of: adipose, adrenal, bladder, blood/lymphatic, bone, bone marrow, brain, cervix, connective, ear, eye, head and neck, heart, intestine, kidney, larynx, liver, lung, lymph, lymph node, mammary, mouth, muscle, nerve, ovary, pancreas, pharynx, pituitary, placenta, prostate, salivary, skin, spleen, stomach, testis, thyroid, tonsil, uterus, and vascular.

9. A screening method for identifying inhibitors of an E2 enzyme component of a mammalian cell ubiquitin-proteasome system (UPS), the method comprising: (a) measuring a binding affinity between ubiquitin and the E2 enzyme in the presence of a candidate inhibitor; and (b) identifying a candidate compound that increase said binding affinity as a potential inhibitor of the E2 enzyme and/or as a potential inhibitor of another enzyme of the UPS.

10. The screening method of claim 9, wherein the E2 enzyme is selected from the group consisting of E2 enzymes listed in Table 1 of the description.

1 1. The screening method of claim 9 or 10, wherein the E2 enzyme is human Cdc34 which amino acid sequence is defined in SEQ ID NO: 1. 12. The screening method of any one of claims 9 to 1 1 , wherein said ubiquitin is human ubiquitin which amino acid sequence is defined in SEQ ID NO: 2.

13. The screening method of any one of claims 9 to 12, wherein the screening method is selected from the group consisting of the following assays: NMR assay, TR- FRET assay, yeast two-hybrid, bead-based protein capture with fluorescent proteins, ITC, Fortebio™, Biacore™, AlphaScreen®, microscale thermophoresis, mass spectrometry based assay, speed screen assay, single molecule spectroscopy assay, in siiico structure-guided molecular docking, proximity scintillation assays (SPA), and BRET assays.

14. The screening method of any one of claims 9 to 12, wherein the ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and wherein measuring a binding affinity comprises observing by NMR peak shifts and/or peak broadening of ubiquitin resonances corresponding to one or more of the following ubiquitin residues: Lys6, Thr7, Leu8, Gln40, Gln41 , Arg42, Leu43, Ile44, Phe45, Gly47, Lys48, Gln49, Leu50, Leu67, His68, Val70, Leu71 , Arg72 and Leu73. 15. The screening method of any one of claims 9 to 12 or 14, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and wherein measuring a binding affinity comprises observing by NMR peak shifts and/or peak broadening of Cdc34 resonances corresponding to one or more of the following Cdc34 residues: Pro49, Asn50, Pro48, Thr,51 , Glu26, Ile128, Leu125, Ser129, Asn132, Thr122, Ser126, Glu133, Leu130, Pro134, Asn135, Ser1 1 1 , Glu1 12, Leu109, Glu108.

16. The screening method of claim 13, wherein the NMR assay comprises the following steps:

- providing labeled ubiquitin;

- providing a catalytic domain of the E2 enzyme (E2cat);

- acquiring a first Heteronuclear Single Quantum Coherence (HSQC) reference spectra of the labeled ubiquitin in the presence of the E2cat;

- acquiring a first HSQC spectrum in presence of a test compound; - detecting chemical shift perturbations or chemical shift intensity changes between the first and second HSQC spectrum;

wherein promotion of molecular interaction between the labeled ubiquitin and E2cat is indicative of a potential inhibitory activity for the test compound. 17. The screening method of claim 16, wherein acquiring the first HSQC spectrum is carried out in presence of a plurality of test compounds and wherein the method further comprises the step of deconvoluting the chemical shift perturbations or chemical shift intensity changes to identify specific compound(s) responsible for said shifts.

18. The screening method of claim of claim 6 or 17, wherein said labeled ubiquitin is 15N-ubiquitin.

19. The screening method of any one of claims 16 to 18, wherein the E2 enzyme comprises amino acids as defined in Figure 7 for human E2s, and wherein the E2cat comprises amino acid residues equivalent Pro7 to Val184 of Cdc34 as defined in the sequence alignment of Figure 7. 20. The screening method of any one of claims 16 to 19, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , wherein the E2cat is the catalytic domain of Cdc34 (Cdc34cat), and wherein said Cdc34cat comprises residues Pro7 to Val 84 of SEQ ID NO: 1.

21 . The screening method of claim 13, wherein the assay is a TR-FRET assay comprising the following steps:

- providing ubiquitin labeled with a first fluorophore;

- providing a E2 fusion protein fused to an antibody-recognizable tag;

- providing an antibody labelled with a second fluorophore recognizing said tag;

- contacting the labeled ubiquitin, the E2 fusion protein and the antibody in the presence or in absence of a test compound; and

- measuring a TR-FRET binding signal in the presence and in the absence of the test compound;

wherein an increase in the TR-FRET binding signal in the presence of the test compound relative to the TR-FRET binding signal in the absence of the test compound is indicative of a potential inhibitory activity for the test compound.

22. The screening method of claim 21 , further comprising a preliminary optimization step of measuring a TR-FRET binding signal in the presence of saturating levels of compound CC0651 as a positive binding control.

23. The screening method of claim 21 or 22, wherein said labeled ubiquitin is fluorescently-labeled ubiquitin.

24. The screening method of claim 13, wherein the assay is an on-bead assay comprising the following steps:

- providing compound-bearing beads comprising one or more test compound coupled to beads;

- providing a catalytic domain of the E2 enzyme (E2cat);

- providing fluorescently-labeled ubiquitin;

- incubating the compound-bearing beads and the fluorescently-labeled ubiquitin in the presence or absence of the E2 protein under conditions allowing the formation of E2-ubiquitin complexes;

- measuring a fluorescence signal on the beads;

wherein a fluorescence signal more intense in presence of compound-bearing beads relative to control beads is indicative of a potential inhibitory activity for the test compound coupled to the bead.

25. The screening method of claim 24, wherein the E2 enzyme comprises amino acids as defined in Figure 7 for human E2s, and wherein the E2cat comprises amino acid residues equivalent Pro7 to Val184 of Cdc34 as per sequence alignment of Figure 7.

26. The screening method of claim 24 or 25, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , wherein the E2cat is the catalytic domain of Cdc34 (Cdc34cat), and wherein said Cdc34cat comprises residues Pro7 to Val184 of SEQ ID NO: 1. 27. The screening method of any one of claims 24 to 26, wherein the compound- bearing beads comprises chemical molecules coupled to beads through a chemically compatible linker, and wherein the control beads are non-coupled beads or beads coupled to a non-inhibitor compound.

28. The screening method of any one of claims 24 to 27, wherein said compound- bearing beads are beads from a combinatorial one bead-one compound (OBOC) library.

29. The screening method of any one of claims 24 to 28, wherein the fluorescently- labeled ubiquitin is a fluorescein-labeled human recombinant ubiquitin.

30. A screening method for identifying inhibitors of an E2 enzyme component of the ubiquitin-proteasome system (UPS) of a subject, comprising:

(a) providing a three-dimensional model of an E2 enzyme in a binding interaction with ubiquitin;

(b) in silico docking a three-dimensional structure of test compounds at an interface between the E2 enzyme and ubiquitin;

(c) selecting test compounds from step (b) increasing binding affinity of the E2 enzyme with ubiquitin;

(d) identifying selected test compounds as potential inhibitors of the E2 enzyme and/or as potential inhibitors of another enzyme part of the UPS.

31. The method of claim 30, wherein said selecting comprises identifying ligand compounds having the highest docking score.

32. The method of claim 30 or 31 , wherein said ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and wherein said in silico docking comprises in silico docking at the following positions on ubiquitin: Gly47Ub, Lys48Ub, Glu51 Ub and Gln49U .

33. The method of any one of claims 30 to 32, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and wherein said in silico docking comprises in silico docking to at least one of the following positions:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C-terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162. 34. The method of claim 30 or 31 , wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , wherein the ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and wherein selecting test compounds increasing binding affinity of human Cdc34 with ubiquitin comprises assessing one or more of the following interactions:

1 ) promotion of electrostatic and H-bond interactions selected from the group consisting of: (i) Asn 32Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc34 side chain and side chains of Gln49Ub, Arg42Ub, and Arg72U , and (iii) the Ser129Cdc34 side chain and the side chains of Gln49Ub and Arg72Ub ; and/or 2) promotion of hydrophobic contacts between a ridge formed by Leu8U , Ile44u , and Val70Ub, and/or a complementary groove formed by Thr122 Cdc34, Leu125 Cdc34, Ser126 Cdc34, Ile128 cdc34 and Ser129 Cdc34 .

35. The method of any one of claims 30 to 34, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , wherein said ubiquitin is human ubiquitin comprising

SEQ ID NO: 2, and wherein the three-dimensional model at step (a) comprises 3D coordinates as defined in PDB ID 4MDK.

36. A method for screening inhibitors of an E2 enzyme component of a mammalian cell ubiquitin-proteasome system (UPS), comprising the following steps:

a) computationally generating a three-dimensional structure of the E2 enzyme in a binding interaction with ubiquitin and computationally generating a three dimensional molecular representation of test compounds;

b) virtual screening of a plurality of test compounds through molecule docking to obtain candidate inhibitors having a minimum docking affinity at an interface between the E2 enzyme and ubiquitin, wherein said docking increases binding affinity of the E2 enzyme with ubiquitin; and

c) testing said candidate inhibitors for in vitro, ex vivo and/or in vivo activity in inhibiting the E2 enzyme and/or inhibiting other enzymes involved in the UPS.

37. The method of claim 36, wherein said ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and wherein said virtual screening comprises assessing a minimum docking affinity of test compounds at an interface between the E2 enzyme and ubiquitin comprising Gly47Ub, Lys48U , Glu51 U and Gln49Ub.

38. The method of claim 36 or 37, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and wherein said virtual screening comprises assessing a minimum docking affinity of test compounds at an interface between the E2 enzyme and ubiquitin comprising:

- a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix α2, β2-β3 linker, C-terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162.

39. The method of any one of claims 36 to 38, wherein said ubiquitin is human ubiquitin comprising SEQ ID NO: 2, wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and wherein the three dimensional molecular structure of the E2 enzyme in a binding interaction with ubiquitin comprises 3D coordinates as defined in PDB ID 4MDK.

40. A method of drug design or drug testing, comprising:

A) uploading in a computer system structural coordinates of a three-dimensional model of human Cdc34 comprising SEQ ID NO: 1 in a binding interaction with human ubiquitin comprising SEQ ID NO: 2, wherein said three-dimensional model comprises one or more of the followings:

1 ) electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc34 side chain and side chains of Gln49Ub, Arg42U , and Arg72U , and (iii) the Ser129Cdc34 side chain and the side chains of Gln49Ub and Arg72Ub ;

2) hydrophobic contacts between a ridge formed by Leu8Ub, Ile44ub, and Val70Ub, and/or a complementary groove formed by Thr122Cdc34, Leu 25Cdc34, Ser126Cdc34, lie I 28cdc34 and Ser129Cdc34 ;

3) a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix α2, β2- β3 linker, C-terminal end of helix a3 and a3- a4 linker;

4) a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, et81 , Ile128, Leu131 and Ile165;

5) a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162;

6) a pocket surface contributed by ubiquitin lined by Gly47U , Lys48Ub,

Glu51 Ub and Gln49Ub ; and

B) virtually screening a plurality of test compounds through molecule docking to obtain candidate inhibitors having a minimum docking affinity at an interface between Cdc34 and ubiquitin;

wherein compounds having a minimum docking affinity are potential candidates for drug design and/or drug testing.

41. The method of claim 40, further comprising the step of testing said potential candidates for in vitro, ex vivo and/or in vivo activity in inhibiting human Cdc34 and/or inhibiting other human enzymes involved in the Ubiquitin-proteasome system (UPS).

42. The method of claim 40 or 41 , wherein said structural coordinates of human ubiquitin and human Cdc34 are as defined in PDB ID 4MDK.

43. A computationally generated three-dimensional model of human Cdc34 comprising SEQ ID NO: 1 in a binding interaction with human ubiquitin comprising SEQ ID NO: 2, wherein said three-dimensional model comprises one or more of the followings:

1) electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc34 side chain and side chains of Gln49U , Arg42Ub, and Arg72Ub, and (iii) the Ser129Cdc34 side chain and the side chains of Gln49U and Arg72Ub ;

2) hydrophobic contacts between a ridge formed by Leu8Ub, Ile44ub, and Val70ub, and/or a complementary groove formed by Thr122Cdc34, Leu125Cdc34, Ser126Cdc34, lie 128Cdc34 and Ser129Cdc34 ;

3) a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C-terminal end of helix a3 and a3- a4 linker;

4) a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

5) a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162; and

6) a pocket surface contributed by ubiquitin lined by Gly47U , Lys48Ub, Glu51 Ub and Gln49Ub ; and

44. The computationally generated three-dimensional model claim 43, wherein said three-dimensional model comprises structural coordinates of human ubiquitin and human Cdc34 are as defined in PDB ID 4MDK. 45. A computer-readable data storage medium comprising a data storage material encoded with the computationally generated three-dimensional model of claim 43 or 44.

