WO2010102281A2

WO2010102281A2 - Inhibitors of anaphase promoting complex activity

Info

Publication number: WO2010102281A2
Application number: PCT/US2010/026505
Authority: WO
Inventors: Randall King; Xing Zeng; Shantanu Gaur
Original assignee: Harvard University
Current assignee: Harvard University
Priority date: 2009-03-06
Filing date: 2010-03-08
Publication date: 2010-09-10
Anticipated expiration: 2011-09-06
Also published as: US9205069B2; WO2010102281A3; US20120115948A1

Abstract

The invention provides an anti-proliferative composition comprising a non-peptide analog of the C-terminal isoleucine-arginine (IR) tail motif of an activator of an anaphase promoting complex (APC). The invention further provides methods of inhibing the ubiquitination activity of the APC by administering compositions of the invention.

Description

INHIBITORS OF ANAPHASE PROMOTING COMPLEX ACTIVITY

GOVERNMENT SUPPORT

[01] This invention was made with U.S. Government support under National Institutes of Health grant ROl GM66492. The Government has certain rights in the invention.

FIELD OF THE INVENTION

[02] This invention relates generally to the fields of oncology and disorders associated with cell division.

BACKGROUND OF THE INVENTION

[03] Conventional antiproliferative agents used in the treatment of cancer are generally grouped as compounds which affect the integrity of nucleic acid polymers, e.g., by binding, alkylating, inducing strand breaks, intercalating between base pairs or affecting enzymes which maintain the integrity and function of DNA and RNA, and compounds that bind to proteins to inhibit enzymatic action (e.g., antimetabolites) or the function of structural proteins necessary for cellular integrity (e.g., antitubulin agents). Other antiproliferative drugs include those that block steroid hormone action for the treatment of hormone- dependent cancer, photochemically activated agents, radiation sensitizers, and protectors. Many of these agents are associated with adverse side effects.

SUMMARY OF THE INVENTION

[04] The invention features an anti-proliferative composition comprising a non-peptide analog of the C-terminal isoleucine-arginine (IR) motif of an activator of an anaphase promoting complex (APC) such as Cdc20 or Cdhl. For example, the consensus sequence for vertebrate cdc20 is I, H, Q, G/S, I, R, and the consensus sequence for cdhl is L, F, T, R, I, R, and conserved region of Cdhl also extends further upstream Preferably, the compound is formulated in a pharmaceutical composition, i.e., in the presence of physiologically- acceptable excipients. A peptide is a polymer of at least two amino acids. A non-peptide compound or IR motif analog is a composition that comprises less than two amino acids; such a compound may include one amino acid or no amino acids. For example, the non- peptide compound or IR motif analog is tosyl-L-arginine methylester (TAME). Other compounds in this class include, tosyl-L-arginine amide (TAA), tosyl-L-lysine methylester (TLME), tosyl-L-arginine (TAOH), acetyl-L-arginine methylester (AAME), Benzoyl-L- arginine amide (BAA), tosyl-L-arginine t-butyl-ester (TATE), or Benzoyl-L-arginine methylester (BAME).

[05] Members of this class of compounds inhibits degradation of a substrate of an APC in a cell such as a human cell or population of cells characterized as aberrantly proliferating. The compounds are useful to treat cellular proliferative disorders such as cancers, e.g., skin cancer, viral induced hyperproliferative HPV-papiloma, HSV-shingles, colon cancer, bladder cancer, breast cancer, melanoma, ovarian carcinoma, prostate carcinoma, or lung cancer as well as psoriasis and eczema. The compounds may be also useful in the context of in vitro fertilization, because they enhance the ability of these early embryos to properly segregate DNA during mitosis.

[06] The compound contacts a component of a tetratricopeptide repeats (TPR) subcomplex of an APC. For example, a TPR subcomplex is composed of APC3/Cdc27, APC6, APC7, and APC8. The compound induces a cell cycle checkpoint such as the spindle assembly checkpoint (SAC). The compound contacts at least one amino acid of said APC3 polypeptide of SEQ ID NO: 1, 2, or 3. In other examples, the compound contacts at least one amino acid of the APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7; at least one amino acid of the APC7 polypeptide of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19; at least one amino acid of the APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25; at least one amino acid of the APC3 polypeptide of SEQ ID NO: 1, 2, or 3 and at least one amino acid of the APC7 polypeptide of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. The compound further further contacts at least one amino acid of the APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7; at least one amino acid of the APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25; or at least one amino acid of the APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7 and contacts at least one amino acid of the APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.

[07] A method for inhibiting a ubiquitination activity of an APC is carried out by administering an effective amount of an analog of the C-terminal IR motif of an activator of an APC to a cell to inhibit the degradation of a substrate of an APC or to to induce a cell cycle checkpoint. The analog contacts a component of a tetratricopeptide repeats (TPR) subcomplex of an APC such as APC3/Cdc27, APC6, APC7, or APC8. [08] An IR tail analog that inhibits APC is useful to identify other antiproliferative agents the mechanism of which involve the same or similar contact regions of the APC. Accordingly, a method of identifying an antiproliferative agent is carried out by contacting an APC complex with a candidate compound in the presence of TAME (and in the absence of TAME as a control. A decrease in binding of the candidate compound to the APC in the presence of TAME compared to the level of binding in its absence indicates that the compound interferes with the APC in a manner analogous to TAME and therefore inhibits cell proliferation. [09] A method of screening for a small molecule inhibitor of an APC includes the steps of

(a) identifying a test compound, wherein the test compound is an analog of an IR motif;

(b) combining either the test compound or a control compound with an activated APC and at least one labeled substrate of APC; and (c) determining the amount of labeled substrate present following the addition of either the test compound or the control compound. An increase in the amount of the labeled substrate following the addition of the test compound compared to the amount of labeled substrate present following the addition of the control compound indicates that the test compound inhibits an activity of APC. The control compound is not an analog of an IR motif. The combining step occurs in vivo, in vitro, or ex vivo. An example of a labeled substrate is cyclin B-luciferase.

[10] The compositions describes herein are useful in treating a cell cycle disorder. The compounds are administered to a subject in need thereof in an effective amount to inhibit an activity of an anaphase promoting complex (APC). For example, the compounds activate a cell cycle checkpoint such as the SAC. Optionally, a second chemotherapeutic compound is administered.

[11] One advantage of APC inhibitors is that they are much less toxic than other antiproliferative agents since they do not disrupt other processes (eg, damage DNA or microtubules).

[12] Publications, U.S. patents and applications, GENBANK™/NCBI accession numbers, and all other references cited herein, are herby incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[13] Figure IA is a schematic diagram representing the structure of TAME and AAME.

[14] Figure IB is a Western Blot showing that TAME but not AAME establishes mitotic arrest in Xenopus extract as indicated by stabilization of Cdc27 phosphorylation and cyclin

B.

[15] Figure 1C is a Western Blot showing that TAME inhibits activation of APC-Cdc20 but does not directly inhibit the ligase activity. APC was immunoprecipitated from mitotic extract and tested in an in vitro ubiquitination assay. Adding 200 μM TAME but not AAME into the extract inhibited APC activity. Adding 200 μM TAME or AAME into the ubiquitination reaction system did not inhibit APC.

[16] Figure ID is a Western Blot showing that TAME inhibits activation of APC-Cdhl but does not directly inhibit the ligase activity. Interphase extract was pre-incubated with 200 μM TAME or AAME for 30 minutes before addition of recombinant Cdhl. APC was then immunoprecipitated and tested in an in vitro ubiquitination assay. Pre-incubation with TAME but not AAME inhibited APC activity. Adding 200 μM TAME or AAME into the ubiquitination reaction system did not inhibit APC.

