WO2014094138A1 - Procédés de criblage pour l'identification de composés inhibiteurs de l'activité d'enzymes e2 par la stabilisation de complexes ubiquitine-e2 non covalents et applications pharmaceutiques associées aux inhibiteurs d'e2 - Google Patents

Procédés de criblage pour l'identification de composés inhibiteurs de l'activité d'enzymes e2 par la stabilisation de complexes ubiquitine-e2 non covalents et applications pharmaceutiques associées aux inhibiteurs d'e2 Download PDF

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WO2014094138A1
WO2014094138A1 PCT/CA2013/001079 CA2013001079W WO2014094138A1 WO 2014094138 A1 WO2014094138 A1 WO 2014094138A1 CA 2013001079 W CA2013001079 W CA 2013001079W WO 2014094138 A1 WO2014094138 A1 WO 2014094138A1
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cdc34
ubiquitin
enzyme
compound
human
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Michael Tyers
Frank Sicheri
Derek Frank Joseph CECCARELLI
Hao Huang
Stephen Orlicky
Albert Martinus Van Der Sloot
Daniel St-Cyr
Susan Jennifer MOORE
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Universite De Montreal
Mount Sinai Hospital
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/25—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving enzymes not classifiable in groups C12Q1/26 - C12Q1/66
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • 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).
  • UPS ubiquitin-proteasome system
  • the ubiquitin-proteasome system 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.
  • 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.
  • Cdc34 a dedicated E2 enzyme
  • CC0651 a small molecule inhibitor of Cdc34, which at the time represented the first described E2 enzyme inhibitor (Ceccarelli et al. Cell (201 1 ), 145: 1075-1087).
  • CC0651 stabilizes the CDK inhibitor p27 in cultured cells and inhibits the proliferation of human cancer cell lines (Ceccarelli, 201 1 ).
  • 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 ).
  • 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.
  • subsequent published work incorrectly predicted that CC0651 would inhibit the Cdc34-ubiquitin interaction (Pruneda et al. Mol Cell (2012), 47:933-942).
  • 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 .
  • 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:
  • 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:
  • 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.
  • 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:
  • 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:
  • 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
  • the compound makes direct contact with the following amino acids: Gly47 Ub , Lys48 U , Glu51 Ub and Gln49 Ub .
  • a cancer 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.
  • a neurological disorder including but not limited to 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.
  • an infectious disease 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.
  • a metabolic disorder such as diabetes or hemochromatosis
  • a genetic disorder such as CF or mental retardation
  • a reproductive disorder such as infertility.
  • 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.
  • UPS ubiquitin-proteasome system
  • 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.
  • 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.
  • 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 .
  • 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.
  • 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;
  • 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.
  • the compound is selected from the following compounds:
  • the present invention also concerns screening methods.
  • the present invention relates to a screening method for identifying inhibitors of an E2 enzyme component of a mammalian cell ubiquitin-proteasome system (UPS).
  • 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.
  • the screening method is a TR-FRET assay which comprisesthe following steps:
  • the screening method is an on-bead assay comprising the following steps:
  • E2 cat providing a catalytic domain of the E2 enzyme (E2 cat );
  • 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).
  • UPS ubiquitin-proteasome system
  • 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.
  • 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.
  • FIGURE 1 CC0651 potentiates the interaction between Cdc34 and 15 N-Ub.
  • FIGURE 2 CC0651 potentiates the interaction between 15 N-Cdc34 and ubiquitin.
  • 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.
  • 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 .0 ⁇ ) and the Fo-Fc map surrounding CC0651 (contoured at 2.5 ⁇ ). Prime-and-switch generated electron density map of the CC0651 -Cdc34A-ubiquitin complex contoured at 1 .0 ⁇ ( ⁇ ).
  • 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 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.
  • 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.
  • CCLs Cullin-RING ligases
  • FIGURE 9 Expression of CRL network components in human cancer.
  • FIGURE 11 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.
  • 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 -
  • FIG. 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.
  • FIG. 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.
  • 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).
  • 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.
  • 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.
  • the subject is a mammal. More preferably, the subject is a human, such as a human patient in need of treatment.
  • UPS Ultraquitin-proteasome system
  • 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.
  • DABs deubiqutinating enzymes
  • UBDs ubiquitin binding domains
  • 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.
  • E2 enzymes mediate the conjugation of ubiquitin to substrates either directly or indirectly and thereby control protein stability and interactions.
  • Table 1 hereinafter provides some information about each one of them.
  • 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 UniProtTM 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 .
  • Cdc34B 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 UniProtTM database under accession number Q712K3 or NP_060281 .2 (gene sequence is available at the NCBI database under accession number BC004862).
  • 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.
  • 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).
  • conserved residues on the E2 enzyme i.e. E2 residues that are conserved with analogous residues on Cdc34.
  • 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 UniProtTM 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • this interface on the E2 is called the donor ubiquitin binding surface, and on ubiquitin it is called the Ile44 hydrophobic surface.
  • 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 .
  • 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.
  • the E2 is human Cdc34 and the binding pocket is as defined herein.
  • the inventors are the first to characterized 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.
  • UPS ubiquitin-proteasome system
  • 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 ).
  • 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.
  • cancers examples 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.
  • 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.
  • 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.
  • the compound binds at an interface between the E2 enzyme and ubiquitin.
  • stabilisation occurs because the compound acts as a molecular bridge between E2 enzyme and ubiquitin.
  • stabilisation occurs because the compound increases the binding affinity between the E2 enzyme and ubiquitin.
  • 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.
  • E2 e.g. Cdc34
  • labeled ubiquitin e.g. 15 N-ubiquitin
  • 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.
  • 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.
  • 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 .
  • 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 .
  • the compound is used in combination with one or more of the following inhibitors: MLN4924 (by Millenium Pharmaceuticals), Bortezomib (VelcadeTM), Carfilzomib (by Onyx Pharmaceuticals).
  • 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.
  • 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.
  • 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.
  • the term “treating” can include increasing a subject's life expectancy and/or delay before additional treatments are required (e.g. joint replacement surgery).