46. A computer system comprising: a representation of the computationally generated three-dimensional model of claim 43 or 44; and a user interface to view the representation. 47. A computer-assisted method for identifying inhibitors of an E2 enzyme part of a mammalian cell ubiquitin-proteasome system (UPS), comprising the following steps:

- loading into a computer's memory a first set of data corresponding to a three- dimensional model of the E2 enzyme in a binding interaction with ubiquitin; - loading into a computer's memory a second set of data corresponding to three- dimensional structure of test compounds;

- computing said first and second set of a data to obtain docking affinity of the test compounds for at an interface between the E2 enzyme and ubiquitin;

- computing said first and second set of a data to obtain binding affinity of the E2 enzyme with ubiquitin in presence or absence of a test compound; and

- selecting test compounds increasing binding affinity of the E2 enzyme with ubiquitin for subsequent in vitro, ex vivo and/or in vivo testing of inhibition of the E2 enzyme and/or testing of inhibition of other enzymes involved in the cell ubiquitin- proteasome system (UPS).

48. The method of claim 47, wherein said ubiquitin is human ubiquitin comprising SEQ ID NO: 2 and wherein the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1.

49. The method of claim 48, wherein said three-dimensional model comprises structural coordinates of human ubiquitin and human Cdc34 are as defined in PDB ID 4MDK.

50. An E2 enzyme inhibitor, wherein said inhibitor stabilizes non-covalent E2- ubiquitin- complexes, and wherein said compound makes direct contact with human ubiquitin comprising SEQ ID NO: 2 at the following amino acids: Gly47U , Lys48Ub, Glu51 U and Gln49U . 51 . The E2 enzyme inhibitor of claim 50, wherein said E2-ubiquitin complex comprises a binding pocket, wherein the E2-ubiquitin complex is a Cdc34-ubiquitin complex, wherein said Cdc34 comprises SEQ ID NO : 1 , and wherein said Cdc34- ubiquitin complex comprises one or more of the following:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C-terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162. 52. An inhibitor of E2 catalytic activity, wherein said inhibitor comprises a chemical structure that (i) fits within a binding pocket formed by a non-covalent E2-ubiquitin- complex and (ii) stabilizes a E2-ubiquitin interaction.

53. The inhibitor of claim 52, wherein said ubiquitin is human ubiquitin which amino acid sequence is defined in SEQ ID NO: 2 and wherein said compound makes direct contact with human ubiquitin at the following amino acids: Gly47Ub, Lys48Ub, Glu51Ub and Gln49Ub.

54. The inhibitor of claim 53, wherein the E2 is human Cdc34 which amino acid sequence is defined in SEQ ID NO: 1, and wherein said binding pocket comprises one or more of the following:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix a2, β2-β3 linker, C-terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162.

55. A method of inhibiting E2 catalytic activity, comprising: contacting an inhibitor in presence of an E2 enzyme and ubiquitin, wherein said inhibitor comprises a chemical structure that (i) fits within a pocket formed by a non-covalent E2-ubiquitin-complex and (ii) stabilizes a E2-ubiquitin interaction.

56. A method of inhibiting E2-mediated ubiquitination reactions comprising: contacting an inhibitor in presence of an E2 enzyme and ubiquitin, wherein said inhibitor comprises a chemical structure that (i) fits within a binding pocket formed by a non- covalent E2-ubiquitin-complex and (ii) stabilizes a E2-ubiquitin interaction.

57. The method of claim 55 or 56, wherein the E2 is human Cdc34 which amino acid sequence is defined in SEQ ID NO: 1 , and wherein said ubiquitin is human ubiquitin which amino acid sequence is defined in SEQ ID NO: 2.

58. The method of claim 57, wherein said compound makes direct contact with human ubiquitin at the following amino acids: Gly47U , Lys48Ub, Glu51Ub and Gln49Ub.

59. The method of claim 57 or 58, wherein said binding pocket comprises one or more of the following:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C-terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165; - a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162.

60. The inhibitor of any one of claims 52 to 54, or the method of any one of claims 1 to 8 or 55 to 59, with the proviso that said compound is other than CC0651 , CC7094, CC9933, CC0040, CC9653, CC9807, CC9652, CC8993, CC9430, CC9504, CC9535, CC9566, CC9833.

61. A method of inhibiting the activity of an E2 enzyme within the ubiquitin- proteasome system (UPS) of a subject in need thereof, the method comprising contacting said E2 enzyme with a compound stabilizing non-covalent ubiquitin-E2 complexes, wherein said stabilizing inhibits said E2 activity.

62. A method of inhibiting proliferation and survival of a mammalian cell, the method comprising contacting the cell with a compound stabilizing non-covalent ubiquitin-E2 complexes within said cell, wherein said stabilizing inhibits proliferation and survival of said cell. 63. The method of claim 60 or 62, wherein said compound binds at an interface between the E2 enzyme and ubiquitin.

64. The method of claim 63, wherein said compound acts as a molecular bridge between E2 enzyme and ubiquitin.

65. The method of any one of claims 60 to 64, wherein said stabilizing comprises increasing binding affinity between the E2 enzyme and ubiquitin.

66. The method of claim 65, wherein the binding affinity between the E2 enzyme and ubiquitin is increased by at least 5, 10, 15, 20, 25 fold or more as evidenced by measurements of Ec50 in presence and in absence of the compound.

67. The method of any one of claims 60 to 66, wherein the compound makes direct contacts with ubiquitin on at least one of the following amino acids: Gly47u , Lys48ub,

Glu51 Ub and Gln49Ub.

68. The method of any one of claims 60 to 67, wherein the E2 enzyme is human Cdc34 and wherein the compound promotes electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc3 side chain and side chains of Gln49Ub, Arg42Ub, and Arg72U , and (iii) the Ser129Cdc34 side chain and the side chains of Gln49Ub and Arg72U .

69. The method of any one of claims 60 to 68, wherein the E2 enzyme is human Cdc34 and wherein the compound promotes hydrophobic contacts between a ridge formed by Leu8U , Ile44ub, and Val70Ub, and/or a complementary groove formed by Thr122 Cdc34, Leu125 Cdc34, Ser126 Cdc34, He I28 cdc34 and Ser129 Cdc34.

70. The method of any one of claims 60 to 67, wherein the E2 enzyme is selected from the group consisting of the E2 enzymes listed in Table 1 of the description.

71. The method of claim 70, wherein the E2 enzyme is an E2 enzyme interacting with cullin-RING ligases (CRLs) E3 enzymes.

72. The method of claim 71 , wherein the E2 enzyme is human Cdc34 (Ube2R1).

73. The method of any one of claims 60 to 72, wherein inhibiting of the activity of the E2 enzyme and/or wherein inhibiting proliferation and survival of the mammalian cell is desirable for treating a cancer, a neurological disorder, an immunological disorder, an infectious disease, diabetes, hemochromatosis, cystic fibrosis, mental retardation, and/or infertility.

74. The method of claim 73, wherein the cancer is selected from the group consisting of: adipose, adrenal, bladder, blood/lymphatic, bone, bone marrow, brain, cervix, connective, ear, eye, head and neck, heart, intestine, kidney, larynx, liver, lung, lymph, lymph node, mammary, mouth, muscle, nerve, ovary, pancreas, pharynx, pituitary, placenta, prostate, salivary, skin, spleen, stomach, testis, thyroid, tonsil, uterus, and vascular.

75. The method of any one of claims 61 to 74, wherein said compound is used in combination with one or more of the following inhibitors: MLN4924, Bortezomib (Velcade™), Carfilzomib.

76. A treatment method comprising administering to a subject in need thereof a compound which stabilizes non-covalent ubiquitin-E2 complexes within cells of the subject, wherein said compound is a compound that makes direct contacts with human ubiquitin comprising SEQ ID NO:2 at the following amino acids: Gly47Ub, Lys48U , Glu51 U and Gln49Ub.

77. A treatment method comprising administering to a subject in need thereof a compound which stabilizes non-covalent ubiquitin-Cdc34 complexes within cells of said subject, wherein said compound is selected from the following compounds:

- a compound by which stabilization of the non-covalent ubiquitin-Cdc34 complexes is observable by crystallography or is observable by NMR using peak shifts and/or peak broadening of ubiquitin resonances, wherein one or more of the following ubiquitin residues are observed: Lys6, Thr7, Leu8, Gln40, Gln41 , Arg42, Leu43, Ile44, Phe45, Gly47, Lys48, Gln49, Leu50, Leu67, His68, Val70, Leu71 , Arg72 and Leu73;

- a compound by which stabilization of the non-covalent ubiquitin-Cdc34 complexes is observable by crystallography or is observable by NMR using peak shifts and/or peak broadening of Cdc34 resonances, wherein one or more of the following Cdc34 residues are observed: Pro49, Asn50, Pro48, Thr51 , Glu26, Ile128, Leu125, Ser129, Asn132, Thr122, Ser126, Glu133, Leu130, Pro134, Asn135, Ser1 1 1 , Glu1 12, Leu109, Glu108. 78. A method of inhibiting cell proliferation in a subject, comprising administering to the subject a compound which stabilizes non-covalent ubiquitin-Cdc34 complexes within cells of said subject, wherein said compound is selected from the following compounds:

1 ) compounds that promote electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132Cdc34 side chain and backbone NH of Gln49Ub, (ii) the Glu133Cdc34 side chain and side chains of Gln49u , Arg42Ub, and Arg72Ub, and (iii) the Ser129Cdc34 side chain and the side chains of Gln49U and Arg72U ;

2) compounds that promote hydrophobic contacts between a ridge formed by Leu8U , Ile44u , and Val70Ub, and/or a complementary groove formed by Thr122Cdc34, Leu 25C c34, Ser126C c34, lie I 28cdc34 and Ser129Cdc34. 79. The method of any one of claims 61 to 78 wherein the subject is afflicted with one or more of the following diseases: a cancer, a neurological disorder, an immunological disorder, an infectious disease, diabetes, hemochromatosis, cystic fibrosis, mental retardation, and/or infertility.

80. The method of any one claims 61 to 79 wherein the subject is a human subject.

81. The method of any one of claims 61 to 80, with the proviso that said compound is other than CC0651 , CC7094, CC9933, CC0040, CC9653, CC9807, CC9652, CC8993, CC9430, CC9504, CC9535, CC9566, CC9833.

Description:
SCREENING METHODS TO IDENTIFY COMPOUNDS INHIBITING THE ACTIVITY OF E2 ENZYMES BY STABILIZATION OF NON-COVALENT UBIQUITIN-E2 COMPLEXES AND PHARMACEUTICAL APPLICATIONS RELATED TO E2 INHIBITORS

RELATED APPLICATIONS The present application claims priority to US provisional 61/740,600 (filed December 21 , 2012) and US provisional 61/914,274 (filed December 10, 2013), the content of which is incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to field of medicine. The present invention concerns inhibitors, pharmaceutical compositions, screening methods for identifying inhibitors, and therapeutic methods for inhibiting enzymes involved in the cell ubiquitin-proteasome system (UPS).

BACKGROUND OF THE INVENTION

The ubiquitin-proteasome system (UPS) controls virtually all aspects of cellular function and is perturbed either directly or indirectly in all human cancers. The UPS is based on the canonical E1 -E2-E3 enzyme cascade that activates and transfers ubiquitin to substrate proteins, thereby marking substrates for degradation by the 26S proteasome and/or altering their interactions with other proteins. E2 enzymes control the flux of activated ubiquitin from E1 enzymes to E3 enzymes to the substrates, and therefore lie at a crucial but still unexplored nexus in the UPS hierarchy. The cullin-RING (CRL) ubiquitin ligases are the largest class of E3 enzymes in humans and likely target hundreds if not thousands of substrates for degradation. These modular "super- enzymes" are composed of catalytic core complexes, based on different cullin scaffolds, and large cohorts of substrate-specific recruitment factors, many of which feature prominently in cancer, including Skp2, beta-TrCP and Fbw7. Notably, promising recent pre-clinical studies indicate that a pan-inhibitor of CRL activity called MLN4924, which inhibits cullin neddylation and activation, may be an efficacious anti-cancer agent (Soucy et al Nature (2009) 458: 732-736; Soucy et al Genes Cancer (201 1 ) 1 : 708-716). The CRLs act in concert with a dedicated E2 enzyme called Cdc34, which is recruited by the RING domain subunit to effect catalytic transfer of the thioester-linked ubiquitin on the E2 to substrate lysine residues. Recently, a group including the present inventors identified and characterized a small molecule inhibitor of Cdc34, called CC0651 , which at the time represented the first described E2 enzyme inhibitor (Ceccarelli et al. Cell (201 1 ), 145: 1075-1087). Like MLN4924, CC0651 stabilizes the CDK inhibitor p27 in cultured cells and inhibits the proliferation of human cancer cell lines (Ceccarelli, 201 1 ). The structure of the CC0651 -Cdc34 complex shows that CC0651 binds a cryptic pocket on the Cdc34 surface that is far removed from the active site cysteine but did not explain the mechanism of inhibition (Ceccarelli, 201 1 ). However, Ceccarelli et al. did not disclose the mechanism of action of CC0651 and it was unknown before the present invention that CC0651 could stabilizes the Cdc34-ubiquitin interaction. In fact, subsequent published work incorrectly predicted that CC0651 would inhibit the Cdc34-ubiquitin interaction (Pruneda et al. Mol Cell (2012), 47:933-942). Further details about the E2 enzyme inhibition by stabilization with ubiquitin were later published by Hung et al. ((Huang et al, Nat Chem Biol. 2013 Dec 15. doi: 10.1038/nchembio.1412. [Epub ahead of print])

The clinical success of the proteasome inhibitor Bortezomib (marketed as Velcade™ by Millennium Pharmaceuticals) has set the stage for more specific UPS inhibitors. Accordingly, there is a need for screening methods for identifying specific UPS inhibitors, including inhibitors of E2 enzymes such as Cdc34, an E2 enzyme implicated in many cancers. There is also a need for methods, compounds and pharmaceutical compositions for inhibiting the UPS, particularly E2 enzymes, in the treatment of cancers, neurological disorders, immunological disorders, infectious diseases, metabolic disorders and genetic disorders.