[17] Figure 2 A is a graph of the results of a luciferase assay showing the dose-dependent inhibition of cyclin B-luciferase degradation in mitotic extract by TAME. Different concentrations of TAME were added to mitotic Xenopus extract containing a cyclin B- luciferase reporter. Samples were collected at 60 minutes and the remaining reporter level was measured by luminescence. Interphase extract was used as a negative control.

[18] Figure 2B is a series of schematic diagrams representing the structures of TAME derivatives.

[19] Figure 2C is a graph showing the results of a luciferase assay in which the derivatives shown in figure 2B were tested at 200 μM

[20] Figure 3 is a graph showing the counts per minute (cpm) of all protein precipitated from Xenopus extract supplemented with DMSO or 200 μM TAME and baculovirus- expressed and purified ³⁵S-labeled Cyclin B, which was first ubiquitinated by APC in an in vitro ubiquitination system. At indicated time points, all protein was precipitated and the level of radioactivity in supernatant was measured by scintillation counting. MG262 was used as a positive control.

[21] Figure 4 is a ubiquitization assay in which recombinant Cdhl was added into interphase extract with or without preincubation with 200 μM TAME for 1.5 h followed by

APC immunoprecipitation.

[22] Figure 5A TAME inhibits Cdc20 association with mitotic APC in a dose-dependent manner. APC was immunoprecipitated from mitotic extract supplemented with different concentrations of TAME as indicated and the amount of Cdc27 and Cdc20 was analyzed by

Western blot. Numbers indicate the relative amount of Cdc20 normalized to that of Cdc27.

[23] Figure 5B TAME inhibits Cdhl association with interphase APC in a dose-dependent manner. Interphase extract was pre-incubated with different concentrations of TAME as indicated for 30 min before addition of recombinant Cdhl. APC was then immunoprecipitated and the amount of Cdc27 and Cdhl was analyzed by Western blot. Numbers indicate the relative amount of Cdhl normalized to that of Cdc27. [24] Figure 5C Cdc20 binding to mitotic APC is highly dynamic. APC-Cdc20 was first immunoprecipitated from mitotic extract and then resuspended in extract containing 200 μM TAME or AAME. Beads were re-isolated, washed and blotted for Cdc27 and Cdc20. [25] Figure 5D Cdc20 binding to interphase APC is not dynamic. APC-Cdc20 was first immunoprecipitated from interphase extract and then resuspended in extract containing 200 μM TAME or AAME. Beads were re-isolated, washed and blotted for Cdc27 and Cdc20. [26] Figure 5E APC-Cdc20 is a stable complex in XB buffer. APC-Cdc20 was first immunoprecipitated from interphase or mitotic extract and then resuspended in XB containing 200 μM TAME or AAME or 1% DMSO. Beads were re-isolated, washed and blotted for Cdc27 and Cdc20.

[27] Figure 5F Cdhl binding to interphase APC is not dynamic. Recombinant Cdhl was added into interphase extract. APC-Cdhl was then immunoprecipitated and resuspended in extract containing 200 μM TAME or AAME. Beads were re-isolated, washed and blotted for Cdc27 and Cdhl.

[28] Figure 6A TAME inhibits the interaction between APC and an IR peptide resin. Top panel: Iodoacetyl resin was coupled with cysteine, a 21aa C-terminal peptide of Cdhl or a 19aa peptide missing the C-terminal IR tail. The resin was incubated with interphase extract and the amount of Cdc27 bound on resin was analyzed. Bottom panel: Iodoacetyl resin coupled with the 21aa IR peptide was incubated with interphase extract supplemented with DMSO, 200 μM TAME or AAME and the amount of Cdc27 bound on resin was analyzed. [29] Figure 6B IR peptide labeled with a photoactive crosslinker crosslinks to a specific subset of APC subunits. Left: Coomassie stain of APC immunopurified from interphase extract with Cdc27 antibody. Right: APC subunits that got crosslinked by the labeled IR peptide visualized by streptavidin-HRP blot.

[30] Figure 6C TAME inhibits IR peptide crosslinking to APC subunits. Crosslinking was performed in the presence of different concentrations of TAME or AAME as indicated. [31] Figure 6D ³H TAME directly binds to Xenopus APC. Interphase extract was subjected to three rounds of immunoprecipitation with Cdc27 antibody. The amount of Cdc27 pulled down at each round was shown by Western blot. 200 nM of H TAME was added into extract that had undergone zero, one or two rounds of immunoprecipitation before another round of Cdc27 immunoprecipitation. The specific counts associated with the beads were determined by scintillation counting. [32] Figure 6E Unlabeled TAME but not AAME can compete with ³H TAME for binding to Xenopus APC. 200 nM ³H TAME was added into interphase extract with different concentrations of unlabeled TAME or AAME as indicated before Cdc27 immunoprecipitation. The specific counts associated with the beads were determined by scintillation counting.

[33] Figure 6F ³H TAME directly binds to human APC. The same experiment was done as described in 3C using asynchronous HeIa cell lysate.

[34] Figure 7 A Cdc27 but not Apc4 is crosslinked by labeled IR peptide labeled with a photoactive crosslinker. Crosslinking assay was performed with interphase and mitotic APC and crosslinked subunits were visualized by streptavidin-HRP blot. The shifted band represents mitotic phosphorylation of Cdc27.

[35] Figure 7B Crosslinking is IR tail specific. Crosslinking assay was performed in the presence of excess unlabeled IR peptide (lane 2), no UV illumination (lane 3) or with labeled

ΔIR peptide.

[36] Figure 8 The ability of unlabeled TAME derivates to compete with ³H TAME for binding to APC agrees with the structure-activity relationship. 200 nM ³H TAME was added into interphase extract with 10 μM of unlabeled TAME derivatives as indicated before Cdc27 immunoprecipitation. The specific counts associated with the beads were determined by scintillation counting. Their relative ability to compete largely agrees with their relative activity in the luciferase assay as shown in Figure 2C.

DETAILED DESCRIPTION

[37] The invention provides the first reported small molecule inhibitor of the APC. The Anaphase-Promoting Complex (APC) is a multisubunit ubiquitin ligase that regulates the timing of mitotic exit and the Gl /S transition. In prometaphase, the APC is activated by Cdc20, leading to ubiquitination and degradation of Nek2A and cyclin A. However, ubiquitination of other APC substrates is inhibited by the Spindle Assembly Checkpoint (SAC) until chromosomes have achieved proper bipolar attachment to the mitotic spindle. Once the SAC is satisfied, ubiquitination and degradation of securin and cyclin B lead to chromosome segregation and mitotic exit. In telophase, another APC activator, Cdhl, replaces Cdc20 and maintains APC activity during Gl.

[38] Cdc20 and Cdhl activate the APC and are important for recruiting substrates to the APC. The activator proteins share several evolutionarily conserved motifs, including an N- terminal C-box (comprising the consensus sequence DRFYIPXR and SEQ ID NO: 26), seven WD40 repeats (also known as WD or beta-transducin repeats of about 40 amino acids, often terminating in a WD dipeptide and containing 4-16 repeating units that together form a circular beta-propellar structure), and a C-terminal IR tail (a C-terminal region including one or more IR dipeptide motifs). Whereas the WD40 domain may interact simultaneously with substrates and the APC, the C-box and the IR tail are specifically involved in APC binding. The IR tail of Cdhl interacts with multiple APC subunits, including Cdc27 and Apc7. Deletion of the IR tail of Cdhl compromises its ability to activate human APC in vitro, and is lethal in budding yeast lacking Sicl. However, deletion of IR tail of Cdc20 does not affect the viability of wild-type budding yeast, and thus, does not seem to be strictly required for APC activation. Instead, the IR tail may be important for regulating Cdc20 abundance, as Cdc20ΔIR accumulates to higher levels than the wild-type protein. Thus while the IR tail seems to be critical for Cdhl recruitment and activation of the APC, the specific role of Cdc20's IR tail in APC binding and activation remains unclear.

