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Antitumor Activity of an Allosteric Inhibitor of Centromere-Associated

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Antitumor Activity of an Allosteric Inhibitor of Centromere-Associated Antitumor activity of an allosteric inhibitor of SEE COMMENTARY centromere-associated protein-E Kenneth W. Wooda,1, Latesh Lada, Lusong Luob, Xiangping Qiana, Steven D. Knightb, Neysa Nevinsb, Katjusa Brejca, David Suttonb, Aidan G. Gilmartinb, Penelope R. Chuaa, Radhika Desaia, Stephen P. Schauera, Dean E. McNultyb, Roland S. Annanb, Lisa D. Belmonta, Carlos Garciaa, Yan Leea, Melody A. Diamondb, Leo F. Faucetteb, Michele Giardiniereb, ShuYun Zhangb, Chiu-Mei Sunb, Justin D. Vidalb, Serge Lichtsteinera, William D. Cornwellb, Joel D. Greshockb, Richard F. Woosterb, Jeffrey T. Finera, Robert A. Copelandb, Pearl S. Huangb, David J. Morgans, Jr.a, Dashyant Dhanakb, Gustave Bergnesa, Roman Sakowicza, and Jeffrey R. Jacksonb aCytokinetics Inc., South San Francisco, CA 94080; and bGlaxoSmithKline, Collegeville, PA 19426 Communicated by Don W. Cleveland, University of California at San Diego, La Jolla, CA, January 2, 2010 (received for review December 2, 2009) Centromere-associatedprotein-E (CENP-E) is a kinetochore-associated Depletion of CENP-E from cultured human cells using anti- mitotic kinesin that is thought to function as the key receptor sense oligonucleotides or RNAi causes prolonged cell-cycle delay responsible for mitotic checkpoint signal transduction after interac- in mitosis that is characterized by an intact bipolar mitotic spindle tion with spindle microtubules. We have identified GSK923295, an with several chromosomes clustered close to either spindle pole allosteric inhibitor of CENP-E kinesin motor ATPase activity, and (10, 11). Studies combining detailed light and electron micro- mapped the inhibitor binding site to a region similar to that bound by scopy, inhibitors of KSP/Eg5 and Aurora kinase, and siRNA- loop-5 inhibitors of the kinesin KSP/Eg5. Unlike these KSP inhibitors, mediated depletion of CENP-E showed that loss of CENP-E which block release of ADP and destabilize motor-microtubule correlated with a failure of monooriented chromosomes to con- interaction, GSK923295 inhibited release of inorganic phosphate gress to the spindle midzone (12). Microinjection of antibody and stabilized CENP-E motor domain interaction with microtubules. directed against CENP-E (13, 14) and expression of N-terminal Inhibition of CENP-E motor activity in cultured cells and tumor deletion mutants of CENP-E lacking 800 or more N-terminal xenografts caused failure of metaphase chromosome alignment residues, including the motor domain (9, 13), produced a similar CELL BIOLOGY and induced mitotic arrest, indicating that tight binding of CENP-E failure of the minority of chromosomes to achieve metaphase to microtubules is insufficient to satisfy the mitotic checkpoint. alignment. Collectively, these studies indicate that CENP-E plays Consistent with genetic studies in mice suggesting that decreased an important role in a cooperative process of chromosome con- CENP-E function can have a tumor-suppressive effect, inhibition of gression to the metaphase plate and in mitotic checkpoint sat- CENP-E induced tumor cell apoptosis and tumor regression. isfaction in human cells. However, these studies do not specifically address the role of CENP-E kinesin motor function in chromo- cancer | checkpoint | kinesin | inhibitor | mitosis some alignment or mitotic checkpoint signaling. Our interest in identifying an inhibitor of CENP-E motor entromere-associated protein-E (CENP-E; kinesin-7) is a function was stimulated not only by its essential and exclusive Ckinetochore-associated kinesin motor protein with an essential mitotic roles but also by the observation that partial loss of CENP- and exclusive role in metaphase chromosome alignment and sat- E function was associated with decreased tumor incidence in mice isfaction of the mitotic checkpoint (1). CENP-E is a likely candi- (15). Mice heterozygous for CENP-E exhibited decreased inci- date to integrate the mechanics of kinetochore–microtubule dence of spontaneous liver tumors, decreased incidence of tumors interaction with the mitotic checkpoint signaling machinery induced by the carcinogen 9,10-dimethyl-1,2-benzanthracene responsible for restraining cell-cycle progression into anaphase. (DMBA), and decreased incidence of tumors arising after CENP-E is a large dimeric protein consisting of an N-terminal homozygous deletion of the tumor suppressor p19ARF. The anti- kinesin motor domain tethered to a globular C-terminal domain tumor effect associated with reduced CENP-E function suggests through an extended coiled-coil rod domain (2, 3). The C-terminal, that inhibition of CENP-E kinesin function might be an effective noncatalytic region of CENP-E is