SUMMARY OF THE INVENTION According to a first aspect, the present invention relates to an isolated and purified E2- ubiquitin complex comprising a binding pocket into which inhibitors of E2 enzymatic activity fit. More particularly, the invention concerns an isolated and purified E2-ubiquitin complex comprising a binding pocket, wherein ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and wherein the binding pocket comprises Gly47 Ub , Lys48 Ub , Glu51 Ub and Gln49 U . In one embodiment the E2-ubiquitin complex is a Cdc34-ubiquitin complex, the Cdc34 comprises SEQ ID NO : 1 , and the Cdc34-ubiquitin complex comprises one or more of the following:

- a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix α2, β2-β3 linker, C terminal end of helix a3 and α3-α4 linker; - a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162. According to a related aspect, the the present invention concerns an isolated and purified stabilized non-covalent ubiquitin-Cdc34 complex, comprising Cdc34 of SEQ ID NO : 1 and ubiquitin of SEQ ID NO: 2, the complex further comprising a compound that fits within a stabilized binding pocket of the ubiquitin-Cdc34 complex and wherein the stabilized binding pocket comprises:

1 ) electrostatic and H-bond interactions selected from the group consisting of:

(i) Asn132 Cdo34 side chain and backbone NH of Gln49 Ub , (ii) the Glu133 Cdc34 side chain and side chains of Gln49 U , Arg42 U , and Arg72 U , and (iii) the Ser129 Cdc34 side chain and the side chains of Gln49 U and Arg72 Ub ; and

2) promotion of hydrophobic contacts between a ridge formed by Leu8 U , Ile44 ub , and Val70 U , and/or a complementary groove formed by Thr122 Cdc34 , Leu125 Cdc34 , Ser126 Cdc34 , Ne128 Cdc34 and Ser129 Cdc34 .

Another aspect of the invention concerns an isolated and purified E2-ubiquitin complex comprising on E2 a donor ubiquitin binding surface and on ubiquitin a Ile44 hydrophobic surface. Another aspect of the invention concerns a method of inhibiting Cdc34 catalytic activity. In one embodiment, the method comprises fitting a compound into a binding pocket of a Cdc34-ubiquitin complex, wherein the Cdc34 comprises SEQ ID NO : 1 , wherein the ubiquitin comprises SEQ ID NO: 2, and wherein the binding pocket comprises Gly47 U , Lys48 U , Glu51 Ub and Gln49 U ; and one or more of the following:

- a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix α2, β2-β3 linker, C terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162.

In another embodiment, the method of inhibiting Cdc34 catalytic activity comprises contacting Cdc34 comprising SEQ ID NO : 1 and ubiquitin comprising SEQ ID NO: 2 with a compound that stabilizes non-covalent ubiquitin-Cdc34 complex, wherein the compound fits within a stabilized binding pocket of the ubiquitin-Cdc34 complex and wherein said stabilized binding pocket comprises:

1 ) electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132 Cdc34 side chain and backbone NH of Gln49 Ub , (ii) the Glu133 Cdc34 side chain and side chains of Gln49 U , Arg42 Ub , and Arg72 U , and (iii) the Ser129 Cdc34 side chain and the side chains of Gln49 U and Arg72 U ; and

2) promotion of hydrophobic contacts between a ridge formed by Leu8 U , Ile44 ub , and Val70 Ub , and/or a complementary groove formed by Thr122 Cdc34 , Leu 125 Cdc34 , Ser126 Cdc34 , Ne128 Cdc34 and Ser129 Cdc34 . In some embodiments of the methods of inhibiting Cdc34 catalytic activity, the compound makes direct contact with the following amino acids: Gly47 Ub , Lys48 U , Glu51 Ub and Gln49 Ub . These methods may be useful in the treatmen of various diseases, including but not limited to a cancer, a neurological disorder, an immunological disorder, an infectious disease, a metabolic disorder such as diabetes or hemochromatosis, a genetic disorder such as CF or mental retardation, and/or a reproductive disorder such as infertility.

According to another aspect, the present invention relates to a method of inhibiting the activity of an E2 enzyme within the ubiquitin-proteasome system (UPS) of a subject in need thereof, the method comprising contacting said E2 enzyme with a compound stabilizing non-covalent ubiquitin-E2 complexes, wherein said stabilizing inhibits said E2 activity.

According to a another aspect, the present invention relates to a method of inhibiting proliferation and survival of a mammalian cell, the method comprising contacting the cell with a compound stabilizing non-covalent ubiquitin-E2 complexes within the cell, wherein the stabilizing inhibits proliferation and survival of the cell.

According to a another aspect, the present invention relates to a treatment method comprising administering to a subject in need thereof a compound which stabilizes non- covalent ubiquitin-E2 complexes within cells of the subject. In one embodiment, the compound is a compound that makes one or more direct contacts with ubiquitin at the following amino acids: Gly47 Ub , Lys48 U , Glu51 Ub and Gln49 Ub . According to another aspect, the present invention relates to a treatment method comprising administering to a subject in need thereof a compound which stabilizes non- covalent ubiquitin-Cdc34 complexes within cells of the subject. According to some embodiments, the compound is selected from the following compounds: - a compound by which stabilization of the non-covalent ubiquitin-Cdc34 complex is observable by crystallography or is observable by NMR using peak shifts and/or peak broadening of ubiquitin resonances, wherein one or more of the following ubiquitin residues are observed: Lys6, Thr7, Leu8, Gln40, Gln41 , Arg42, Leu43, Ile44, Phe45, Gly47, Lys48, Gln49, Leu50, Leu67, His68, Val70, Leu71 , Arg72 and Leu73;

- a compound by which stabilization of the non-covalent ubiquitin-Cdc34 complex is observable by crystallography or is observable by NMR using peak shifts and/or peak broadening of Cdc34 resonances, wherein one or more of the following Cdc34 residues are observed: Pro49, Asn50, Pro48, Thr51 , Glu26, Ile128, Leu125, Ser129, Asn132, Thr122, Ser126, Glu133, Leu130, Pro134, Asn135, Ser1 1 1 , Glu1 12, Leu109, Glu108.

According to another aspect, the present invention relates to a method of inhibiting cell proliferation in a subject comprising administering to the subject a compound which stabilizes non-covalent ubiquitin-Cdc34 complexes within cells of the subject. According to some embodiments, the compound is selected from the following compounds:

1 ) compounds that promote electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132 Cdc34 side chain and backbone NH of Gln49 U , (ii) the G | u 1 33 cdc34 sjde chain and sjde chains of Gln49 U , Arg42 Ub , and Arg72 Ub , and (iii) the Ser1 2g cdc34 sjde chain and the sjde chains of Gln49 U and Arg72 U ;

2) compounds that promote hydrophobic contacts between a ridge formed by Leu8 Ub , Ile44 ub , and Val70 U , and/or a complementary groove formed by Thr122 Cdc34 , Leu125 Cdc34 , Ser126 Cdc34 , lie I 28 cdc34 and Ser129 Cdc34 .

The present invention also concerns screening methods. According to a first aspect, the present invention relates to a screening method for identifying inhibitors of an E2 enzyme component of a mammalian cell ubiquitin-proteasome system (UPS). In one embodiment, the method comprises (a) measuring a binding affinity between ubiquitin and the E2 enzyme in the presence of a candidate inhibitor; and (b) identifying a candidate compound that an increase in said binding affinity identifies said candidate compound as a potential inhibitor of the E2 enzyme and/or as a potential inhibitor of another enzyme of the UPS.

According to one embodiment, the screening method is a TR-FRET assay which comprisesthe following steps:

- providing ubiquitin labeled with a first fluorophore;

- providing a E2 fusion protein fused to an antibody-recognizable tag;

- providing an antibody labelled with a second fluorophore recognizing said tag; - contacting the labeled ubiquitin, the E2 fusion protein and the antibody in the presence or in absence of a test compound; and

- measuring a TR-FRET binding signal in the presence and in the absence of the test compound;

wherein an increase in the TR-FRET binding signal in the presence of the test compound relative to the TR-FRET binding signal in the absence of the test compound is indicative of a potential inhibitory activity for the test compound.

According to another embodiment, the screening method is an on-bead assay comprising the following steps:

- providing compound-bearing beads comprising one or more test compound coupled to beads;

- providing a catalytic domain of the E2 enzyme (E2 cat );

- providing fluorescently-labeled ubiquitin;

- incubating the compound-bearing beads and the fluorescently-labeled ubiquitin in the presence or absence of the E2 protein under conditions allowing the formation of E2-ubiquitin complexes;

- measuring a fluorescence signal on the beads;

wherein a fluorescence signal more intense in presence of compound-bearing beads relative to control beads is indicative of a potential inhibitory activity for the test compound coupled to the bead.

The present invention also concerns in silico screening methods, including methods for for identifying inhibitors of a E2 enzyme component of the ubiquitin-proteasome system (UPS).

Other aspects the present invention concern a computationally generated three- dimensional structure of a three-dimensional model of an E2 enzyme in a binding interaction with ubiquitin, and methods for screening inhibitors of an E2 enzyme and methods of drug design or testing. In preferred embodiments, these aspects are based on the use of a three-dimensional model of an E2 enzyme in a binding interaction with ubiquitin, and more particularly a three-dimensional model which comprises particular electrostatic and H-bond interactions, hydrophobic contacts, pockets and other binding interactions and environments as defined herein. The screening methods described herein may be used to identify new active compounds (i.e. compound that were not known to be stabilize E2-ubiquiting interaction), to confirm activity, and/or to optimize (e.g. SAR) the activity of potentially useful compounds.

Additional aspects of the invention concerns screening methods (in vitro and in silico) using such isolated and purified E2-ubiquitin complex.

Additional features of the invention will be apparent from review of the disclosure, figures, and description of the invention below.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1. CC0651 potentiates the interaction between Cdc34 and 15 N-Ub. (A)

Superposition of the 1 H, 15 N-HSQC spectra of 15 N-Ub:Cdc34 cat (black) and 15 N- Ub:Cdc34 cat :CC0651 (grey) at the indicated mole ratios. Right inset, peak intensity change versus residue number for the compared HSQC spectra on the left. (B-E) Peak intensity change versus residue number for H, 5 N-HSQC spectra: (B) 15 N-Ub versus 15 N-Ub:Cdc34 cat . (C) 15 N-Ub versus 15 N-Ub:CC0651. (D) 15 N-Ub versus 15 N-Ub:Cdc34 FL . (E) 15 N-Ub:Cdc34 FL versus 15 N-Ub:Cdc34 FL :CC0651. (F-H) Interaction surfaces with labeled residues on ubiquitin for Cdc34 cat as induced by: (F) CC0651 , (G) disulfide tethered UBC1 (PDB identification code 1 FXT), and (H) Cdc34 FL . Interaction surfaces in (F) and (H) were obtained using the peak intensity change cut-off shown as dashed line in panels A and D.

FIGURE 2. CC0651 potentiates the interaction between 15 N-Cdc34 and ubiquitin. (A-

C) Superposition of H, 15 N-HSQC spectra at the indicated mole ratios for (A) 15 N- Cdc34 cat (black) versus 15 N-Cdc34 cat :CC0651 (grey), (B) 15 N-Cdc34 cat :Ub (black) versus 15 N-Cdc34 cat :Ub:CC0651 (grey), (C) 15 N-Cdc34 cat (black) versus 15 N-Cdc34 cat :Ub (grey). (D-E) Chemical shift perturbation for three representative resonances of (D) 15 N-Cdc34 cat or (E) 15 N-Cdc34 cat :Ub as a function of titrated CC0651. Calculated EC 50 value is the mean of the three displayed profiles (left panels) for which one representative resonance peak is shown (right panels). (F) CC0651 titration analysis of E2 enzyme binding to ubiquitin using a TR-FRET assay. Data presented as mean +/- S.E.M. n=2.

FIGURE 3. Crystal structure of a CC0651 -Cdc34-ubiquitin complex. (A) Ribbons (left) and slabbed surface (right) representation of the CC0651 -Cdc34-Ub complex with subunits labeled. (B) Stereo view of the Ub-Cdc34 binding interface. Contact residues are shown as sticks. More detailed views are shown Figure 6. (C) Reciprocal interaction surfaces on ubiquitin (left) and Cdc34 (right). Interaction residues on ubiquitin and on Cdc34 are labeled. Complementary interacting triad of hydrophobic residues on ubiquitin and pentad of residues on Cdc34 are demarcated by black boxes. Residues that form an inter-molecular salt bridge are indicated by circles. (D) Binding orientation of ubiquitin to Cdc34 induced by CC0651 is similar to previously characterized E2-ubiquitin and E2- SUMO interactions. Overlays of the CC0651 -Cdc34-ubiquitin complex with the indicated Ubc1 -Ub covalent complex (PDB code 1 FXT), UbcH5A/B-Ub complex (PDB ID code 4AP4; PDB ID code 4AUQ) and UbcH9-SUMO complex (PDB ID code 1Z5S) are shown. Superpositions were performed using the E2 coordinates. Rotation angles relate the orientations of ubiquitin and SUMO subunits. (E-F) Representative electron density maps. Unbiased electron density map of the CC0651 -Cdc34A-ubiquitin complex before inclusion of CC0651 coordinates into the refinement model (A). Shown superimposed are the 2Fo-Fc map surrounding the protein atoms of ubiquitin and Cdc34A (contoured at 1 .0σ) and the Fo-Fc map surrounding CC0651 (contoured at 2.5σ). Prime-and-switch generated electron density map of the CC0651 -Cdc34A-ubiquitin complex contoured at 1 .0σ (Β).