[39] Phenotypic chemical genetic screens identify small molecules that are useful for dissecting the regulation of complex systems as well as novel targets for drug development. However, determining the precise mechanism of action of compounds discovered in phenotypic screens can be challenging. Method of the invention identify APC as the target of a small molecule discovered in a phenotypic screen for inhibitors of cyclin proteolysis in Xenopus extracts and use this compound as a tool to study APC regulation in Xenopus extracts and human cells.

[40] The methods of the invention are used to show that TAME, a compound identified previously in a high-throughput screen for inhibitors of cyclin proteolysis in Xenopus extracts (Verna, R. et al. 2004. Science 306(5693): 117), blocks APC activation by competitively inhibiting the association of the IR tail of Cdc20 and Cdhl with the APC. The data provided indicate that the IR tail of Cdc20 is critical for binding to the APC in mitotic Xenopus extract. The requirement for an IR-dependent recruitment mechanism stems from the dynamic nature of Cdc20 binding to mitotic APC. There is an activity in mitotic Xenopus extracts that actively dissociates Cdc20 from the APC, thereby sensitizing the system to the inhibitory effects of TAME. Because Cdhl binding to interphase APC is not dynamic, TAME is a less effective inhibitor of Cdhl association with interphase APC in the Xenopus system. [41] A key aspect of TAME function is that it can inhibit APC activation without activating the spindle checkpoint. For example, TAME blocks APC activation in the Xenopus system which lacks an intact spindle checkpoint. However in mammalian cells, a cell-permeable derivative of the compound arrests cells in mitosis through two mechanisms: direct APC inhibition, and by preventing inactivation of the spindle checkpoint. [42] The discovery of TAME' s mechanism of action sheds new light on how Cdc20 binding to the APC is regulated. It has been found that Cdc20 associates with interphase APC in Xenopus extracts, conflicting with previous reports suggesting that APC must be mitotically phosphorylated to interact with Cdc20 (Kraft, C. et al. 2003. EMBO J.22(24):6598; Kramer, E.R. et al. 2000. MoI Biol Cell. 11(5): 1555). One difference is that association of endogenous Xenopus Cdc20 rather than exogenous protein was examined in the present experiments (Kramer, E.R. et al. 2000. MoI Biol Cell. 11(5): 1555). Factors present in extracts or cells, such as chaperones involved in folding of Cdc20, may therefore be important for efficient loading of Cdc20 onto interphase APC. However, in agreement with others, it was observed that the steady state level of Cdc20 bound to the APC increases during mitosis Fang, G. et al. 1998. Genes Dev. 12(12):1871; Kallio, M. et al. 1998. J Cell Biol 141(6), 1393; Kramer, E.R. et al. 1998. Curr Biol. 8(22),1207). Provided the results of the experiments described herein demonstrating that the rate of Cdc20 dissociation increases in mitosis, there must also be a corresponding increase in the rate of Cdc20 association with the APC during mitosis, which may require mitotic phosphorylation of APC or Cdc20 (Kraft, C. et al. 2003. EMBO J.22(24):6598; Kramer, E.R. et al. 2000. MoI Biol Cell. 11(5): 1555). The observed dissociation phenomenon also explains why Cdc20 association with kinetochores during mitosis is also highly dynamic, and why Cdc20 exists predominantly in a high molecular weight complex during interphase with a free pool of Cdc20 appearing in mitosis.

[43] Despite a common biochemical target, TAME induces mitotic arrest through distinct mechanisms in Xenopus extract and mammalian cells. Whereas mitotic arrest in Xenopus extracts is a consequence of inhibition of Cdc20 association with the APC, the arrest in mammalian cells depends partially on the SAC, and occurs despite only a 30% reduction in Cdc20 bound to the APC.

APC Proteins

[44] Pharmaceutical compositions and compounds of the invention comprise an analog of the C-terminal IR motif of an activator of an APC. Thus, analogs of the invention contact one or proteins of the APC. In one aspect of the invention, analogs bind one or proteins of the APC either reversibly or irreversibly. In another aspect of the invention, one or more analogs bind one or proteins of the APC either reversibly or irreversibly. Alternatively or in addition, analogs of the invention contact or bind multiple proteins of the APC. In a preferred embodiment of the invention, analogs contact or bind discrete three dimentional surfaces that are exposed to the C-terminal IR motif of an activator of an APC. Contemplated exposed three dimentional surfaces are comprised of sequences that are either contiguous or not contiguous within the amino acid sequences provided below.

[45] Essential amino acids required for binding to a C-terminal IR motif of an activator of an APC or analog of the invention thereof are determined by deletion or substitution analysis, wherein the elminiation of binding between an analog or APC activator and at least one APC protein indicates that the eliminated or substituted amino acid is essential to the binding of that analog or activator of the APC. Methods of deleting and substituting amino acids within the APC polypeptide sequences below and determining the binding efficacy of an activator of the APC or analog thereof can be performed by routine methods known to those of ordinary skill in the art. Finally, all polynucleotide sequences that encode the polypeptide sequences provided herein, are encompassed by the invention. [46] Human APC3/CDC27, isoform 1, is encoded by the following amino acid sequence

(NCBI Accession No. NP OOl 107563 and SEQ ID NO: 1):

1 mtvlqepvqa aiwqalnhya yrdavflaer lyaevhseea lfllatcyyr sgkaykayrl 61 lkghscttpq ckyllakccv dlsklaegeq llsggvfnkq kshddivtef gdsacftlsl 121 lghvycktdr lakgsecyqk slslnpflws pfeslceige kpdpdqtfkf tslqnfsncl 181 pnscttqvpn hslshrqpet vltetpqdti elnrlnless nskyslntds svsyidsavi 241 spdtvplgtg tsilskqvqn kpktgrsllg gpaalspltp sfgilpletp spgdgsylqn 301 ytntppvidv pstgapskkt frvlqsvaπ gqtgtksvfs qsgnsrevtp llaqtqssgp 361 qtsttpqvls ptitsppnal prrssrlfts dssttkensk klkmkfppki pnrktksktn 421 kggitqpnm dsleitklds siisegkist ltpqiqafnl qkaaaeglms llremgkgyl 481 alcsynckea imlshlpsh hyntgwvlcq igrayfelse ymqaerifse vrπenyrve 541 gmeiysttlw hlqkdvalsv lskdltdmdk nspeawcaag ncfslqrehd laikffqrai 601 qvdpnyayay tllghefvlt eeldkalacf rnairvnprh ynawyglgmi yykqekfsla 661 emhfqkaldi npqssvllch lgvvqhalkk sekaldtlnk aividpknpl ckfhrasvlf 721 anekyksalq eleelkqivp keslvyflig kvykklgqth lalmnfswam dldpkgannq 781 ikeaidkryl pddeepitqe eqimgtdesq essmtdaddt qlhaaesdef

[47] Human APC3/CDC27, isoform 2, is encoded by the following amino acid sequence (NCBI Accession No. NP 001247 and SEQ ID NO: 2):