  • an aspect of the invention concerns a screening method for identifying inhibitors of any E2 enzyme in the mammalian ubiquitin-proteasome system (UPS).
  • 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, FortebioTM, BiacoreTM, 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.
  • 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.
  • the E2 enzyme is selected from E2 enzymes listed in Table 1.
  • 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.
  • resonance shifts may correspond to conserved residues in other E2 enzymes, including but not limited to those illustrated in Figure 7.
  • the E2 enzyme is Cdc34 and the assay is an NMR assay which comprises the following steps:
  • ubiquitin labeled with 15 N-amonium supplemented bacteria culture media for instance ubiquitin labeled with 15 N-amonium supplemented bacteria culture media
  • E2 cat providing a catalytic domain of the E2 enzyme (E2 cat );
  • HSQC Heteronuclear Single Quantum Coherence
  • E2 cat is indicative of a potential inhibitory activity for the test compound.
  • 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.
  • the labeled ubiquitin is 15 N-ubiquitin.
  • 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.
  • the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1
  • the E2 cat is the catalytic domain of Cdc34 (Cdc34 cat )
  • Cdc34 cat comprises residues Pro7 to Val184 of SEQ ID NO: 1.
  • the assay is a TR-FRET assay which comprises the following steps:
  • ubiquitin labeled with a first fluorophore for instance ubiquitin labeled with fluorescein or a similar probe with similar excitation and emission spectra
  • 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 InvitrogenTM);
  • a second fluorophore e.g. Terbium, Europium
  • said tag e.g. Terbium anti-his antibody
  • 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.
  • saturating levels e.g. 100 micromolar
  • the assay is an on-bead assay which comprises the following steps:
  • 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
  • E2 cat providing a catalytic domain of the E2 enzyme (E2 cat );
  • fluorescently-labeled ubiquitin e.g. recombinant ubiquitin bearing a non- native cysteine residue at its N-terminus which has been derivatized with fluorescein iodoacetamide];
  • 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];
  • 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.
  • the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1
  • the E2 cat is the catalytic domain of Cdc34 (Cdc34 cat )
  • the Cdc34 cat comprises residues Pro7 to Val184 of SEQ ID NO: 1.
  • 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 as TentagelTM beads having a diameter of about 100 ⁇ , 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.
  • OBOC combinatorial one bead- one compound
  • 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.
  • 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:
  • step (c) selecting test compounds from step (b) increasing binding affinity of the E2 enzyme with ubiquitin;
  • test compounds are selected test compounds as potential inhibitors of the E2 enzyme and/or as potential inhibitors of another enzyme part of the UPS.
  • the selecting step comprises identifying ligand compounds having the highest docking score.
  • the ubiquitin is human ubiquitin comprising SEQ ID NO: 2
  • the in silico docking comprises in silico docking at the following position: Gly47 Ub , Lys48 Ub , Glu51 Ub and Gln49 Ub .
  • 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 .
  • the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1
  • the in silico docking comprises in silico docking at a site that involves at least one of the following positions:
  • 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.
  • the X-ray crystal structure of Cdc34 in complex with Ubiquitin and CC0651 provided herein 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.
  • the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1
  • the ubiquitin is human ubiquitin comprising SEQ ID NO: 2
  • selecting test compounds increasing binding affinity of human Cdc34 with ubiquitin comprises assessing one or more of the following interactions:
  • 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
  • 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;
  • UPS mammalian cell ubiquitin-proteasome system
  • the ubiquitin is human ubiquitin comprising SEQ ID NO: 2
  • 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 .
  • the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1
  • the virtual screening comprises assessing a minimum docking affinity of test compounds at an interface between the E2 enzyme and ubiquitin comprising:
  • the ubiquitin is human ubiquitin comprising SEQ ID NO: 2
  • the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1
  • 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:
  • 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).
  • UPS Ubiquitin-proteasome system
  • 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.
  • the three-dimensional model comprises one or more of the followings:
  • 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 ;
  • 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.
  • 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:
  • the ubiquitin is human ubiquitin comprising SEQ ID NO: 2 and the E2 enzyme is human Cdc34 comprising SEQ ID NO: 1.
  • 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).
  • 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.
  • UPS enzyme classes E1 , E3, DUB, UBA
  • SUMO ubiquitin-like modifiers
  • 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.
  • Example 1 Inhibition of the Cdc34 ubiquitin conjugating enzyme by trapping of a non-covalent ubiquitin intermediate
  • the ubiquitin-proteasome system governs the dynamics of the proteome.
  • UPS ubiquitin-proteasome system
  • 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.
  • 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 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).
  • CC0651 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.
  • 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).
  • CSP chemical shift perturbation
  • HSQC heteronuclear single quantum coherence
  • 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.
  • Cdc34 FL unlabeled full length Cdc34
  • Fig. 1 D the acidic tail interacts with covalently linked ubiquitin
  • Fig. 1 D The combination of Cdc34 FL and CC0651 caused more extensive peak broadening and/or disappearance
  • TR-FRET time resolved Forster energy transfer
  • Table 2 provides X-ray data collection and refinement statistics (20).
  • the 3D coordinates of the crystal structure are available at Protein Data BankTM (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.
  • 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).
  • regions of CC0651 that were highly solvent exposed in the CC0651 -Cdc34 binary complex (79) were shielded by ubiquitin in the ternary complex.
  • 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.
  • 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 ⁇ 2- ⁇ 3, helices 3 0 -a2, and helix a1 and strand ⁇ 1 and strands ⁇ 2 ⁇ 3.
  • the reciprocal contact surface on ubiquitin was composed of strands ⁇ 1 , ⁇ 3, and ⁇ 4 and intervening linkers joining ⁇ 1 - ⁇ 2 and ⁇ 3- ⁇ 4 (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).
  • the binding mode of ubiquitin to Cdc34 induced by CC0651 was highly pronounced 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).
  • 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).
  • sensitivity of the Cdc34 GIU133Ar9 mutant to CC0651 was restored by substitution of a charge reversal (jb Arg 2GIU mutant (Fig. 4B).
  • 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.
  • 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).
  • 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.
  • ubiquitin-Cdc34 interface 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.
  • FRET-based assay Screening of inhibitors and or in vitro characterization of candidate inhibitors may be facilitated by using a FRET-based assay.
  • 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
  • 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).
  • HTP high-throughput
  • 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.
  • 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.
  • 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 YASARATM 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 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.
  • 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.
  • FIG. 12 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.
  • 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).
  • NMRPipe a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6, 277-293 (1995).