FIGURE 4. Mutations in Cdc34 that disrupt interaction with ubiquitin impair sensitivity to CC0651. (A) Peak intensity change versus residue number for the 1 H, 5N-HSQC spectra of 15 Nubiquitin: Cdc34cat versus 15 N-ubiquitin:Cdc34cat:CC0651 for wild type Cdc34 and the indicated mutants. (B) Sensitivity of the SCF Cdc4 Sid ubiquitination reaction to CC0651 using wild type Cdc34 and ubiquitin or the indicated point mutants.

FIGURE 5 shows the amino acid sequence of human Cdc34 (SEQ ID NO.: 1 ), the amino acid sequence of human ubiquitin (SEQ ID NO.:2), and the amino acid sequence of human Cdc34B (SEQ ID NO.:3). FIGURE 6. Notable contact features of the CC0651 -Cdc34-Ub complex. Stereo views are shown for (A) contacts between ubiquitin and CC0651 , (B) hydrogen bonding network between ubiquitin and Cdc34 and (C) hydrophobic contacts between ubiquitin and Cdc34.

FIGURE 7. Structure-based sequence alignments for human E2 enzymes and for ubiquitin. (A) Residue numbers and secondary structure elements for human Cdc34 are indicated above the aligned sequences. A region of disorder in the Cdc34 structure is indicated by a dashed line. Cdc34 residues comprising the ubiquitin contact interface are shaded. Residues that contact CC0651 are highlighted by stars. (B) Residue numbers and secondary structure elements for human ubiquitin are indicated above the aligned sequences of wild type ubiquitin and SUMO-1. Residues that contact Cdc34 are shaded. Residues that contact CC0651 are highlighted by stars.

FIGURE 8. (A) E2 enzyme mediated transfer of a cysteine-linked donor Ub to the lysine residue of an acceptor ubiquitin. Weak affinity sites, called the donor site and the acceptor site, engage and orient the donor and acceptor Ub, respectively. CC0651 stabilizes the interaction between the donor site on Cdc34A and ubiquitin. (B) SCF architecture and reaction mechanics. E1 enzyme activates and transfers Ub to the catalytic cysteine of the Cdc34 E2 enzyme. The charged Cdc34 is positioned by the SCF E3 complex to conjugate Ub to a substrate lysine or to lysine 48 of a previously conjugated Ub moiety. Conjugation of Nedd8 to the cullin is required for SCF activation, and is mediated in part by Dcn1 and Ubc12 and reversed by the COP9/Signalosome. (C) Neddylation of the cullin releases the Rbx1 RING domain on a flexible tether to allow charged E2 to access the bound substrate. A similar Cullin-RING architecture as in SCF complexes underpins other related Cullin-RING ligases (CRLs) that are based on other cullin homologs, all of which use Cdc34 as the preferred E2 enzyme.

FIGURE 9. Expression of CRL network components in human cancer. (A)

Expression heatmap for indicated genes in various human cancer tissue types from Genevestigator V3 database using Human133_2: Human Genome 47k array data. Note that available probes for CUL3, CUL4B and SKP2 were non-single gene targets. (B) Percentage of cancer tissue samples showing positive staining for the indicated proteins, derived from Human Protein Atlas Database. (C) CDC34A and CDC34B EST expression in normal human tissue, derived from UniGene EST Profile Viewer. FIGURE 10. Assays for in vitro characterization of Cdc34 inhibitor analogs. (A)

Schematic of FRET assay. (B) FRET signal upon titration of CC0651 with Cdc34A (Ube2R1 ) or Cdc34B (Ube2R2). (C) Ubiquitination assay using fluorescently-labeled Ub, visualized at 473 nm with a Typhoon imager. (D) quantification of poly-Ub in (C) normalized to DMSO only controls. FIGURE 11. Conservation of the donor ubiquitin interaction surface on E2 enzymes. (A) Superposition of four E2 enzyme structures coloured according to grey scale labeling below. (B) Surface representation of Cdc34A with bound CC0651 shown in sticks and bound ubiquitin shown as black C-alpha trace. Cdc34A surface coloured in white with ubiquitin contact residues coloured black and grey for weakly conserved (<50% identity) and strongly conserved (>50% identity) nature across the E2 family.

FIGURE 12. Structures and activity of SAR derivatives of CC0651. (A-C) Chemical structures (top) and activities of the indicated derivatives were measured in a Sid -

Cdc4

SCF ubiquitination assay (middle) and a Cdc34A-ubiquitin TR-FRET binding assay (bottom) according to Example 1 . Data is represented as the mean ± s.e.m., n = 2. Bn, benzyl moiety.

Figure 13. The CC0651 binding pocket. CC0651 is displayed in thick dark gray sticks. Amino acid residues of Cdc34 and ubiquitin enclosing the binding site are shown as thin sticks, with Cdc34 residues in light grey and ubiquitin residues in dark grey. Ubiquitin residue numbers are labeled with the prefix "Ubi", while Cdc34 residues numbers are labeled without a prefix.

Figure 14. Superposition and binding energies. (A) Superposition of CC0651 as observed in the Cdc34-ubiquitin-CC0651 crystal structure (dark grey thick sticks) with two docked CC0651 conformers (light grey thin sticks). (B) Superposition of CC0651 (dark grey thick sticks) with the non-binding compound #5 from Figure 12. (C) Predicted binding energies (in kcal/mol) as calculated by the in silico-docking algorithm of highest scoring conformers of compounds shown in Figure 12. Higher binding energies indicate a predicted higher affinity interaction.

DETAILED DESCRIPTION OF THE INVENTION A) General overview of the invention

The present invention stems from the analysis by the present inventors of the mechanism of action of a small molecule called CC0651 , which was reported as the first inhibitor of an E2 enzyme (Ceccarelli et al 201 1 , Cell 145: 1075). Using NMR and X-ray crystallographic approaches, the present application shows that CC0651 acts as a "ubiquitin trap" that stabilizes the critical non-covalent donor ubiquitin interaction with the E2. The data presented in the Examples provides a snapshot of this catalytic intermediate, which has previously been refractive to visualization at atomic resolution by X-ray crystallographic methods, and demonstrates how the ternary Cdc34- ubiquitin-CC0651 complex interferes with the ubiquitination reaction. These findings demonstrate a means to develop novel mechanism-based inhibitors of many if not all E2 enzymes, and suggest more generally that many other weak non-covalent ubiquitin interfaces in the UPS may be targeted for stabilization by small molecules in an analogous fashion.

B) Definitions

For the purpose of the present invention the following terms are defined below. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a compound" includes one or more of such compounds and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

As used herein the term "subject" includes living organisms with cells in which there is a ubiquitin-proteasome system (UPS). The term "subject" includes animals (e.g., mammals, e.g., cats, dogs, horses, pigs, cows, goats, sheep, rodents, e.g., mice or rats, rabbits, squirrels, bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans)), as well as and transgenic species thereof. Preferably, the subject is a mammal. More preferably, the subject is a human, such as a human patient in need of treatment.

"Ubiquitin-proteasome system" or "UPS" as used herein refers to the cellular quality control system that controls the stability, interactions and localization of many thousands protein across virtually all cellular processes. The UPS governs the dynamics of the proteome via ubiquitin and cascade of involving E1→E2→E3 and enzymes, as well as deubiqutinating enzymes (DUBs), ubiquitin binding domains (UBDs) and proteasome subunits. The inventors estimate that there are about 1232 known UPS genes in humans.

"E2 enzyme" or "E2" as used herein this term refers to the enzymes that lie at a crucial nexus in the UPS hierarchy, between the E1 ubiquitin activating enzymes and the E3 ubiquitin ligases. In the UPS, E2 enzymes (E2s) mediate the conjugation of ubiquitin to substrates either directly or indirectly and thereby control protein stability and interactions. There are about 38 known human E2s. Table 1 hereinafter provides some information about each one of them.

Table 1 : Human E2 enzymes

21 M1 P61081 Ubc12 NEDD8

22 N1 P61088 Ubc13 Cell cycle

DNA repair

23 NL Q5JXB2

24 0 Q9C0C9 E2-230K

25 Q1 Q7Z7E8 Embryonic

development

26 Q2 Q8VWN8 Cell cycle Breast cancer

apoptosis Squamous cell carcinoma

27 R1 P49427 CDC34 Cell cycle Acute lymphoblastic

leukaemia

Herpes virus infection

28 R2 Q712K3 CDC34b Cell cycle

29 S1 Q16763 E2EPF Cell cycle Esophageal squamous cell carcinoma

30 T1 Q9NPD8 DNA repair Breast, lung cancer

Fanconi anemia

31 U Q5WX9

32 V1 Q13404 apoptosis

33 V2 Q15819 DNA repair

Cell cycle

Differentiation

34 V3 Q8IX04 UELVD Colon cancer

35 W1 Q96B02

36 Z Q9H832 Apoptosis

37 AKTIP/FHS Vesicle trafficking

Q9H8T0 Apoptosis

38 BIRC6 Q9NR09 Apollon Cell cycle

apoptosis

"Cdc34" or "Ube2R1 " or "Ubiquitin-conjugating enzyme E2 R1" or "Cdc34A" is the principal E2 enzyme for the CRL enzymes. The amino acid sequence (236 residues) of human Cdc34 is provided in Figure 5 (SEQ ID NO: 1 ) and this sequence can be found in the UniProt™ database under accession number P49427 or NPJD04350 (gene sequence is available at the NCBI database under accession number NM_004359.1 ). Reference in this application to various positions of the Cdc34 protein correlates with the positions of the amino acids set forth in SEQ ID NO: 1 . A highly similar isoform of Cdc34, referred to as "Cdc34B" or "Ube2R2" also operates in conjunction with the CRL enzymes, particularly in tissues where it is more highly expressed that Cdc34A (see for instance Figure 9C for Cdc34A vs Cdc34B expression). Cdc34B shares 80% identity with Cdc34A. The amino acid sequence (238 residues) of human Cdc34B is provided in Figure 5 (SEQ ID NO: 3) and this sequence can be found in the UniProt™ database under accession number Q712K3 or NP_060281 .2 (gene sequence is available at the NCBI database under accession number BC004862). According to some embodiments, Cdc34B may be used instead of Cdc34 for inhibition, treatment and screening applications. In some methods, for instance in screening, Cdc34B may be used before or after Cdc34.

Although extensive reference is made herein to Cdc34, those skilled in the art will understand that the present invention (compounds, screening methods, etc.) is not limited to Cdc34 only. Because of the conservation of the ubiquitin binding surface (in topology if not entirely in precise primary amino acid sequence) on multiple human E2s, it may be possible to use any other E2 enzyme according to the invention. For instance, compounds of the invention may allow specific stabilization of a non-covalent complex between ubiquitin and another specific E2 enzyme that is not Cdc34, and this stabilization may be observable by crystallography or may be observable by NMR using peak shifts and/or peak broadening of E2 resonances using the same residues on ubiquitin and one or more of conserved residues on the E2 enzyme (i.e. E2 residues that are conserved with analogous residues on Cdc34). For instance, those skilled in the art can readily identify amino acid residues of other E2s equivalent to particular residues in Cdc34 by using the sequence alignment provided in Figure 7. The compounds of the invention may also promote analogous interactions between the same residues in ubiquitin and analogous conserved residues on E2 enzymes other than Cdc34.

"Ubiquitin" refers to the small regulatory protein, which by virtue of covalent attachment to substrate proteins, is involved in directing proteins to compartments in the cell, including the 26S proteasome which destroys and recycles proteins, and/or in controlling different enzyme activities and protein localizations. The amino acid sequence of human ubiquitin is provided in Figure 5 (SEQ ID NO: 2) and this sequence can be found in the UniProt™ database under accession number P0CG47 (UBB) or P0CG48 NP_066289.2 (UBC) (gene sequence is available at the NCBI database under accession number NM_021009.5). Reference in this application to various positions of the ubiquitin protein correlates with the positions of the amino acids set forth in SEQ ID NO: 2.

As used herein, an "inhibitor" or a "compound stabilizing non-covalent ubiquitin-E2 complexes" refers to any compound or inhibitor capable of binding selectively to a complex formed by the non-covalent and typically very low affinity association of an E2 enzyme and ubiquitin, the compound being capable of stabilizing or trapping the binding of E2 and ubiquitin. Although the invention is mostly concerned with "non-colalent" ubiquitin-E2 binding, the above expression encompasses other types of E2-ubiquitin complexes where both proteins are connected, for instance by a labile thioester bond between the C-terminus of ubiquitin and the catalytic cysteine (residue 92) of Cdc34. As such, as used herein, reference to "stabilizing non-covalent ubiquitin-E2 complexes" should not be interpreted as limited to free ubiquitin and E2 enzyme in solution. In some embodiments, the binding of the compound to the ubiquitin-E2 complex increases the binding affinity between E2 and ubiquitin, thereby solidifying that binding interaction to the point where it is detectable by biophysical and enzymatic methods. Preferably, the compound binds simultaneously to E2 and to ubiquitin at an interface between both proteins, which takes the form of a binding pocket for the compound. In some embodiments, this interface on the E2 is called the donor ubiquitin binding surface, and on ubiquitin it is called the Ile44 hydrophobic surface. In some embodiments, the compound binds simultaneously to the E2 enzyme Cdc34 and ubiquitin at an interface between Cdc34 and ubiquitin. In some embodiments, the compound binds simultaneously to an E2 enzyme that is not Cdc34 and ubiquitin at an interface or pocket between the E2 and ubiquitin. In particular embodiments, the E2 enzyme that is not Cdc34 is to be selected from the E2s listed in Table 1 and/or the E2s listed in Figure 7. In some embodiments, the compound or inhibitor makes direct contact with human ubiquitin comprising SEQ ID NO: 2 at the following amino acids: Gly47 Ub , Lys48 U , Glu51 Ub and Gln49 U . In some embodiments, the inhibitor comprises a chemical structure that (i) fits within a binding pocket formed by a non-covalent E2-ubiquitin-complex and (ii) stabilizes a E2-ubiquitin interaction. In some embodiments the E2 is human Cdc34 and the binding pocket is as defined herein.