1 mtvlqepvqa aiwqalnhya yrdavflaer lyaevhseea lfllatcyyr sgkaykayrl 61 lkghscttpq ckyllakccv dlsklaegeq llsggvfnkq kshddivtef gdsacftlsl 121 lghvycktdr lakgsecyqk slslnpflws pfeslceige kpdpdqtfkf tslqnfsncl 181 pnscttqvpn hslshrqpet vltetpqdti elnrlnless nskyslntds svsyidsavi 241 spdtvplgtg tsilskqvqn kpktgrsllg gpaalspltp sfgilpletp spgdgsylqn 301 ytntppvidv pstgapskks vaπgqtgtk svfsqsgnsr evtpilaqtq ssgpqtsttp 361 qvlsptitsp pnalprrssr lftsdssttk enskklkmkf ppkipnrktk sktnkggitq 421 pnmdsleit kldssiiseg kistitpqiq afnlqkaaae glmsllremg kgylalcsyn 481 ckeainilsh lpshhyntgw vlcqigrayf elseymqaer lfsevrπen yrvegmeiys 541 ttlwhlqkdv alsvlskdlt dmdknspeaw caagncfslq rehdiaikff qraiqvdpny 601 ayaytllghe fvlteeldka lacfrnairv nprhynawyg lgmiyykqek fslaemhfqk 661 aldmpqssv llchigvvqh alkksekald tlnkaividp knplckfhra svlfanekyk 721 salqeleelk qivpkeslvy fligkvykkl gqthlalmnf swamdldpkg annqikeaid 781 krylpddeep ltqeeqimgt desqessmtd addtqlhaae sdef [48] Human APC3/CDC27 is encoded by the following amino acid sequence (NCBI Accession No. AAHl 1656 and SEQ ID NO: 3):

1 mtvlqepvqa aiwqalnhya yrdavflaer lyaevhseea lfllatcyyr sgkaykayrl 61 lkghscttpq ckyllakccv dlsklaegeq llsggvfnkq kshddivtef gdsacftlsl 121 lghvycktdr lakgsecyqk slslnpflws pfeslceige kpdpdqtfkf tslqnfsncl 181 pnscttqvpn hslshrqpet vltetpqdti elnrlnless nskyslntds svsyidsavi 241 spdtvplgtg tsilskqvqn kpktgrsllg gpaalspltp sfgilpletp spgdgsylqn 301 ytntppvidv pstgapskkt frvlqsvaπ gqtgtksvfs qsgnsrevtp llaqtqssgp 361 qtsttpqvls ptitsppnal prrssrlfts dssttkensk klkmkfppei pnrktksktn 421 kggitqpnm dsleitklds siisegkist ltpqiqafnl qkaaaeglms llremgkgyl 481 alcsynckea imlshlpsh hyntgwvlcq igrayfelse ymqaerifse vrπenyrve 541 gmeiysttlw hlqkdvalsv lskdltdmdk nspeawcaag ncfslqrehd laikffqrai 601 qvdpnyayay tllghefvlt eeldkalacf rnairvnprh ynawyglgmi yykqekfsla 661 emhfqkaldi npqssvllch lgvvqhalkk sekaldtlnk aividpknpl ckfhrasvlf 721 anekyksalq eleelkqivp keslvyflig kvykklgqth lalmnfswam dldpkgannq 781 ikeaidkryl pddeepitqe eqimgtdesq essmtdaddt qlhaaesdef

[49] Human APC6/CDC16 is encoded by the following amino acid sequence (NCBI Accession No. AAH10875 and SEQ ID NO: 4):

1 meepmkrlf ekylkdesgf kdpssdwems qssikssicl lrgkiydald nrtlatysyk 61 ealkldvycf eafdlltshh mltaqeekel leslplsklc neeqellrfl fenklkkynk 121 psetvipesv dglqenldvv vslaerhyyn cdfkmcyklt svvmekdpfh asclpvhigt 181 lvelnkanel fylshklvdl ypsnpvswfa vgcyylmvgh kneharryls kattlektyg 241 pawiayghsf avesehdqam aayftaaqlm kgchlpmlyi gleygltnns klaerffsqa 301 lsiapedpfv mhevgvvafq ngewktaekw fldalekika ignevtvdkw epllnnlghv 361 crklkkyaea ldyhrqalvl lpqnastysa igyihslmgn fenavdyfht alglrrddtf 421 svtmlghcie myigdseayi gadikdklkc ydfdvhtmkt lkmisppwd frefevekqt 481 aeetgltple tsrktpdsrp sleetfeiem nesdmmlets msdhst

[50] Human APC6/CDC16 is encoded by the following amino acid sequence (NCBI Accession No. NP 001072113 and SEQ ID NO: 5):

1 mnlerlrkrv rqyldqqqyq salfwadkva slsreepqdi ywlaqclylt aqyhraahal 61 rsrkldklye acrylaarch yaakehqqal dvldmeepm krlfekylkd esgfkdpssd 121 wemsqssiks sicllrgkiy daldnrtlat ysykealkld vycfeafdll tshhmltaqe 181 ekelleslpl sklcneeqel lrflfenklk kynkpsetvi pesvdglqen ldvvvslaer 241 hyyncdfkmc ykltsvvmek dpfhasclpv higtlvelnk anelfylshk lvdlypsnpv 301 swfavgcyyl mvghknehar rylskattle ktygpawiay ghsfaveseh dqamaayfta 361 aqlmkgchlp mlyigleygl tnnsklaerf fsqalsiape dpfvmhevgv vafqngewkt 421 aekwfldale kikaignevt vdkwepllnn lghvcrklkk yaealdyhrq alvlipqnas 481 tysaigyihs lmgnfenavd yfhtalglrr ddtfsvtmlg hciemyigds eayigadikd 541 klkcydfdvh tmktlkniis ppwdfrefev ekqtaeetgl tpletsrktp dsrpsleetf 601 eiemnesdmm letsmsdhst

[51] Human APC6/CDC16 is encoded by the following amino acid sequence (NCBI Accession No. NP 003894 and SEQ ID NO: 6):

[52] Human APC6/CDC16 is encoded by the following amino acid sequence (NCBI Accession No. Q13042 and SEQ ID NO: 7):

[53] Human APC7 is encoded by the following amino acid sequence (NCBI Accession

No. AAH98362 and SEQ ID NO: 8):

1 nvidhvrdma aaglhsnvrl lssllltmsn nnpelfsppq kyqllvyhad slfhdkeyrn 61 avskytmalq qkkalsktsk vrpstgnsas tpqsqclpse levkykmaec ytmlkqdkda 121 iaildgipsr qrtpkmmml anlykkagqe rpsvtsykev lrqcplalda llgllslsvk 181 gaevasmtmn viqtvpnldw lsvwikayaf vhtgdnsrai sticslekks llrdnvdllg 241 sladlyfrag dnknsvlkfe qaqmldpyli kgmdvygyll aregrledve nlgcrlfnis 301 dqhaepwvvs gchsfyskry sralylgaka lqlnsnsvqa lllkgaalrn mgrvqeaiih 361 freairlapc rldcyeglie cylasnsire amvmannvyk tlganaqtlt llatvcledp 421 vtqekaktll dkaltqrpdy lkavvkkael lsreqkyedg iallrnalan qsdcvlhπl 481 gdflvavney qeamdqysia lsldpndqks legmqkmeke esptdatqee dvddmegsge 541 egdlegsdse aaqwadqeqw fgmq

[54] Human APC7 is encoded by the following amino acid sequence (NCBI Accession No. AAH98264 and SEQ ID NO: X):

[55] Human APC7 is encoded by the following amino acid sequence (NCBI Accession

No. AAH98295 and SEQ ID NO: 10):