not only sufficient to specify approach for the treatment of cancers. localization to kinetochores, but it also mediates interaction of We report here the identification and characterization of CENP-E with the serine/threonine kinase BubR1, a key effector GSK923295, a first-in-class, specific, allosteric inhibitor of CENP- of mitotic checkpoint signaling that forms complexes with E kinesin motor function, and describe the effects of this unique the checkpoint proteins Cdc20, Bub3, and Mad2 to inhibit the mode of CENP-E inhibition on chromosome alignment, mitotic ubiquitin ligase activity of the anaphase promoting complex checkpoint satisfaction, and human tumor-cell viability in vitro APC/CCDC20 (4–7). The combined interaction of CENP-E with and in vivo. microtubules and a key regulator of APC/CCDC20 has led to the hypothesis that CENP-E functions as the key kinetochore micro- tubule receptor responsible for silencing mitotic checkpoint signal Author contributions: K.W.W., L. Lad, L. Luo, X.Q., S.D.K., N.N., K.B., D.S., A.G.G., P.R.C., L.D.B., J.D.V., S.L., W.D.C., J.D.G., R.F.W., J.T.F., R.A.C., P.S.H., D.J.M., D.D., G.B., R.S., and J.R.J. designed transduction after capture of spindle microtubules. This hypothesis research; L. Lad, L. Luo, N.N., K.B., A.G.G., P.R.C., R.D., S.P.S., D.E.M., R.S.A., L.D.B., C.G., Y.L., was further strengthened by the finding that CENP-E could stim- M.A.D., L.F.F., M.G., S.Z., C.-M.S., and S.L. performed research; X.Q., S.D.K., and S.L. contributed ulate the kinase activity of BubR1 in a microtubule-sensitive new reagents/analytic tools; K.W.W., L. Lad, L. Luo, X.Q., S.D.K., N.N., K.B., D.S., A.G.G., P.R.C., manner (8, 9). In vitro, the addition of CENP-E to BubR1 resulted R.D., S.P.S., D.E.M., R.S.A., L.D.B., C.G., Y.L., M.A.D., L.F.F., M.G., S.Z., C.-M.S., J.D.V., W.D.C., J.D.G., R.F.W., J.T.F., R.A.C., P.S.H., D.J.M., D.D., G.B., R.S., and J.R.J. analyzed data; and K.W.W. in a stimulation of BubR1 kinase activity. The addition of micro- and L. Lad wrote the paper. tubules suppressed this stimulatory activity, an effect thought to be Conflict of interest statement: All authors are current or former employees of Cytokinetics mediated by the CENP-E kinesin motor domain. Although the or GlaxoSmithKline and may hold stock in these companies. importance of CENP-E interaction with BubR1 and the role of See Commentary article on page 5699. BubR1-mediated phosphorylation in mitotic checkpoint function 1To whom correspondence should be addressed. E-mail: [email protected]. remain unclear, CENP-E remains a prominent candidate to play a This article contains supporting information online at www.pnas.org/cgi/content/full/ key role in mitotic checkpoint signal transduction. 0915068107/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.0915068107 PNAS | March 30, 2010 | vol. 107 | no. 13 | 5839–5844 Downloaded by guest on September 29, 2021 Results Table 1. Sensitivity of human, murine, and canine CENP-E motor Biochemical Mechanism of Action of GSK923295. We screened a domains to GSK923295 and reduction in sensitivity of human library of structurally diverse, small organic compounds for CENP-E after mutation of I182 and T183 to corresponding murine inhibition of CENP-E microtubule-stimulated ATPase activity. residues Medicinal chemistry optimization of initial inhibitors gave rise to a Species Mutation GSK923295 Ki (nM) series of high affinity, stereoselective, specific and drug-like inhibitors of CENP-E with potent on-target activity against human Human wt 3.2 ± 0.2 cancer cells, culminating in the identification of GSK923295 and Canine wt 1.6 ± 0.1 related compounds (Fig. 1 and Table S1) (16). Murine wt 67 ± 5 Using steady-state and presteady-state kinetic methodologies Human I182L T183A 61 ± 8 ± and equilibrium binding, we defined the biochemical potency and Human I182L 14 0.2 ± mechanism of GSK923295 inhibition of CENP-E motor domain Human T183A 13 0.2 (Fig. 1, Table 1, Fig. S1, and Tables S1–S3). Analysis of the rates Ki values are ± SD (n = 4). Sequence comparison of human, canine, and of CENP-E ATP hydrolysis observed in the presence of varying murine CENP-E motor domains is shown in Fig. S4 and characterization of concentrations of inhibitor and substrates [ATP and microtubules mutant enzymes in Table S3. (MT)] indicated that GSK923295 is uncompetitive with both ATP and MT, inhibiting CENP-E MT-stimulated ATPase activity with a Ki of 3.2 ± 0.2 nM (Table 1, Tables S1 and S2, and Fig. S1). examined the interaction of CENP-E motor domain with MT in Among a diverse group of seven kinesins tested, GSK923295 was the presence and absence of GSK923295 under various nucleo- found to be highly selective for CENP-E (Fig. S2). tide conditions by copelleting of CENP-E motor domain with MT The greater potency of GSK923295 against microtubule- (Fig. 1C).
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