C) Three-dimensional structure of ubiquitin-E2 complexes

The inventors are the first to caracterized the stabilization the non-covalent donor ubiquitin interaction with the E2 and to have revealed such stabilization using NMR and X-ray crystallographic approaches. The stabilization was observed in the presence of a small molecule (CC0651 ), which binds at an interface between the E2 enzyme and ubiquitin.

E2 enzymes lie at a crucial nexus in the ubiquitin-proteasome system (UPS) hierarchy as they exhibit specific interactions with E1 enzymes, E3 enzymes, deubitquinating enzymes and substrates. Because E2 enzymes are crucial for the UPS, selective stabilization of E2 enzyme-ubiquitin complexes opens new avenues of prevention and treatment of various diseases because inhibition of E2 activities may be desirable for the treatment of many UPS-related diseases, including but not limited to treating a cancer, a neurological disorder, an immunological disorder, an infectious disease, a metabolic disorder such as diabetes or hemochromatosis, a genetic disorder such as cystic fibrosis or mental retardation and/or a reproductive disorder such as infertility. Accordingly, the invention encompasses E2 inhibitors, screening methods, methods for identifying, testing or optimizing therapeutic compounds, treatment methods using such compouds, methods of uses for inhibiting the activity of E2 enzymes, including those E2s listed in Table 1. A subgroup of E2 enzyme according to the invention are E2 enzymes interacting with cullin-RING ligases (CRLs) E3 enzymes (Figure 8). One particular E2 enzyme is Cdc34 (Ube2R1 ). Notably, Cdc34 and/or other CRL components are often differentially expressed at the mRNA or protein level in different cancer types (Fig. 9A- C). Accordingly, the invention encompasses methods of inhibiting proliferation and survival of mammalian cells. Examples of cancers that may be treated according to the invention include, but is not limited to, adipose, adrenal, bladder, blood/lymphatic, bone, bone marrow, brain, cervix, connective, ear, eye, head and neck, heart, intestine, kidney, larynx, liver, lung, lymph, lymph node, mammary, mouth, muscle, nerve, ovary, pancreas, pharynx, pituitary, placenta, prostate, salivary, skin, spleen, stomach, testis, thyroid, tonsil, uterus, and vascular cancers.

Related aspects concerns treatment methods which comprises administering to a subject in need thereof a compound which stabilizes non-covalent ubiquitin-E2 complexes within cells of the subject and/or a compound which stabilizes non-covalent ubiquitin-Cdc34 complexes within cells of the subject.

In one embodiment, the method comprises contacting the E2 enzyme with a compound stabilizing non-covalent ubiquitin-E2 complexes, wherein the stabilized ubiquitin complex inhibits the E2 activity. In some embodiments, the compound binds at an interface between the E2 enzyme and ubiquitin. In some embodiments stabilisation occurs because the compound acts as a molecular bridge between E2 enzyme and ubiquitin. In some embodiments stabilisation occurs because the compound increases the binding affinity between the E2 enzyme and ubiquitin. In some embodiments, the binding affinity between the E2 enzyme and ubiquitin is increased by at least 5, 10, 15, 20, 25 fold or more in presence of the compound, as evidenced by measurements of EC50, IC50 or Kd values with or without the compound. This principle is illustrated by the CC0651 derivatives shown in Figure 12, which demonstrate a correlation between stabilization of the Cdc34-ubiquitin interaction and inhibition of Cdc34 catalytic activity.

The stabilization of the ubiquitin-E2 complex in presence of the compound may be observable by crystallography, or by NMR since the compound-induced interaction between E2 (e.g. Cdc34) and labeled ubiquitin (e.g. 15 N-ubiquitin) will be reflected in part by the specific resonance peaks of the labeled ubiquitin molecule affected. According to some embodiments, the stabilizing is observable by crystallography or is observable by NMR using peak shifts and/or peak broadening of ubiquitin resonances, wherein one or more of the following ubiquitin contact residues are observed: Lys6, Thr7, Leu8, Gln40, Gln41 , Arg42, Leu43, Ile44, Phe45, Gly47, Lys48, Gln49, Leu50, Leu67, His68, Val70, Leu71 , Arg72 and Leu73. According to some embodiments the E2 enzyme is Cdc34 and the stabilizing is observable by crystallography or is observable by NMR using peak shifts and/or peak broadening of Cdc34 resonances, wherein one or more of the following Cdc34 residues are observed: Pro49, Asn50, Pro48, Thr51 , Glu26, Ile128, Leu125, Ser129, Asn132, Thr122, Ser126, Glu133, Leu130, Pro134, Asn135, Ser1 1 1 , Glu1 12, Leu109, Glu108.

In some embodiments, the E2 enzyme is human Cdc34 and the compound promotes electrostatic and H-bond interactions selected from: (i) Asn132 Cdc34 side chain and backbone NH of Gln49 Ub , (ii) the Glu133 Cdc34 side chain and side chains of Gln49 Ub , Arg42 Ub , and Arg72 Ub , and (iii) the Ser129 Cdc34 side chain and the side chains of Gln49 Ub and Arg72 Ub . In some embodiments, the E2 enzyme is human Cdc34 and the compound promotes hydrophobic contacts between a ridge formed by Leu8 U , Ile44 u , and Val70 U , and/or a complementary groove formed by Thr122 Cdo34 , Leu 125 Cdc34 , Ser 26 Cdc34 , lie 28 Cdc3 and Ser129 Cdc34 .

According to some embodiments of the therapeutic or treatment methods of the invention, the compound is used in combination with one or more of the following inhibitors: MLN4924 (by Millenium Pharmaceuticals), Bortezomib (Velcade™), Carfilzomib (by Onyx Pharmaceuticals). In some embodiments, the compound or inhibitor is any suitable compound, other than CC0651 , CC7094, CC9933, CC0040, CC9653, CC9807, CC9652, CC8993, CC9430, CC9504 CC9535, CC9566, CC9833. The chemical structure of these compounds can be found in Ceccarelli et al. Cell (201 1 ), 145, 1075-1087.

As used herein, the terms "treatment" or "treating" of a subject includes the application or administration of a suitable compound, or composition of the invention as defined herein to a subject (or application or administration of a compound or composition of the invention to a cell or tissue from a subject) with the purpose of delaying, stabilizing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indicia of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, slowing disease progression or severity, stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, or improving a subject's physical or mental well-being. In some embodiments, the term "treating" can include increasing a subject's life expectancy and/or delay before additional treatments are required (e.g. joint replacement surgery). D) Screening methods and assays

Knowledge that is possible to inhibit activity of the E2 enzyme Cdc34A by stabilizing the critical non-covalent donor ubiquitin interaction also opens new avenues for screening therapeutic inhibitors of other E2s and also other enzymes of the ubiquitin-proteasome system (UPS). That knowledge also opens avenues for screening methods based on the catalytic domain of Cdc34 and catalytic domain of other E2 enzymes. Accordingly, an aspect of the invention concerns a screening method for identifying inhibitors of any E2 enzyme in the mammalian ubiquitin-proteasome system (UPS). In one embodiment the method comprises measuring a binding affinity between ubiquitin and the E2 enzyme in the presence of a candidate inhibitor, wherein an increase in the binding affinity identifies the candidate compound as a potential inhibitor of the E2 enzyme and/or as a potential inhibitor of another enzyme of the UPS.

Various screening methods, techniques and assays may be used according to the invention. Examples includes, but are not limited to, NMR assay, TR-FRET assay, yeast two-hybrid, bead-based protein capture with fluorescent proteins, ITC, Fortebio™, Biacore™, AlphaScreen®, microscale thermophoresis, mass spectrometry based assays, speed screen assay, single molecule spectroscopy assay, in silico structure- guided screens, proximity scintillation assays (SPA), and BRET assays.

The increase in the binding affinity of the ubiquitin-E2 complex in presence of the compound may be directly observable by crystallography or by NMR. In some embodiments measuring a binding affinity comprises observing by NMR peak shifts and/or peak broadening of ubiquitin resonances in the presence of an E2, corresponding to one or more of the following ubiquitin residues: Lys6, Thr7, Leu8, Gln40, Gln41 , Arg42, Leu43, Ile44, Phe45, Gly47, Lys48, Gln49, Leu50, Leu67, His68, Val70, Leu71 , Arg72 and Leu73. In some embodiments, the E2 enzyme is selected from E2 enzymes listed in Table 1. In some embodiments, the E2 enzyme is human Cdc34 and measuring a binding affinity comprises observing by NMR peak shifts and/or peak broadening of Cdc34 resonances corresponding to one or more of the following Cdc34 residues: Pro49, Asn50, Pro48, Thr51 , Glu26, Ile128, Leu125, Ser129, Asn132, Thr122, Ser126, Glu133, Leu130, Pro134, Asn135, Ser1 1 1 , Glu1 12, Leu109, Glu108. In some embodiments, resonance shifts may correspond to conserved residues in other E2 enzymes, including but not limited to those illustrated in Figure 7.

According to a particular embodiment, the E2 enzyme is Cdc34 and the assay is an NMR assay which comprises the following steps:

- providing labeled ubiquitin (for instance ubiquitin labeled with 15 N-amonium supplemented bacteria culture media);

- providing a catalytic domain of the E2 enzyme (E2 cat );

- acquiring a first Heteronuclear Single Quantum Coherence (HSQC) reference spectra of the labeled ubiquitin in the presence of the E2 cat [This spectrum would correspond to a non-interacting state between said proteins];

- acquiring a first HSQC spectrum in presence of a test compound;

- detecting chemical shift perturbations or chemical shift intensity changes between the first and second HSQC spectrum;

wherein promotion of molecular interaction between the labeled ubiquitin and

E2 cat is indicative of a potential inhibitory activity for the test compound.

According to a particular embodiment of that method the first HSQC spectrum is carried out in presence of a plurality of test compounds and the method further comprises the step of deconvoluting the chemical shift perturbations or chemical shift intensity changes to identify specific compound(s) responsible for said shifts.

According to a particular embodiment, the labeled ubiquitin is 15 N-ubiquitin.

According to a particular embodiment the E2 enzyme comprises amino acids as defined in Figure 7 for human E2s, and wherein the E2 cat comprises amino acid residues equivalent to Pro7 to Val184 of Cdc34 as defined in the sequence alignment of Figure 7. According to a more particular embodiment, the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , the E2 cat is the catalytic domain of Cdc34 (Cdc34 cat ), and Cdc34 cat comprises residues Pro7 to Val184 of SEQ ID NO: 1. According to another particular embodiment, the assay is a TR-FRET assay which comprises the following steps:

- providing ubiquitin labeled with a first fluorophore (for instance ubiquitin labeled with fluorescein or a similar probe with similar excitation and emission spectra); - providing a, E2 fusion protein comprising an antibody-recognizable tag (e.g. an E2 fusion protein with a poly histidine tag that is recognizable by the Terbium anti-his antibody commercialized by Invitrogen™);

- providing an antibody labelled with a second fluorophore (e.g. Terbium, Europium) recognizing said tag (e.g. Terbium anti-his antibody);

- contacting the labeled ubiquitin, the E2 fusion protein and the antibody in the presence or in absence of a test compound; and

- measuring a TR-FRET binding signal in the presence and in the absence of a test compound;

wherein an increase in the TR-FRET binding signal in the presence of the test compound relative to the TR-FRET binding signal in the absence of the test compound is indicative of a potential inhibitory activity for the test compound.

According to a particular embodiment, that method further comprises a preliminary optimization step of measuring a TR-FRET binding signal in the presence of saturating levels (e.g. 100 micromolar) of compound CC0651 as a positive binding control.

According to another particular embodiment, the assay is an on-bead assay which comprises the following steps:

- providing compound-bearing beads comprising one or more test compound coupled to beads [e.g. a diverse library of chemical molecules coupled to microbeads through a PEG linker or any other type of linker) and control beads

[e.g. non-coupled beads or beads coupled to a non-inhibitor];

- providing a catalytic domain of the E2 enzyme (E2 cat );

- providing fluorescently-labeled ubiquitin [e.g. recombinant ubiquitin bearing a non- native cysteine residue at its N-terminus which has been derivatized with fluorescein iodoacetamide];

- incubating the compound-bearing beads and the fluorescently-labeled ubiquitin in the presence or absence of the E2 protein under conditions allowing the formation of E2-ubiquitin complexes [rationale: the E2-ubiquitin complexes will be captured by the compound-bearing beads by virtue of the test compound on the bead) (e.g. incubation of the three components in either a crude cell lysate or buffer solution with purified ubiquitin and E2];

- measuring a fluorescence signal on the beads [e.g. as a ring of fluorescence around the bead perimeter due to capture of the fluorescent ubiquitin];

wherein a fluorescence signal more intense in presence of compound-bearing beads relative to control beads is indicative of a potential inhibitory activity for the test compound coupled to the bead.