1 nvidhvrdma aaglhsnvrl lssllltmsn nnpelfsppq kyqllvyhad slfhdkeyrn 61 avskytmalq qkkalsktsk vrpstgnsas tpqsqclpse levkykmaec ytmlkqdkda 121 iaildgipsr qrtpkmmml anlykkagqe rpsvtsykev lrqcplalda llgllslsvk 181 gaevasmtmn viqtvpnldw lsvwikayaf vhtgdnsrai sticslekks llrdnvdllg 241 sladlyfrag dnknsvlkfe qaqmldpyli kgmdvygyll aregrledve nlgcrlfnis 301 dqhaepwvvs gchsfyskry sralylgaka lqlnsnsvqa lllkgaalrn mgrvqeaiih 361 freairlapc rldcyeglie cylasnsire amvmannvyk tlganaqtlt llatvcledp 421 vtqekaktll dkaltqrpdy lkavvkkael lsreqkyedg iallrnalan qsdcvlhπl 481 gdflvavney qeamdqysia lsldpndqks legmqkmeke esptdatqee dvddmegsge 541 egdlegsdse aaqwadqeqw fgmq [56] Human APC7 is encoded by the following amino acid sequence (NCBI Accession o. AAH99732 and SEQ ID NO: 11):

[57] Human APC7 is encoded by the following amino acid sequence (NCBI Accession o. AAI41849 and SEQ ID NO: 12):

1 mnvidhvrdm aaaglhsnvr llssllltms nnnpelfspp qkyqllvyha dslfhdkeyr 61 navskytmal qqkkalskts kvrpstgnsa stpqsqclps eievkykmae cytmlkqdkd 121 aiaildgips rqrtpkmmm lanlykkagq erpsvtsyke vlrqcplald ailgllslsv 181 kgaevasmtm nviqtvpnld wlsvwikaya fvhtgdnsra isticslekk sllrdnvdll 241 gsladlyfra gdnknsvlkf eqaqmldpyl lkgmdvygyl laregrledv enlgcrlfni 301 sdqhaepwvv sgchsfyskr ysralylgak aiqlnsnsvq alllkgaalr nmgrvqeaii 361 hfreairlap crldcyegli ecylasnsir eamviannvy ktlganaqtl tllatvcled 421 pvtqekaktl ldkaltqrpd yikavvkkae llsreqkyed giallrnala nqsdcvlhπ 481 lgdflvavne yqeamdqysi alsldpndqk slegmqkmek eesptdatqe edvddmegsg 541 eegdlegsds eaaqwadqeq wfgmq

[58] Human APC7 is encoded by the following amino acid sequence (NCBI Accession o. AAIl 1799 and SEQ ID NO: 13):

1 nvidhvrdma aaglhsnvrl lssllltmsn nnpelfsppq kyqllvyhad slfhdkeyrn 61 avskytmalq qkkalsktsk vrpstgnsas tpqsqclpse levkykmaec ytmlkqdkda 121 iaildgipsr qrtpkmmml anlykkagqe rpsvtsykev lrqcplalda llgllslsvk 181 gaevasmtmn viqtvpnldw lsvwikayaf vhtgdnsrai sticslekks llqdnvdllg 241 sladlyfrag dnknsvlkfe qaqmldpyli kgmdvygyll aregrledve nlgcrlfnis 301 dqhaepwvvs gchsfyskry sralylgaka lqlnsnsvqa lllkgaalrn mgrvqeaiih 361 freairlapc rldcyeglie cylasnsire amvmannvyk tlganaqtlt llatvcledp 421 vtqekaktll dkaltqrpdy lkavvkkael lsreqkyedg iallrnalan qsdcvlhπl 481 gdflvavney qeamdqysia lsldpndqks legmqkmeke esptdatqee dvddmegsge 541 egdlegsdse aaqwadqeqw fgm

[59] Human APC7 is encoded by the following amino acid sequence (NCBI Accession o. AAI48237 and SEQ ID NO: 14):

1 mnvidhvrdm aaaglhsnvr llssllltms nnnpelfspp qkyqllvyha dslfhdkeyr 61 navskytmal qqkkalskts kvrpstgnsa stpqsqclps eievkykmae cytmlkqdkd 121 aiaildgips rqrtpkmmm lanlykkagq erpsvtsyke vlrqcplald ailgllslsv 181 kgaevasmtm nviqtvpnld wlsvwikaya fvhtgdnsra isticslekk sllqdnvdll 241 gsladlyfra gdnknsvlkf eqaqmldpyl lkgmdvygyl laregrledv enlgcrlfni 301 sdqhaepwvv sgchsfyskr ysralylgak aiqlnsnsvq alllkgaalr nmgrvqeaii 361 hfreairlap crldcyegli ecylasnsir eamvmannvy ktlganaqtl tllatvcled 421 pvtqekaktl ldkaltqrpd yikavvkkae llsreqkyed giallrnala nqsdcvlhπ 481 lgdflvavne yqeamdqysi alsldpndqk slegmqkmek eesptdatqe edvddmegsg 541 eegdlegsds eaaqwadqeq wfgm

[60] Human APC7 is encoded by the following amino acid sequence (NCBI Accession o. NP 057322 and SEQ ID NO: 15):

1 mnvidhvrdm aaaglhsnvr llssllltms nnnpelfspp qkyqllvyha dslfhdkeyr 61 navskytmal qqkkalskts kvrpstgnsa stpqsqclps eievkykmae cytmlkqdkd 121 aiaildgips rqrtpkmmm lanlykkagr erpsvtsyke vlrqcplald ailgllslsv 181 kgaevasmtm nviqtvpnld wlsvwikaya fvhtgdnsra isticslekk sllrdnvdll 241 gsladlyfra gdnknsvlkf eqaqmldlyl lkgmdvygyl laregrledv enlgcrlfni 301 sdqhaepwvv sgchsfyskr ysralylgak aiqlnsnsvq alllkgaalr nmgrvqeaii 361 hfreairlap crldcyegli ecylasnsir eamvmannvy ktlganaqtl tllatvcled 421 pvtqekaktl ldkaltqrpd yikavvkkae llsreqkyed giallrnala nqsdcvlhri 481 lgdflvavne yqeamdqysi alsldpndqk slegmqkmek eesptdatqe edvddmegsg 541 eegdlegsds eaaqwadqeq wfgmq

[61] Human APC7 is encoded by the following amino acid sequence (NCBI Accession

No. Q9UJX3 and SEQ ID NO: 16):

1 mnvidhvrdm aaaglhsnvr llssllltms nnnpelfspp qkyqllvyha dslfhdkeyr 61 navskytmal qqkkalskts kvrpstgnsa stpqsqclps eievkykmae cytmlkqdkd 121 aiaildgips rqrtpkmmm lanlykkagq erpsvtsyke vlrqcplald ailgllslsv 181 kgaevasmtm nviqtvpnld wlsvwikaya fvhtgdnsra isticslekk sllrdnvdll 241 gsladlyfra gdnknsvlkf eqaqmldpyl lkgmdvygyl laregrledv enlgcrlfni 301 sdqhaepwvv sgchsfyskr ysralylgak aiqlnsnsvq alllkgaalr nmgrvqeaii 361 hfreairlap crldcyegli ecylasnsir eamvmannvy ktlganaqtl tllatvcled 421 pvtqekaktl ldkaltqrpd yikavvkkae llsreqkyed giallrnala nqsdcvlhri 481 lgdflvavne yqeamdqysi alsldpndqk slegmqkmek eesptdatqe edvddmegsg 541 eegdlegsds eaaqwadqeq wfgmq

[62] Human APC7 is encoded by the following amino acid sequence (NCBI Accession

No. AAF05754 and SEQ ID NO: 17):