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Abstract

La présente invention utilise la stabilisation de l'interaction de l'ubiquitine donneuse non covalente avec des enzymes E2, y compris l'interaction Cdc34-ubiquitine. La présente invention concerne un complexe E2-ubiquitine isolé et purifié comportant une poche de liaison dans laquelle des inhibiteurs de l'activité enzymatique d'E2 s'adaptent. L'invention concerne également divers aspects associés à cette poche de liaison comprenant des inhibiteurs, des compositions pharmaceutiques, des procédés de criblage pour l'identification d'inhibiteurs, et des méthodes thérapeutiques pour l'inhibition d'enzymes impliquées dans le système cellulaire ubiquitine-protéasome (UPS).
PCT/CA2013/001079 2012-12-21 2013-12-20 Procédés de criblage pour l'identification de composés inhibiteurs de l'activité d'enzymes e2 par la stabilisation de complexes ubiquitine-e2 non covalents et applications pharmaceutiques associées aux inhibiteurs d'e2 WO2014094138A1 (fr)

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US20160108406A1 (en) * 2014-10-08 2016-04-21 University Of Iowa Research Foundation Method of regulating cftr expression and processing
CN110232954A (zh) * 2019-05-10 2019-09-13 江苏理工学院 基于迁移学习的分子对接方法和系统
CN115181069A (zh) * 2022-07-15 2022-10-14 天津医科大学眼科医院 一种防治近视的化合物

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160108406A1 (en) * 2014-10-08 2016-04-21 University Of Iowa Research Foundation Method of regulating cftr expression and processing
CN110232954A (zh) * 2019-05-10 2019-09-13 江苏理工学院 基于迁移学习的分子对接方法和系统
CN115181069A (zh) * 2022-07-15 2022-10-14 天津医科大学眼科医院 一种防治近视的化合物
CN115181069B (zh) * 2022-07-15 2023-04-11 天津医科大学眼科医院 一种防治近视的化合物

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