According to a particular embodiment of the on-bead assay, the E2 enzyme comprises amino acids as defined in Figure 7 for human E2s, and the E2 cat comprises amino acid residues equivalent Pro7 to Val184 of Cdc34 as per sequence alignment of Figure 7. According to a more particular embodiment, the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , the E2 cat is the catalytic domain of Cdc34 (Cdc34 cat ), and the Cdc34 cat comprises residues Pro7 to Val184 of SEQ ID NO: 1. In one particular embodiment, the fluorescently-labeled ubiquitin is a fluorescein-labeled human recombinant ubiquitin. Many differetent types of beads may be used according to the invention. The compound- bearing beads may comprise chemical molecules coupled to the beads through a chemically compatible linker (e.g. PEG). The beads may be microbeads such such as Tentagel™ beads having a diameter of about 100 μηη, or any other bead format compatible with generation of a combinatorial library. The control beads may be non- coupled beads (i.e. no chemical compound) or beads coupled to a non-inhibitor compound.

The compound-bearing beads may be beads obtained from a combinatorial one bead- one compound (OBOC) library.

In other embodiments, the analogous NMR or TR-FRET assays are carried out in a similar manner but with any one of the E2 enzymes listed in Table 1.

E) In silico screening

Knowledge of the 3D structures of an E2 enzyme in a binding interaction with ubiquitin, with or without an inhibitor bound to such E2-ubiquitin complex, opens new avenues for screening in silico additional inhibitors, especially therapeutically useful E2 inhibitors as well as therapeutic inhibitors of other enzymes of the ubiquitin-proteasome system (UPS). Accordingly, the invention further relates to a screening method for identifying inhibitors of an E2 enzyme component of the ubiquitin-proteasome system (UPS) of a subject, comprising:

(a) providing a three-dimensional model of an E2 enzyme in a binding interaction with ubiquitin;

(b) in silico docking a three-dimensional structure of test compounds at an interface between the E2 enzyme and ubiquitin;

(c) selecting test compounds from step (b) increasing binding affinity of the E2 enzyme with ubiquitin;

(d) identifying selected test compounds as potential inhibitors of the E2 enzyme and/or as potential inhibitors of another enzyme part of the UPS.

In one embodiment, the selecting step comprises identifying ligand compounds having the highest docking score. In one embodiment, the ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and the in silico docking comprises in silico docking at the following position: Gly47 Ub , Lys48 Ub , Glu51 Ub and Gln49 Ub . In one embodiment the in silico docking comprises in silico docking at a pocket enclosed by one or more of Gly47 Ub , Lys48 Ub , Glu51 Ub and Gln49 Ub . In some embodiments the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and the in silico docking comprises in silico docking at a site that involves at least one of the following positions:

- a pocket on Cdc34 formed by α1-β1 linker, C-terminal end of helix α2, β2-β3 linker, C-terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162.

In other embodiments the E2 enzyme is an E2 enzyme other than Cdc34 and in silico docking is performed in a pocket enclosed by one or more side chain and/or backbone atoms of ubiquitin residues Gly47, Lys48, Glu49, Glu51 , Arg54 and one or more side chain and/or backbone atoms equivalent to Cdc34 residues Gly27, Phe28, Ile45, Phe46, Pro48, Tyr52, Tyr53, Phe58, Phe77, Met81 , His83, Ile128, Leu131 , Asn132, Tyr148, Trp151 , Tyr161 , Thr162, Ile165 or other residues structurally equivalent to any other Cdc34 residues described above. Amino acid residues of other E2s equivalent to residues in Cdc34 can be identified using a sigle or multiple sequence alignment (e.g. the multiple sequence alignment of Figure 7) or a structure based alignment (e.g. the structure based alignment of Figure 11). Suitable 3D models for in silico docking can be obtained by X-ray crystallography or by homology based modeling.

In most cases, 3D models of other E2s obtained by X-ray crystallography (with or without in complex with ubiquitin) in the absence of an inhibitor similar to CC0651 will not have a pocket large enough to fit a drug like inhibitor and thus be of limited use for in silico modeling. Molecular dynamics simulations of X-ray crystal structures of E2s and E2s followed by structural clustering of the trajectory can identify conformations of the E2 suitable for in silico docking. In the absence of suitable X-ray crystal structures or of E2 structures not in complex with ubiquitin, suitable 3D models can be obtained using homology based modeling. In particular, the X-ray crystal structure of Cdc34 in complex with Ubiquitin and CC0651 provided herein (c.f. PDB ID 4MDK as defined hereinafter) can be used as a template for homology based modeling of other E2-ubiquitin complexes as it has the advantage that these other E2s will then be also modeled with the "open pocket" conformation suitable for in silico docking. These modeled conformation can subsequently also be refined by molecular dynamics simulations if necessary.

In some embodiments the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , the ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and selecting test compounds increasing binding affinity of human Cdc34 with ubiquitin comprises assessing one or more of the following interactions:

1 ) promotion of electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132 Cdc34 side chain and backbone NH of Gln49 Ub , (ii) the Glu133 Cdc34 side chain and side chains of Gln49 Ub , Arg42 Ub , and Arg72 Ub , and (iii) the Ser129 Cdc34 side chain and the side chains of Gln49 U and Arg72 U ; and/or

2) promotion of hydrophobic contacts between a ridge formed by Leu8 Ub , Ile44 ub , and Val70 Ub , and/or a complementary groove formed by Thr122 Cdc34 , Leu125 Cdc34 , Ser126 Cdc34 , Ile128 cdc34 and Ser129 Cdc34 .

According to a particular embodiment, the three-dimensional model at step (a) comprises 3D coordinates as definedin PDB ID 4MDK.

The invention further relates to a method for screening inhibitors of an E2 enzyme component of the mammalian cell ubiquitin-proteasome system (UPS), comprising the following steps: a) computationally generating a three-dimensional structure of the E2 enzyme in a binding interaction with ubiquitin and computationally generating a three dimensional molecular representation of test compounds;

b) virtual screening of a plurality of test compounds through molecule docking to obtain candidate inhibitors having a minimum docking affinity at an interface between the

E2 enzyme and ubiquitin, wherein the docking increases binding affinity of the E2 enzyme with ubiquitin; and

c) testing the candidate inhibitors for in vitro, ex vivo and/or in vivo activity in inhibiting the E2 enzyme and/or inhibiting other enzymes involved in the UPS. According to a particular embodiment, the ubiquitin is human ubiquitin comprising SEQ ID NO: 2, and the virtual screening comprises assessing a minimum docking affinity of test compounds at an interface between the E2 enzyme and ubiquitin comprising Gly47 U , Lys48 Ub , Glu51 Ub and Gln49 Ub .

According to another particular embodiment, the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and the virtual screening comprises assessing a minimum docking affinity of test compounds at an interface between the E2 enzyme and ubiquitin comprising:

- a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix α2, β2-β3 linker, C terminal end of helix a3 and α3-α4 linker;

- a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

- a mixed polar and hydrophobic environment on Cdc34 created by Asn50, Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162.

According to another particular embodiment, the ubiquitin is human ubiquitin comprising SEQ ID NO: 2, the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1 , and the three dimensional molecular structure of the E2 enzyme in a binding interaction with ubiquitin comprises 3D coordinates as defined in PDB ID 4MDK.

The invention further relates to a method of drug design or drug testing, comprising:

A) uploading in a computer system structural coordinates of a three-dimensional model of human Cdc34 comprising SEQ ID NO: 1 in a binding interaction with human ubiquitin, the three-dimensional model comprising one or more of the followings:

1 ) electrostatic and H-bond interactions selected from the group consisting of:

(i) Asn132 Cdc34 side chain and backbone NH of Gln49 U , (ii) the Glu133 Cdc34 side chain and side chains of Gln49 U , Arg42 U , and Arg72 Ub , and (iii) the Ser129 Cdc34 side chain and the side chains of Gln49 U and Arg72 U ;

2) hydrophobic contacts between a ridge formed by Leu8 Ub , lle44 U , and Val70 U , and/or a complementary groove formed by Thr122 Cdc34 , Leu125 C dc34 , Ser126 Cdc34 , lie 128 Cdc34 and Ser129 Cdc3 ;

3) a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix a 2, β2-β3 linker, C-terminal end of helix a3 and a3- a4 linker;

4) a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58,

Phe77, Met81 , Ile 28, Leu131 and Ile165;

5) a mixed polar and hydrophobic environment on Cdc34 created by Asn50,

Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162;

6) a pocket surface contributed by ubiquitin lined by Gly47 U , Lys48 Ub , Glu51 Ub and Gln49 U ; and

B) virtually screening a plurality of test compounds through molecule docking to obtain candidate inhibitors having a minimum docking affinity at an interface between Cdc34 and ubiquitin;

wherein compounds having a minimum docking affinity are potential candidates for drug design and/or drug testing.

In one embodiment, the method further comprises the step of testing the potential candidates for in vitro, ex vivo and/or in vivo activity in inhibiting human Cdc34 and/or inhibiting other human enzymes involved in the Ubiquitin-proteasome system (UPS). In one particular embodiment, the structural coordinates of human ubiquitin and human Cdc34 are as defined in PDB ID 4MDK.

The invention further relates to a computationally generated three-dimensional model of human Cdc34 comprising SEQ ID NO: 1 in a binding interaction with human ubiquitin comprising SEQ ID NO: 2. In one embodiment the three-dimensional model comprises one or more of the followings:

1 ) electrostatic and H-bond interactions selected from the group consisting of: (i) Asn132 Cdc34 side chain and backbone NH of Gln49 U , (ii) the Glu133 Cdc34 side chain and side chains of Gln49 U , Arg42 Ub , and Arg72 U , and (iii) the Ser129 Cdc34 side chain and the side chains of Gln49 U and Arg72 Ub ;

2) hydrophobic contacts between a ridge formed by Leu8 U , Ile44 u , and Val70 U , and/or a complementary groove formed by Thr122 Cdo34 , Leu125 Cdc34 , Ser126 Cdc34 , lie

1 2 g Cdc34 Se r1 29 Cdc34 3) a pocket on Cdc34 formed by α1 -β1 linker, C-terminal end of helix a 2, β2-β3 linker, C-terminal end of helix a3 and a3- a4 linker;

4) a hydrophobic pocket on Cdc34 lined by Phe28, Ile45, Pro48, Tyr53, Phe58, Phe77, Met81 , Ile128, Leu131 and Ile165;

5) a mixed polar and hydrophobic environment on Cdc34 created by Asn50,

Tyr52, Pro134, Tyr148, Trp151 , Tyr161 , and Thr162; and

6) a pocket surface contributed by ubiquitin lined by Gly47 Ub , Lys48 U , Glu51 Ub and Gln49 Ub . The invention further relates to a computer-readable data storage medium comprising a data storage material encoded with the computationally generated three-dimensional model defined above. The invention further relates to a computer system comprising: a representation of the computationally generated three-dimensional model defined above; and a user interface to view the representation. In one particular embodiment, the three- dimensional model comprises structural coordinates of human ubiquitin and human Cdc34 are as defined in PDB ID 4MDK.

The invention further relates to a computer-assisted method for identifying inhibitors of an E2 enzyme part of a mammalian cell ubiquitin-proteasome system (UPS), comprising the following steps:

- loading into a computer's memory a first set of data corresponding to the three- dimensional model of the E2 enzyme in a binding interaction with ubiquitin;

- loading into a computer's memory a second set of data corresponding to three- dimensional structure of test compounds;

- computing said first and second set of a data to obtain docking affinity of the test compounds for at an interface between the E2 enzyme and ubiquitin;

- computing said first and second set of a data to obtain binding affinity of the E2 enzyme with ubiquitin in presence or absence of a test compound; and

- selecting test compounds increasing binding affinity of the E2 enzyme with ubiquitin for subsequent in vitro, ex vivo and/or in vivo testing of inhibition of the E2 enzyme and/or testing of inhibition of other enzymes involved in the cell ubiquitin- proteasome system (UPS).

In a particular embodiment, the ubiquitin is human ubiquitin comprising SEQ ID NO: 2 and the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1. In a particular embodiment, the three-dimensional model comprises structural coordinates of human ubiquitin and human Cdc34 are as defined in PDB ID 4MDK. Several virtual and chemical libraries of molecules are commercially available and may be used to identify putative inhibitors according to the invention. Exemplary compounds that may be used in such screening methods includes without limitation, interfering proteins or peptides, antibodies or antibody fragments or small chemical organic molecules (i.e. having preferably a molecular weight of less than 2000 Daltons, more preferably less than 1000 Daltons, even more preferably less than 500 Daltons).

According to the invention, similar principles may be applicable for the stabilization of other non-covalent interactions of ubiquitin in the UPS with other UPS enzyme classes (E1 , E3, DUB, UBA) and/or with other ubiquitin-like modifiers (SUMO, Nedd8, etc). Accordingly, useful inhibitors would bind to the interface of an UPS enzyme in a non- covalent complex with ubiquitin.

EXAMPLES

The Examples set forth hereinafter provide a three-dimensional (3D) structure or model of a E2 enzyme in a binding interaction with ubiquitin and provide a specific example of a compound stabilizing non-covalent ubiquitin-E2 complexes.

Also provided are exemplary screening methods for identifying specific UPS inhibitors, including inhibitors of E2 enzymes such as Cdc34.