1 mnvidhvrdm aaaglhsnvr llssllltms nnnpelfspp qkyqllvyha dslfhdkeyr 61 navskytmal qqkkalskts kvrpstgnsa stpqsqclps eievkykmae cytmlkqdkd 121 aiaildgips rqrtpkmmm lanlykkagr erpsvtsyke vlrqcplald ailgllslsv 181 kgaevasmtm nviqtvpnld wlsvwikaya fvhtgdnsra isticslekk sllrdnvdll 241 gsladlyfra gdnknsvlkf eqaqmldlyl lkgmdvygyl laregrledv enlgcrlfni 301 sdqhaepwvv sgchsfyskr ysralylgak aiqlnsnsvq alllkgaalr nmgrvqeaii 361 hfreairlap crldcyegli ecylasnsir eamvmannvy ktlganaqtl tllatvcled 421 pvtqekaktl ldkaltqrpd yikavvkkae llsreqkyed giallrnala nqsdcvlhri 481 lgdflvavne yqeamdqysi alsldpndqk slegmqkmek eesptdatqe edvddmegsg 541 eegdlegsds eaaqwadqeq wfgmq [63] Human APC7 is encoded by the following amino acid sequence (NCBI Accession

No. AAH02941 and SEQ ID NO: 18):

1 dnknsvlkfe qaqmldpyli kgmdvygyll aregrledve nlgcrlfnis dqhaepwvvs 61 gchsfyskry sralylgaka lqlnsnsvqa lllkgaalrn mgrvqeaiih freairlapc 121 rldcyeglie cylasnsire amvmannvyk tlganaqtlt llatvcledp vtqekaktll 181 dkaltqrpdy lkavvkkael lsreqkyedg iallrnalan qsdcvlhπl gdflvavney 241 qeamdqysia lsldpndqks legmqkmeke esptdatqee dvddmegsge egdlegsdse 301 aaqwadqeqw fgmq

[64] Human APC7 is encoded by the following amino acid sequence (NCBI Accession No. AAH09498 and SEQ ID NO: 19):

1 ecytmlkqdk daiaildgip srqrtpkmm mlanlykkag qerpsvtsyk evlrqcplal 61 dailgllsls vkgaevasmt mnviqtvpnl dwlsvwikay afvhtgdnsr aisticslek 121 ksllrdnvdl lgsladlyfr agdnknsvlk feqaqmldpy likgmdvygy llaregrled 181 venlgcrlfn lsdqhaepwv vsgchsfysk rysralylga kaiqlnsnsv qalllkgaal 241 rnmgrvqeai ihfreairla pcrldcyegl iecylasnsi reamvmannv yktlganaqt 301 ltllatvcle dpvtqekakt lldkaltqrp dyikavvkka ellsreqkye dgiallrnal 361 anqsdcvlhr llgdflvavn eyqeamdqys lalsldpndq kslegmqkme keesptdatq 421 eedvddmegs geegdlegsd seaaqwadqe qwfgmq [65] Human APC8/CDC23 is encoded by the following amino acid sequence (NCBI Accession No. AAF05755 and SEQ ID NO: 20):

1 mvpvavtaav apvlsinsdf sdlreikkql lliagltrer gllhsskwsa elafslpalp 61 laelqppppi teedaqdmda ytlakayfdv keydraahfl hgcnskkayf lymysrylsg 121 ekkkddetvd slgplekgqv knealrelrv elskkhqare ldgfglylyg vvlrkldlvk 181 eaidvfveat hvlplhwgaw lelcnlitdk emlkflslpd twmkefflah lytelqliee 241 alqkyqnlid vgfskssyiv sqiavayhm rdidkalsif nelrkqdpyr ienmdtfsnl 301 lyvrsmksel sylahnlcei dkyrvetccv ignyyslrsq hekaalyfqr alklnprylg 361 awtlmgheym emkntsaaiq ayrhaievnk rdyrawyglg qtyeilkmpf yclyyyrrah 421 qlrpndsrml valgecyekl nqlveakkcy wrayavgdve kmalvklakl heqlteseqa 481 aqcyikyiqd lyscgeiveh leestafryl aqyyfkcklw deastcaqkc cafndtreeg 541 kallrqilql rnqgetptte vpapfflpas lsanntptrr vfplnlssvt p

[66] Human APC8/CDC23 is encoded by the following amino acid sequence (NCBI

Accession No. AAH10944 and SEQ ID NO: 21):

1 mvpvavtaav apvlsinsdf sdlreikkql lliagltrer gllhsskwsa elafslpalp 61 laelqppppi teedaqdmda ytlakayfdv keydraahfl hgcnskkayf lymysrylvr 121 ailkchsafs etsifrtngk vksfk

[67] Human APC8/CDC23 is encoded by the following amino acid sequence (NCBI

Accession No. AAH05258 and SEQ ID NO: 22):

[68] Human APC8/CDC23 is encoded by the following amino acid sequence (NCBI

Accession No. AAH17713 and SEQ ID NO: 23):

1 mvpvavtaav apvlsinsdf sdlreikkql lliagltrer gllhsskwsa elafslpalp 61 laelqppppi teedaqdmda ytlakayfdv keydraahfl hgcnskkayf lymysrylsg 121 ekkkddetvd slgplekgqv knealrelrv elskkhqare ldgfglylyg vvlrkldlvk 181 eaidvfveat hvlplhwgaw lelcnlitdk emlkflslpd twmkefflah lytelqliee 241 alqkyqnlid vgfskssyiv sqiavayhm rdidkalsif nelrkqdpyr ienmdtfsnl 301 lyvrsmksel sylahnlcei dkyrvetccv ignyyslrsq hekaalyfqr alklnprylg 361 awtlmgheym emkntsaaiq ayrhaievnk rdyrawyglg qtyeilkmpf yclyyyrrah 421 qlrpndsrml valgecyekl nqlveakkcy wrayavgdve kmalvklakl heqlteseqa 481 aqcyikyiqd lyscgeiveh leestafryl aqyyfkcklw deastcaqkc cafndtreeg 541 kallrqilql rnqgetptte vpapfflpas lsanntptrr vsplnlssvt p

[69] Human APC8/CDC23 is encoded by the following amino acid sequence (NCBI Accession No. NP_004652 and SEQ ID NO: 24):

1 maastsmvpv avtaavapvl smsdfsdlr eikkqlllia gltrergllh sskwsaelaf 61 slpalplael qppppiteed aqdmdaytla kayfdvkeyd raahflhgcn skkayflymy 121 srylsgekkk ddetvdslgp lekgqvknea lrelrvelsk khqareldgf glylygvvlr 181 kldlvkeaid vfveathvlp lhwgawlelc nlitdkemlk flslpdtwmk efflahiyte 241 lqlieealqk yqnlidvgfs kssyivsqia vayhnirdid kalsifnelr kqdpyπenm 301 dtfsnllyvr smkselsyla hnlceidkyr vetccvigny yslrsqheka alyfqralkl 361 nprylgawtl mgheymemkn tsaaiqayrh aievnkrdyr awyglgqtye ilkmpfycly 421 yyrrahqlrp ndsrmlvalg ecyeklnqlv eakkcywray avgdvekmal vklaklheql 481 teseqaaqcy ikyiqdiysc geivehlees tafrylaqyy fkcklwdeas tcaqkccafn 541 dtreegkall rqilqlrnqg etpttevpap fflpaslsan ntptrrvspl nlssvtp [70] Human APC8/CDC23 is encoded by the following amino acid sequence (NCBI Accession No. Q9UJX2 and SEQ ID NO: 25):

EXAMPLES

EXAMPLE 1 : TAME Inhibits APC Activation

[71] A library of 110,000 compounds was screened for inhibitors of APC-dependent proteolysis in mitotic Xenopus egg extract, as previously reported (Verna, R. et al. 2004. Science 306(5693): 117). Tosyl-L-Arginine Methyl Ester (TAME; Fig. IA) was identified as one of the most potent hits, with an IC50 of approximately 12 μM (Fig. 2A). TAME also inhibited cyclin-luciferase proteolysis stimulated by addition of Cdhl to interphase extract, but did not inhibit β-catenin-luciferase proteolysis stimulated by addition of axin (Verna, R. et al. 2004. Science 306(5693): 117), indicating that TAME is not a general inhibitor of the ubiquitin-proteasome system. A series of TAME derivatives were tested to establish a structure-activity relationship (Fig. 2B, C). Acetyl-L-Arginine Methyl Ester (AAME; Fig IA) emerged as the derivative with the lowest activity, and this compound was used as a negative control in subsequent experiments.