Example 1 : Inhibition of the Cdc34 ubiquitin conjugating enzyme by trapping of a non-covalent ubiquitin intermediate

Abstract:

The ubiquitin-proteasome system (UPS) governs the dynamics of the proteome. In addition to the catalytic transfer of ubiquitin via thioester intermediates through the E1 -E2-E3 enzyme cascade, weak non-covalent interactions between UPS enzymes and ubiquitin are critical for catalysis and linkage specificity. The present example show that a small molecule inhibitor of the E2 enzyme Cdc34 traps a non-covalent interaction between ubiquitin and the donor site on the E2, and thereby freezes the catalytic cycle. The inhibitor engages a composite binding pocket formed from Cdc34 and ubiquitin, and inhibits hydrolysis of the ubiquitin thioester. These results demonstrate that stabilization of the prevalent non-covalent enzyme interactions with ubiquitin is a feasible strategy to inhibit the UPS. Introduction

The UPS regulates all cellular processes through precise spatial and temporal control of protein stability, activity and/or localization (1 ), and is frequently dysregulated in cancer and other diseases (2, 3). The conserved E1→E2→E3 enzyme cascade activates and transfers ubiquitin through step-wise thioester linkages for covalent conjugation to free amino groups on substrate proteins. The resultant mono- or poly- ubiquitination of the substrate typically leads to altered protein interactions or destruction by the 26S proteasome, respectively (1 , 4, 5). The E2 enzymes lie at a crucial nexus in the UPS hierarchy as they exhibit specific interactions with E1 enzymes, E3 enzymes, deubiquitinating enzymes and substrates. E2 enzymes contain an essential catalytic cysteine that forms the ubiquitin thioester and an adjacent invariant asparagine residue that stabilizes the oxyanion transition state (6, 7). Weak non-covalent interactions between the E2 and ubiquitin are important for catalysis: the donor site tethers the thioesterified ubiquitin to prevent steric occlusion of the reaction centre and allow efficient attack of the thioester by the incoming substrate nucleophile, whereas the acceptor site orients the incoming ubiquitin to guide formation of the correct chain linkage (8-10). The detailed structural understanding of the ubiquitin transferase reaction has been hampered by the transient and structurally complex nature of these non-covalent catalytic intermediates.

The cullin-RING ligases (CRLs) form the largest family of E3 enzymes and are built on a core cullin-based architecture that recruits many hundreds of substrates through large cohorts of different adaptor proteins (1 1 -13). The Rbx1 RING domain subunit provides the docking site for Cdc34 (Ube2R1 ), which is the principal E2 enzyme for the CRL enzymes (14). Weak electrostatic interactions between the acidic C-terminus of Cdc34 and a basic cleft on the cullin subunit facilitate rapid cycles of E2 loading/unloading in the complex (14) and stabilize the E2-cullin interaction (15). CRL enzyme activity depends on the reversible modification of the cullin subunit by the ubiquitin-like modifier Nedd8, which triggers a conformational release of the Rbx1 subunit and the docked E2 enzyme to allow the E2 to access the bound substrate (16). Global CRL activity has been validated as a cancer target through development of a Nedd8 activating enzyme (NAE1 ) inhibitor called MLN4924 that traps NAE1 in a stable intermediate with Nedd8 and drives all CRLs into inactive non-neddylated forms (17, 18). MLN4924 potently inhibits cancer cell proliferation, primarily through disabling cell cycle, DNA replication and DNA damage/repair functions, and is highly efficacious in pre-clinical cancer models (3).

As a parallel strategy to inhibit CRL activity, a small molecule called CC0651 was recently identified as a specific inhibitor of the human E2 enzyme Cdc34A, also known as Ube2R1 and referred to here as Cdc34 (19). Like MLN4924, CC0651 stabilizes the CDK inhibitor p27 in cultured cells and inhibits the proliferation of human cancer cell lines (19). The structure of the CC0651 -Cdc34 complex shows that CC0651 binds a cryptic pocket on the Cdc34 surface that is far removed from the active site cysteine but did not explain the mechanism of inhibition (19). The present examples shows that CC0651 traps a weak non-covalent interaction of ubiquitin on the donor site of Cdc34 and that stabilization of this ubiquitin-E2 complex is sufficient to impede catalysis.

Results

NMR characterization of interactions between CC0651 , Cdc34 and free ubiquitin. A partial overlap between the CC0651 binding site and a predicted non-covalent donor ubiquitin binding surface on Cdc34 (19) lead the inventors to investigate the interactions between CC0651 , Cdc34 and free ubiquitin. A scheme was developed for the in-house synthesis of CC0651 (20). Nuclear magnetic resonance spectroscopy (NMR) was used to assess the interaction of Cdc34 with 15 N-ubiquitin by chemical shift perturbation (CSP) and peak intensity analysis of the heteronuclear single quantum coherence (HSQC) spectra (20). Strikingly, CC0651 caused a pronounced interaction between ubiquitin and the core catalytic domain of Cdc34 (Cdc34 cat ), which lacks the acidic C-terminal tail (Fig. 1A). Peak shifts and peak broadening of ubiquitin resonances occurred at residues K6, T7, L8, Q40, Q41 , R42, L43, I44, F45, G47, K48, Q49, L50, L67, H68, V70, L71 , R72 and L73. As none of these shifts were evident in the absence of CC0651 (Fig. 1 B) or with ubiquitin alone in the presence of CC0651 (Fig. 1 C), the inventors concluded that CC0651 specifically stabilizes a non-covalent interaction between ubiquitin and the catalytic domain of Cdc34. Addition of unlabeled full length Cdc34 (Cdc34 FL ), including the acidic C-terminal extension, caused a similar pattern of resonance shifts as for the CC0651 -Cdc34-ubiquitin complex (Fig. 1 D), consistent with a previous observation that the acidic tail interacts with covalently linked ubiquitin (21 , 22). The combination of Cdc34 FL and CC0651 caused more extensive peak broadening and/or disappearance (Fig. 1 E), indicating that CC0651 and the acidic tail additively potentiate the interaction of free ubiquitin with Cdc34. The affected residues formed a contiguous surface on ubiquitin that closely matched the previously inferred contact surface for the catalytic domain of E2 enzymes (Fig 1 F) (8, 9, 23-26). Comparison of the interaction surfaces on ubiquitin for Cdc34 cat in the presence of CC0651 and for Cdc34 FL alone with a previously determined disulfide tethered Cdc34 FL -ubiquitin complex demonstrated that the CC0651 -induced complex may mimic a natural interaction between ubiquitin and Cdc34 (Fig. 1 F-H). Collectively, these results demonstrate that CC0651 acts to stabilize a non-covalent interaction between Cdc34 and ubiquitin.

A series of experiments with 15 N-Cdc34 cat showed that CC0651 caused resonance shifts on Cdc34 in both the presence and absence of a four-fold excess of free ubiquitin (Fig. 2A, B), whereas unlabeled ubiquitin alone did not cause any detectable shifts even in large excess (Fig. 2C). Chemical shift changes for three representative peaks at different concentrations of CC0651 was used to estimate the affinity of CC0651 for Cdc34 cat in the presence and absence of free ubiquitin. Concordant with a model where CC0651 and ubiquitin bind cooperatively to Cdc34, quantitation of chemical shifts in the NMR titrations showed that CC0651 bound to Cdc34 cat alone with a EC 50 of 267 μΜ but that in the presence of ubiquitin this affinity was increased to an EC 50 of 19 μΜ (Fig. 2D, E). Independent measurements using a time resolved Forster energy transfer (TR-FRET) assay performed at dilute concentrations of Cdc34 and ubiquitin as compared to NMR experiments (20) showed that CC0651 potentiated binding of Cdc34 FL to ubiquitin with an EC 50 of 14 ± 2 μΜ, whereas the Cdc34B (Ube2R2) isoform, which is insensitive to CC0651 ( 19), did not respond to CC0651 (Fig. 2F). The induced binding effect in the TR- FRET assay was dependent on the acidic tail of Cdc34, consistent with additive effect of the tail observed by NMR. Structure of a CC0651-Cdc34-ubiquitin complex. To investigate the precise mode of binding between CC0651 , Cdc34 and ubiquitin, an equimolar mixture of constituents was crystallized and the X-ray crystal structure was solved at 2.6 A resolution by molecular replacement. Table 2 provides X-ray data collection and refinement statistics (20). The 3D coordinates of the crystal structure are available at Protein Data Bank™ (on the internet at www.rcsb.org) with the identification code 4MDK (herein after "PDB ID 4MDK"), the data information in that file being incorporated herein by reference. The 3D coordinates are also available in Table 3 of US 61 /914,274 which is incorporated herein by reference. See Figure 3E,F for representative electron density maps. The structure revealed that CC0651 engages a composite binding pocket nestled at the periphery of the Cdc34-ubiquitin binding interface composed of residues from both Cdc34 and ubiquitin (Fig. 3A-C, Fig. 6A-C). The ternary complex was formed without any overt structural perturbation to either Cdc34 or to ubiquitin (RMSD of ubiquitin = 0.67A 2 ; RMSD of Cdc34-CC0651 complex = 0.75A 2 ). Notably, regions of CC0651 that were highly solvent exposed in the CC0651 -Cdc34 binary complex (79) were shielded by ubiquitin in the ternary complex. Of a total accessible surface area of 658 A 2 on CC0651 , 528 A 2 (81 %) and 66 A 2 (10%) were buried by contact of CC0651 with Cdc34 and ubiquitin respectively, with only 61 A 2 (9%) of the compound surface exposed to solvent. In addition, the CC0651 contacts with Cdc34 described previously (79) were unchanged by the presence of ubiquitin. The biphenyl ring system of CC0651 made a number of direct contacts with ubiquitin (Fig. 3B, Fig. 6A), including van der Waals contacts with the backbone of Gly47 U , Lys48 Ub , Glu51 U and Gln49 Ub and hydrophobic contact with the apolar portion of the Lys48 Ub side chain. These structural features suggest that CC0651 acts as a molecular bridge between Cdc34 and ubiquitin.

Table 2. Data collection and refinement statistics for the CC0651 -Cdc34A-ubiquitin complex. Data was collected from a single frozen crystal. Highest resolution shell is shown in parenthesis.

Data collection

Space group P1

Cell dimensions

a, b, c ( A) 55.4, 72.2, 75.9

Α, β, γ ( °) 71 .9, 79.6, 81 .6

Resolution (A) 44.6-2.6

Rmeas (%) a 7.9 (45.7)

Ι/σ 1 3.7 ( 1 .2)

Completeness (%) 95.8 (85.5)

Redundancy 2.2 ( 1 .7)

Refinement

Resolution (A) 44.6-2.6

Rwork/Rfree " 20.4/25.9

No. unique reflections 31 377

No. atoms

protein 7545

ligand 166

water 46

Avg. B-factors (A 2 )

protein 39.2

compound 56.9

water 51 .5

R.M.S deviations

Bond lengths (A) 0.008

Bond angles (°) 1 .26

Ramachandran Plot Most favoured regions (%) 98.2

Allowed regions (%) 1.8

Disallowed regions (%) 0.0 The donor ubiquitin-Cdc34 interface. The direct interaction of ubiquitin with Cdc34 accounts for 1092 A 2 out of a total of 2485 A 2 buried surface area in the CC0651 -Cdc34- ubiquitin complex. The contact surface on Cdc34 was composed of helix a2, helix 3 10, and the linkers that join helices α2-α3, helices 3 0 -a2, and helix a1 and strand β1 and strands β2 β3. The reciprocal contact surface on ubiquitin was composed of strands β1 , β3, and β4 and intervening linkers joining β1 -β2 and β3-β4 (see Fig. 3B,C for all contact residues). Notable electrostatic and H-bond interactions included a network between the Asn132 Cdc34 side chain and backbone NH of Gln49 Ub , the Glu133 Cdc34 side chain and side chains of Gln49 U , Arg42 Ub , and Arg72 Ub , and lastly between the Ser129 Cdc34 side chain and the side chains of Gln49 U and Arg72 U (Fig. 3B; Fig. 6B). Notable hydrophobic contacts occurred between a ridge formed by Leu8 U , Ile44 ub , and Val70 Ub , and a complementary groove formed by Thr122 Cdc34 , Leu125 Cdc34 , Ser126 Cdc34 , lie 128 Cdc34 and Ser129 Cdc34 (Fig. 3B,C; Fig. 6C).

The binding mode of ubiquitin to Cdc34 induced by CC0651 was highly reminiscent of donor ubiquitin-E2 interactions deduced previously for Ubc1 (23), for Cdc34 and Ube2S (8, 9), and for UbcH5A/B (24, 25). This similarity extended to the interaction of the ubiquitin-like modifier SUMO with its cognate E2 Ubc9 (27, 28). The contact surfaces on both ubiquitin/SUMO and the E2s are similar and only small rotations (23° to 43°) of ubiquitin/SUMO subunits on their respective E2 surfaces differentiate the structures (Fig. 3D). Quite remarkably though, all E2s engage the same surface of ubiquitin using the same topological surface on the E2s (Fig. 3C-D, Fig. 7 and Fig. 11 ). These features suggest that the donor binding site is topologically well-conserved although there is a degree of variability of residues on different E2s. Figure 3C-D, Figure 7 and Figure 11 illustrate conservation of the ubiquitin binding surface on multiple human E2. Altogether this suggests that similar interfacial pockets will form between ubiquitin and all E2s. The CC0651 -Cdc34-ubiquitin structure validated a previously proposed non-covalent interaction between ubiquitin and Cdc34 inferred from mutational analysis (8). In particular, perturbation of the hydrophobic environment surrounding Ser129 Cdc34 or disruption of a presumptive salt bridge between Glu133 C c34 and Arg42 U compromise Cdc34 function (8). Both of these residues lie at the nexus of interactions between Cdc34 and ubiquitin (Fig. 3B). Consistently, mutation of either Glu133 Cdc34 or Ser129 Cdc34 to Arg (but not the latter to Leu) abolished all CC0651 -dependent resonance shifts and peak intensity changes in the 15 N-Ub HSQC spectrum, thereby demonstrating that the X- ray structure represents a snapshot of the CC0651 -Cdc34-ubiquitin complex observed in solution (Fig. 4A). In contrast to the Cdc34 Ser129Arg mutant, which was completely compromised for function, the Cdc34 G ' u133Arg mutant retained wild type activity when assayed against the substrate Sid and yet displayed insensitivity to CC0651 in vitro (Fig. 4B). However, sensitivity of the Cdc34 GIU133Ar9 mutant to CC0651 was restored by substitution of a charge reversal (jb Arg 2GIU mutant (Fig. 4B). These results demonstrate that the ubiquitin-Cdc34 interface is integral to CC0651 action and that the inhibitor strengthens a native-like interaction of ubiquitin and Cdc34. Structure-guided CC0651 derivatives. To probe features of the CC0651 pocket from the chemical ligand perspective, we undertook a chemical structure-activity relationship (SAR) analysis focused on the core dichlorobiphenyl moiety of CC0651 , as guided by the CC0651-Cdc34-ubiquitin crystal structure (Figure 12). We developed a modified synthetic scheme for CC0651 (See Huang et al, Nat Chem Biol. 2013 Dec 15. doi: 10.1038/nchembio.1412. [Epub ahead of print], for methods and materials details and for compound characterization data) to allow comparisons across a set of six biphenyl derivatives (see Fig. 12 A-C top panels for chemical structures) and analyzed each analog for inhibition of Cdc34A-mediated ubiquitination reactions in vitro (Fig. 12 A-C middle panels) and stabilization of the Cdc34A-ubiquitin interaction in a TR-FRET assay (Fig. 12 A-C bottom panels) (see Huang et al, Nat Chem Biol. 2013 Dec 15. doi: 10.1038/nchembio.1412. [Epub ahead of print], for ubiquitination assay and Cdc34A- ubiquitin binding assay details in addition to Example 2 herein). We observed that both parameters were tightly correlated and that the relative bioactivity of the analogs could be rationalized by their fit (as assessed visually herein or computationally in Example 4) into the binding pocket. In particular, substitution of both chlorine atoms (atomic radius = 1 .75 A) with similarly sized methyl groups (van der Waals 2x radius =2.0 A) was equally well tolerated for inhibitory activity and binding effects (compound 2), whereas substitution with smaller fluorine atoms (atomic radius = 1.47 A) was mildly detrimental (compound 3). Repositioning of one of the two meta methyl groups in analog 2 to the para position (compound 4) impaired inhibitor activity, consistent with an absence of an accommodating pocket in the crystal structure and hence the generation of a steric clash. More substantial substitutions at all three para and meta positions with O-methyl groups (compound 5) which would geneate more extensive steric clashes within the binding pocket abolished all inhibitory and binding activity. Notably, deletion of one chlorine atom and substitution of the other with a bulky benzyoxy group at ring position 3 (compound 6) also abolished activity. Although the original CC0651 -Cdc34A binary structure suggested that a bulky group at this position should be tolerated by projection into solvent, in the CC0651-Cdc34A-ubiquitin ternary structure, the benzyoxy group would be predicted to sterically clash with ubiquitin.

These results validate the utility of the Cdc34-ubiquitin-CC0651 ternary structure as a guide for SAR and further demonstrate that stabilization of the Cdc34A-ubiquitin interaction is an essential aspect of the CC0651 mechanism of action.

These results further demonstrate that there is a direct correlation between stabilization of the Cdc34-ubiquitin interaction and inhibition of Cdc34 catalytic activity.

Specifically, the greater the complemenatriy of fit (as can be computationally assessed in Example 4) between a chemical entitity and the composite binding pocket for CC0651 at the interaction interface between Cdc34 and ubiquitin, i) the greater the induced binding affinity between Cdc34 and ubiquitin, ii) the greater the affinity of the chemical entity for the Cdc34-ubiqutin complex, and iii) the greater the potency of the chemical entitity for inhibiting a ubiquitination reaction that is normally supported by Cdc34.

The results described in this example, demonstrate the utility of the claimed invention in defining a novel binding pocket formed at the E2 enzyme-ubiquitin interface. It is the realm of the present invention to exploit this novel binding pocket to identify, develop and use small molecule inhibitors of E2 enzyme activity by stabilization of the E2-ubiquitin interface.

Discussion

The present analysis of the CC0651 -Cdc34-ubiquitin complex demonstrates that small molecule-mediated stabilization of the weak ubiquitin interaction at the donor surface is sufficient to lock the E2 catalytic cycle. The structure of the CC0651 -Cdc34-ubiquitin ternary complex appears to reflect the native-like docking pose of ubiquitin during the catalytic reaction. The enhanced binding of free ubiquitin to Cdc34 induced by CC0651 is likely due to a combination of factors including direct bridging contacts between CC0651 and ubiquitin, distortion of the Cdc34 structure to improve complementarity of fit with ubiquitin and/or reduction of entropic penalty by restriction of Cdc34 conformer space. In addition to enhancement of interaction affinity between Cdc34 and ubiquitin, catalytic inhibition by CC0651 also arises from perturbation of the intrinsic catalytic transfer function of the E2. The E2~ubiquitin thioester can adopt open and closed (folded-back) conformations, the latter of which represents the catalytically poised conformer ( 10, 24). While the active conformer is evidently locked into place by CC0651 , the inhibitor-bound E2 may lack the necessary strain across the scissile thioester bond, which is presumed to be actuated upon E3 binding (24). The specificity of CC0651 for Cdc34 is explained by the variability of the donor ubiquitin surface on all E2s, which despite the conserved overall topology, exhibits considerable sequence variation across the E2 family (Fig. 7 and Fig. 11 ) (30). This variability, and the occurrence of likely partial binding pockets in other E2s ( 19), suggests that it is possible to exploit stabilization of the donor ubiquitin-E2 interaction as a general means to isolate specific small molecule inhibitors of many E2 enzymes. This in principle positions the E2 enzyme family as a new drug target class in the UPS, despite the lack of an overt catalytic cleft in the E2 structure. We note that the ternary structure reveals additional crevasses at the ubiquitin-Cdc34 interface (Fig. 3A) that may be exploited to further increase the affinity of CC0651 . Weak non-covalent interactions of ubiquitin occur not only with many E2 enzymes (6, 7), but also with E1 enzymes (35), E3 enzymes (36), deubiquitinating enzymes (37), and a host of ubiquitin binding domains (37). These weak interactions serve to modulate catalytic output, orient ubiquitin for efficient formation of different chain types, and enable recognition of different types of modification. In total, ubiquitin must form many hundreds of unique non-covalent interfaces with the UPS enzyme hierarchy. By analogy to CC0651 , it is envisonable according to the teachings of the present invention to identify unique small molecule inhibitors that selectively stabilize non-covalent interactions of ubiquitin, or for that matter other ubiquitin-like modifiers, with any target of interest within the UPS or related ubiquitin-like modifier systems. Example 2: FRET-based assay

Screening of inhibitors and or in vitro characterization of candidate inhibitors may be facilitated by using a FRET-based assay. One particular example is a FRET-based assay that monitors the interaction between fluorescein-labeled Ub and a terbium- labeled version of Cdc34 (Fig. 10 A-B), which detects interactions independent of effects on catalytic function. This assay format may be applied to any other E2 enzyme that is tagged with a poly-histidine sequence.

Results from this binding assay could then be compared to inhibitory effects in a quantitative ubiquitination assay that monitors the rate of incorporation of fluorescently- labeled Ub (Fig. 10C-D). The candidate inhibitors may then be assayed in cell lines to identify alterations that improve potency in vivo, bioavailability and on-target effects. X-ray structure determination of ternary complexes with optimized compounds may then be used to drive subsequent medicinal chemistry decision points. Example 3: On-bead ubiquitin target capture screens for inhibitors of Cdc34 or other E2 enzymes

The finding that CC0651 forms a ternary complex with ubiquitin and Cdc34 may be exploited in a parallel high-throughput (HTP) screening approach using on-bead capture of target proteins in order to identify beads that harbor a compound that interacts with the target protein of interest (Hintersteiner M. et al. Chem Biol. (2009) 16:724-735). Combinatorial one bead-one compound (OBOC) libraries based on scaffolds selected from in silico docking approaches may be custom synthesized and screened in a confocal imaging assay format to directly identify compounds on beads that bind to the ubiquitin-Cdc34 complex or to any other ubiquitin-E2 enzyme complex. The on-bead screening format is predicated on the capture of a fluorescently labeled target onto individual beads, which are scanned for specific fluorescent signals on either a manual or automated imaging platform. A step-by-step example of an on-bead assay is provided hereinbefore.

As a specific case example, CC0651 may be coupled to beads through a PEG linker (with non-coupled beads as a control), and incubated with fluorescein-labeled recombinant ubiquitin in the presence or absence of Cdc34, in either a crude cell lysate or buffer solution. An intense fluorescence signal from a ubiquitin-fluorescein conjugate would only be observed with the CC0651 bead and not the control bead, and only in in the presence of Cdc34. That is the increased signal in the presence of Cdc34 would demonstrate co-capture of the labeled ubiquitin and Cdc34. Accordingly, such an assay can be used to screen a a library of beads (e.g. a custom synthesized one-bead one- compound library) for compounds that selectively stabilize the interaction of ubiquitin with Cdc34, or any other E2 of interest that is screened in the same manner. Hit compound identities may be then deconvolved by mass spectrometry and the hit compound subjected to rapid and exhaustive SAR through focused secondary on-bead library screens, or by conventional medicinal chemistry. The pursuit of hit compounds from on- bead screens may be guided by biophysical, biochemical and structure-based assays. Example 4. In silico screens for E2 inhibitors

The ternary X-ray crystal structure of Cdc34A in complex with ubiquitin and the CC0651 inhibitor was prepared as template structure ("receptor") for in silico docking of small molecule inhibitors. Amino acid residues that were not visible or only partial visible in the X-ray density were modeled in using the molecular modeling program YASARA™ Structure (version 13.1 1 .26; Krieger E, Koraimann G, Vriend G (2002) Proteins 47, 393- 402), hydrogen atoms were added (assuming a pH of 7.4), and the hydrogen bonding network was optimized, and the atomic coordinates of the CC0651 inhibitor were removed.

Small molecule compound 3D geometries ("ligands") were prepared by converting 1 D smile string format files or 2D sd format files into 3D coordinates by importing the files in Yasara Structure, followed by the "inflate" command, hydrogen addition, atom typing, point charge assignments using Amber2003force field parameters (using the AutoSmile algorithm), energy minimization and a final quantum mechanical (QM) optimization by YASARA's MOPAC implementation. Alternatively, smile files were converted into 3D coordinates using OpenBabel's "gen3d" option and subsequently imported as *.mol2 files in Yasara Structure, followed by cleaning, atom typing and point charge assignment using Amber2003 force field parameters and a final QM based geometry optimization.

Docking of the small molecule compounds was restricted to the pocket enclosed by side chain and/or backbone atoms of ubiquitin residues Gly47, Lys48, Glu49, Glu51 , Arg54 and Cdc34 residues Gly27, Phe28, Ile45, Phe46, Pro48, Tyr52, Tyr53, Phe58, Phe77, Met81 , His83, Ile128, Leu131 , Asn132, Tyr148, Trp151 , Tyr161 , Thr162, Ile165 (Figure 13).

Docking was performed using AutoDock VINA (Trott O, Olson AJ (2010), J.Comput.Chem. 31 ,455-461 ) using default parameters. The ligands were treated as flexible, while the side chain residues of the receptor remained fixed. Alternative implementations of the in silico docking protocol also treated the side chains surrounding the pocket as flexible or included multiple conformations of the receptor (obtained from a molecular dynamics trajectory or from various available crystal structures). For each ligand, 20 to 100 conformers were generated and docked in the binding pocket, and the ligand conformer having the highest docking score (most favorable binding energy) was retained. Typically, ligands from a library of compounds having a more favorable binding energy than the docked conformation of CC0651 would be selected for experimental validation in TR-FRET and ubiquitination assays.

An example in silico docking screen performed with a series of biphenyl compounds (Figure 12) showed that the docking procedure can closely reproduce the binding pose of CC0651 as observed in the X-ray structure of Cdc34 in complex with ubiquitin and CC0651 (Figure 14A). The 3D coordinates of the two docked CC0651 compounds shown were generated using the same procedure as for the other compounds in the series, using the above described procedure. As illustrated (Figure 14B and C), the docking procedure can successfully distinguish between compounds that bind the Cdc34A-ubiquitin complex (compounds 2,3,4 in Figure 12) and compounds that do not bind the Cdc34A-ubiquitin complex (compound 5 in Figure 12).

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Headings are included herein for reference and to aid in locating certain sections. These headings are not intended to limit the scope of the concepts described therein under, and these concepts may be applicable in other sections throughout the entire specification. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, concentrations, properties, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about". At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the present specification and attached claims are approximations that may vary depending upon the properties sought to be obtained. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the embodiments are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors resulting from variations in experiments, testing measurements, statistical analyses and such. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the present invention and scope of the appended claims.