[72] It was next determined whether TAME is sufficient to induce mitotic arrest in Xenopus extract. Purified cyclin Bl/cdc2 complex (MPF) was added to interphase extract in the presence of DMSO, AAME, or TAME. All extracts entered mitosis within 15 min of MPF addition, as indicated by decreased mobility of the APC subunit Cdc27, a consequence of Cdc27 phosphorylation (Fig. IB). In the presence of DMSO or AAME, cyclin Bl was degraded by 30 min, resulting in MPF inactivation and dephosphorylation of Cdc27. In the presence of 200 μM TAME, however, cyclin Bland Cdc27 phosphorylation remained stable, indicating that TAME is capable of inducing mitotic arrest in Xenopus extract. Another APC substrate, cyclin A, was also stabilized by TAME in Xenopus extract.

[73] To identify the molecular target of TAME, it was determined whether TAME interferes with ubiquitination of cyclin Bl, or with the subsequent step of targeting ubiquitinated cyclin Bl to the proteasome. A purified MPF complex containing 35S-labeled cyclin Bl was ubiquitinated by APC in a reconstituted ubiquitination system, and then added to interphase Xenopus extract in the presence or absence of inhibitors. Degradation of pre -ubiquitinated cyclin Bl was equally efficient in the presence of DMSO or TAME, but was strongly inhibited by the proteasome inhibitor MG262 (Fig. 3), indicating that TAME does not inhibit proteasomal degradation of ubiquitinated cyclin Bl.

[74] To test if TAME inhibits APC activity, APC was immunopurified from mitotic Xenopus extract and added the compound directly to a reconstituted ubiquitination assay. Surprisingly, TAME showed no activity in this assay (Fig. 1C), indicating that it is not an inhibitor of the ligase activity per se. However, if TAME was added to mitotic Xenopus extract during immunoprecipitation, the isolated APC showed a dramatic loss of activity (Fig. 1C), suggesting that TAME acts as an inhibitor of APC activation. Because TAME is capable of stabilizing cyclin Bl in interphase extracts treated with Cdhl, it was also determined whether TAME could inhibit interphase APC activated by Cdhl. Again, TAME did not inhibit APC-Cdhl when added directly to a reconstituted assay; however, if TAME was added to interphase extract 30 minutes prior to the addition of Cdhl, considerable reduction in the level of cyclin Bl ubiquitination was observed (Fig. ID). Interestingly, if TAME and Cdhl were added simultaneously, TAME was less effective in blocking APC activation (Fig. 4). Together these findings indicate that TAME is capable of inhibiting activation of the APC mediated by either Cdc20 or Cdhl.

EXAMPLE 2: TAME Inhibits Activator Protein Binding to the APC [75] Because TAME inhibits APC activation, it was next determined whether TAME acts by interfering with activator protein association. APC was immunoprecipitated from mitotic Xenopus extract containing different concentrations of TAME and observed a dose- dependent reduction in the amount of Cdc20 associated with the APC (Fig. 5A). Similarly, TAME inhibited Cdhl binding to the APC, although the reduction was not as dramatic (Fig. 5B).

[76] Current models of APC regulation suggest that Cdc20 associates with the APC weakly during interphase and becomes more strongly associated with the APC as the complex becomes phosphorylated during mitosis (Kraft, C. et al. 2003. EMBO J.22(24):6598; Kramer, E.R. et al. 2000. MoI Biol Cell. 11(5): 1555). However, TAME is an effective inhibitor of cyclin proteolysis and Cdc20 association even when added to mitotic extracts in which Cdc20 is presumably stably bound to the APC. To understand the basis of this discrepancy, the dynamics of Cdc20 association with the APC were measured in interphase and mitotic extracts, using TAME as a tool to block reassociation of Cdc20 with the APC. APC was immunoprecipitated from mitotic extract and then resuspended the complex in mitotic extract containing TAME. It was found that Cdc20 dissociated from the APC very rapidly in mitotic extract, with a half-life of approximately 1 min (Fig. 5C). In contrast, although Cdc20 was capable of binding interphase Cdc20, its association was not dynamic (Fig. 5D). Dissociation of Cdc20 from mitotic APC appears to require an activity in the extract, as Cdc20 binding to mitotic APC remained stable in buffer (Fig. 5E). Finally, it was found that Cdhl binding to interphase APC is also not dynamic (Fig. 5F), explaining why pre -incubation enhances the ability of TAME to block Cdhl binding to the APC. In contrast, the high rate of Cdc20 dissociation from the APC during mitosis appears to render the system highly sensitive to the inhibitory effects of TAME, regardless of the timing of compound addition.

EXAMPLE 3: TAME Binds to the APC and Blocks IR-tail Interactions [77] Because TAME has a striking structural resemblance to Cdc20 and Cdhl 's IR tail, TAME may inhibit the binding of activator proteins to the APC by competing with the binding of the IR tail. To demonstrate this mechanism, a 21 amino acid C-terminal peptide of Cdhl was synthesized, which was previously used to recruit APC from HeLa cell lysate in an IR-dependent manner (Vodermaier, H.C. et al. 2003. Curr Biol. 13(17): 1459). It was found that this peptide could also recruit APC from interphase Xenopus extract in an IR-dependent manner (Fig. 6A). It was found that TAME could efficiently inhibit APC recruitment, whereas DMSO or AAME had no effect (Fig. 6A), suggesting that TAME can inhibit interactions between an IR tail-containing peptide and the APC.

To identify APC subunits that interact with TAME and the IR peptide, the IR peptide was conjugated to a photo-affinity reagent that covalently transfers biotin to proteins in the proximity of the labeled peptide. APC was immunoprecipitated from interphase Xenopus extract, incubated with the labeled peptide, and then induced to crosslink by UV illumination. Crosslinked proteins containing biotin were visualized by streptavidin-HRP blot. To identify specific subunits, the blot was aligned with a coomassie stain of purified APC, in which individual subunits were identified by mass spectrometry. It was found that while Apcl was weakly labeled, proteins in the TPR subcomplex, namely Cdc27, Cdcl6, Cdc23 and Apc7, were strongly crosslinked (Fig. 6B). This result is consistent with previous reports that the TPR subunits mediate Cdhl binding to the APC (Vodermaier, H.C. et al. 2003. Curr Biol. 13(17): 1459; Thornton, B.R. et al. 2006. Genes Dev. 20(4):449). Simliar studies were performed with mitotic APC, in which the mobility of Cdc27 is shifted due to phosphorylation 17 and confirmed that the peptide crosslinked to Cdc27 but not Apc4 (Fig. 7A). Crosslinking to the TPR subunits was strictly UV dependent and could be competed by excess unlabeled IR peptide (Fig. 7B). Furthermore, a labeled ΔIR peptide showed dramatically reduced crosslinking to APC subunits, indicating that crosslinking is IR-specific (Fig. 7B). The ability of TAME to block crosslinking of the IR-containing peptide to the APC was next determined. It was found that 20 μM TAME efficiently inhibited Cdc27 and Cdclβ labeling, but only slightly reduced Cdc23 and Apc7 labeling (Fig. 6C). At 200 μM, TAME strongly inhibited crosslinking to all APC subunits. The differential sensitivity to TAME suggests that different TPR subunits may have distinct affinities for the IR tail or for TAME. However, it is worth noting that the biotin label can be transferred to any subunit within close proximity of the peptide binding site, not necessarily only the subunit to which the IR tail is bound. For example, we observed some biotin transfer to the IgG light chain of the Cdc27 antibody, which could be inhibited by a high concentration of TAME. Nevertheless, the data strongly suggest that TAME antagonizes crosslinking by competing for binding to IR tail binding sites present in the TPR subcomplex of the APC.

[78] To directly determine whether TAME binds to the APC, 3H-TAME was added to interphase Xenopus extract, or to extract that had been partially or completely immunodepleted of the APC. The remaining APC was immunoprecipitated with Cdc27 antibody and the amount of bound TAME was measured by scintillation counting. In parallel, the amount of immunoprecipitated Cdc27 was determined by immunoblot. It was found that specific binding of 3H-T AME correlated with the amount of Cdc27 isolated from the extract (Fig. 6D). Moreover, unlabeled TAME competitively inhibited specific binding of 3H- TAME, whereas AAME did not (Fig. 6E). The ability of other TAME derivatives to compete with 3 H-TAME for APC binding correlated with their ability to inhibit degradation of cyclin B-luciferase in Xenopus extract (Fig. 8). Together these experiments indicate that TAME is capable of binding directly to Xenopus APC.

[79] It was next determined that TAME also binds to human APC. 3H-TAME was added to lysates from asynchronous HeLa cells partially or completely immunodepleted of APC, and again found that the specific binding of 3H-TAME correlated with the amount of APC that was isolated (Fig. 6F). Moreover, cold TAME competed efficiently with specific 3H- TAME binding but AAME did not (Fig. 6F). [80] Taken together, these results indicate that TAME is capable of binding to both Xenopus and human APC, where it antagonizes IR tail interactions to inhibit Cdc20 or Cdhl binding to the APC.

OTHER EMBODIMENTS

[81] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[82] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:CLAIMS

1. An anti-pro liferative composition comprising a non-peptide analog of the C-terminal IR motif of an activator of an anaphase promoting complex (APC).

2. A pharmaceutical composition comprising the compound of claim 1.

3. The compound of claim 1, wherein said analog is tosyl-L-arginine methylester (TAME).

4. The compound of claim 1, wherein said analog is tosyl-L-arginine methylester (TAME), tosyl-L-arginine amide (TAA), tosyl-L-lysine methylester (TLME), tosyl-L-arginine (TAOH), Benzoyl-L-arginine amide (BAA), tosyl-L-arginine t-butyl-ester (TATE), or Benzoyl-L-arginine methylester (BAME).

5. The compound of claim 1, wherein said compound inhibits the degradation of a substrate of an APC in a cell.

6. The compound of claim 1, wherein said cell is human.

7. The compound of claim 1, wherein said compound contacts a component of a tetratricopeptide repeats (TPR) subcomplex of an APC.

8. The compound of claim 7, wherein said component of a TPR subcomplex is APC3/Cdc27, APC6, APC7, or APC8.

9. The compound of claim 1, wherein said compound induces a cell cycle checkpoint.

10. The compound of claim 9, wherein said cell cycle checkpoint is the spindle assembly checkpoint (SAC).

11. The compound of claim 1, wherein said activator of an APC is Cdc20 or Cdhl.

12. The compound of claim 8, wherein said compound contacts at least one amino acid of said APC3 polypeptide of SEQ ID NO: 1, 2 , or 3.

13. The compound of claim 8, wherein said compound contacts at least one amino acid of said APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7.

14. The compound of claim 8, wherein said compound contacts at least one amino acid of said APC7 polypeptide of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

15. The compound of claim 8, wherein said compound contacts at least one amino acid of said APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.

16. The compound of claim 8, wherein said compound contacts at least one amino acid of said APC3 polypeptide of SEQ ID NO: 1, 2, or 3 and contacts at least one amino acid of said APC7 polypeptide of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

17. The compound of claim 16, wherein said compound further contacts at least one amino acid of said APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7.

18. The compound of claim 16, wherein said compound further contacts at least one amino acid of said APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.

19. The compound of claim 16, wherein said compound further contacts at least one amino acid of said APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7 and contacts at least one amino acid of said APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.

20. A method for inhibiting a ubiquitination activity of an APC comprising administering an effective amount of an analog of the C-terminal IR motif of an activator of an APC to a cell to inhibit the degradation of a substrate of an APC.

21. A method for inhibiting a ubiquitination activity of an APC comprising administering an effective amount of an analog of the C-terminal IR motif of an activator of an APC to a cell to induce a cell cycle checkpoint.

22. The method of claim 20 or 21, wherein said analog is tosyl-L-arginine methylester (TAME).

23. The method of claim 20 or 21, wherein said analog is tosyl-L-arginine methylester (TAME), tosyl-L-arginine amide (TAA), tosyl-L-lysine methylester (TLME), tosyl-L- arginine (TAOH), acetyl-L-arginine methylester (AAME), Benzoyl-L-arginine amide (BAA), tosyl-L-arginine t-butyl-ester (TATE), or Benzoyl-L-arginine methylester (BAME).

24. The method of claim 20, wherein said cell is human.

25. The method of claim 20, wherein said analog contacts a component of a tetratricopeptide repeats (TPR) subcomplex of an APC.

26. The method of claim 25, wherein said component of a TPR subcomplex is APC3/Cdc27, APC6, APC7, or APC8.

27. The method of claim 21, wherein said cell cycle checkpoint is the spindle assembly checkpoint (SAC).

28. The method of claim 20 or 21, wherein said activator of an APC is Cdc20 or Cdhl.

29. A method of screening for a small molecule inhibitor of an APC, comprising the steps of:

(a) identifying a test compound, wherein said test compound is an analog of an IR motif;

(b) combining either said test compound or a control compound with an activated APC and at least one labeled substrate of said APC; and

(c) determining the amount of labeled substrate present following the addition of either said test compound or said control compound; wherein an increase in the amount of said labeled substrate following the addition of said test compound compared to the amount of labeled substrate present following the addition of said control compound indicates that said test compound inhibits an activity of APC.

30. The method of claim 29, wherein said control compound is not an analog of an IR motif.

31. The method of claim 29, wherein said combining step occurs in vivo, in vitro, or ex vivo.

32. The method of claim 29, wherein said labeled substrate is cyclin B-luciferase.

33. A method of treating a cell cycle disorder, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 2, wherein said composition inhibits an activity of an anaphase promoting complex (APC).

34. A method of treating a cell cycle disorder, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition of claim 2, wherein said composition activates a cell cycle checkpoint.

35. The method of claim 34, wherein said cell cycle checkpoint is the spindle assembly checkpoint (SAC).

36. The method of claim 33 or 34, wherein said composition comprises a second chemotherapeutic compound.

37. The method of claim 26, wherein said compound contacts at least one amino acid of said APC3 polypeptide of SEQ ID NO: 1, 2 , or 3.

38. The method of claim 26, wherein said compound contacts at least one amino acid of said APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7.

39. The method of claim 26, wherein said compound contacts at least one amino acid of said APC7 polypeptide of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

40. The method of claim 26, wherein said compound contacts at least one amino acid of said APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.

41. The method of claim 26, wherein said compound contacts at least one amino acid of said APC3 polypeptide of SEQ ID NO: 1, 2, or 3 and contacts at least one amino acid of said APC7 polypeptide of SEQ ID NO: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19.

42. The method of claim 41, wherein said compound further contacts at least one amino acid of said APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7.

43. The method of claim 41, wherein said compound further contacts at least one amino acid of said APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.

44. The method of claim 41, wherein said compound further contacts at least one amino acid of said APC6 polypeptide of SEQ ID NO: 4, 5, 6, or 7 and contacts at least one amino acid of said APC8 polypeptide of SEQ ID NO: 20, 21, 22, 23, 24, or 25.