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Centromere‐associated protein E (CENP‐E) is a kinesin motor protein essential for mitosis and a new target for anticancer agents with less side effects. To rationally design anticancer drug candidates based on structure, it is important to determine the three‐dimensional structure of the CENP‐E motor domain bound to its inhibitor. Here, we report the first crystal structure of the CENP‐E motor domain in complex with a non‐hydrolysable ATP analogue, adenylyl‐imidodiphosphate (AMPPNP). Furthermore, the structure is compared with the ADP‐bound form of the CENP‐E motor domain as well as the AMPPNP‐bound forms of other kinesins. This study indicates that helix α4 of CENP‐E participates in the slow binding of CENP‐E to microtubules. These results will contribute to the development of anticancer drugs targeting CENP‐E. [ABSTRACT FROM AUTHOR]

Centromere‐associated protein E (CENP‐E) plays an essential role in mitosis and is a target candidate for anticancer drugs. However, it is difficult to design small‐molecule inhibitors of CENP‐E kinesin motor ATPase activity owing to a lack of structural information on the CENP‐E motor domain in complex with its inhibitors. Here, the CENP‐E motor domain was crystallized in the presence of an ATP‐competitive inhibitor and the crystal structure was determined at 1.9 Å resolution. In the determined structure, ADP was observed instead of the inhibitor in the nucleotide‐binding site, even though no ADP was added during protein preparation. Structural comparison with the structures of previously reported CENP‐E and those of other kinesins indicates that the determined structure is nearly identical except for several loop regions. However, the retention of ADP in the nucleotide‐binding site of the structure strengthens the biochemical view that the release of ADP is a rate‐limiting step in the ATPase cycle of CENP‐E. These results will contribute to the development of anticancer drugs targeting CENP‐E and to understanding the function of kinesin motor domains. [ABSTRACT FROM AUTHOR]

Kinesin centromere-associated protein E (CENP-E) has emerged as a potential target for the development of anticancer drugs due to its involvement in the mitotic progression of the cell cycle. Although several CENP-E inhibitors have been reported, more knowledge of chemical structures and inhibitory mechanisms is necessary for developing CENP-E inhibitors. Here, we describe the identification of new CENP-E inhibitors. Screening of a small-molecule chemical library identified benzo[ d ]pyrrolo[2,1- b ]thiazole derivatives, including 1 , as compounds with inhibitory activity against the microtubule-stimulated ATPase of the CENP-E motor domain. Among the mitotic kinesins examined, 1 selectively inhibited the kinesin ATPase activity of CENP-E. In a steady-state ATPase assay, 1 exhibited ATP-competitive behavior, which was different from the CENP-E inhibitor GSK923295. Compound 1 inhibited the proliferation of tumor-derived HeLa and HCT116 cells more efficiently than that of non-cancerous WI-38 cells. The inhibition of cell proliferation was attributed to the ability of 1 to induce apoptotic cell death. The compound showed antimitotic activity, which caused cell cycle arrest at mitosis via interference with proper chromosome alignment. We identified 1 and its derivatives as the lead compounds that target CENP-E, thus providing a new opportunity for the development of anticancer agents targeting kinesins. Image 1 • New CENP-E inhibitors with benzo[ d ]pyrrolo[2,1- b ]thiazole structure were discovered. • One of the inhibitors prevents the CENP-E motor domain from binding to ATP. • It preferentially inhibits proliferation of tumor cells but not non-cancerous cells. • It causes a defect in chromosome alignment, thereby inducing mitotic arrest. [ABSTRACT FROM AUTHOR]

1,3a,6a-Triazapentalene is a compact fluorescent chromophore. In this study, triazapentalene was used to modify a series of biphenyl-type inhibitors of kinesin spindle protein (KSP) to develop fluorescent probes for the intracellular visualization of this protein. Microscopic studies demonstrated that these novel triazapentalene-labeled compounds exhibited inhibitory activity towards KSP in cultured cells and provided important information concerning the intracellular distribution. [ABSTRACT FROM AUTHOR]

Abstract: Structure–activity relationship studies of diaryl amine-type KSP inhibitors were carried out. Diaryl amine derivatives with a pyridine ring or urea group were less active when compared with the parent carboline and carbazole derivatives. Optimization studies of a lactam-fused diphenylamine-type KSP inhibitor revealed that the aniline NH group and 3-CF3 phenyl group were indispensable for potent KSP inhibition. Modification with a seven-membered lactam-fused phenyl group and a 4-(trifluoromethyl)pyridin-2-yl group improved aqueous solubility while maintaining potent KSP inhibitory activity. From these studies, we identified novel diaryl amine-type KSP inhibitors with a favorable balance of potency and solubility. [Copyright &y& Elsevier]

[Display omitted] Kinesin-5 has received considerable attention as a new target for mitosis. Various small-molecule compounds targeting kinesin-5 have been developed in the last few decades. However, the differences in the cellular effects of kinesin-5 inhibitors remain poorly understood. Here, we used two different kinesin-5 inhibitors, biphenyl-type PVZB1194 and S ‐trityl‐ L ‐cysteine-type PVEI0021, to examine their effects on molecular events involving kinesin-5. Our biochemical study of kinesin-5 protein-protein interactions showed that PVZB1194-treated kinesin-5 interacted with TPX2 microtubule nucleation factor, Aurora-A kinase, receptor for hyaluronan-mediated motility, and γ-tubulin, as did untreated mitotic kinesin-5. However, PVEI0021 prevented kinesin-5 from binding to these proteins. In mitotic HeLa cells recovered from nocodazole inhibition, kinesin-5 colocalized with these binding proteins, along with microtubules nucleated near kinetochores. By acting on kinesin-5 interactions with chromatin-associated microtubules, PVZB1194, rather than PVEI0021, not only affected the formation of dispersed microtubule clusters but also enhanced the stability of microtubules. In addition, screening for mitotic inhibitors working synergistically with the kinesin-5 inhibitors revealed that paclitaxel synergistically inhibited HeLa cell proliferation only with PVZB1194. In contrast, the Aurora-A inhibitor MLN8237 exerted a synergistic anti-cell proliferation effect when combined with either inhibitor. Together, these results have provided a better understanding of the molecular action of kinesin-5 inhibitors and indicate their usefulness as molecular tools for the study of mitosis and the development of anticancer agents. [ABSTRACT FROM AUTHOR]

The kinesin spindle protein (KSP) is a mitotic kinesin involved in the establishment of a functional bipolar mitotic spindle during cell division. It is considered to be an attractive target for cancer chemotherapy with reduced side effects. Based on natural product scaffold-derived fused indole-based inhibitors and known biphenyl-type KSP inhibitors, various carboline and carbazole derivatives were synthesized and biologically evaluated. β-Carboline and lactam-fused carbazole derivatives exhibited remarkably potent KSP inhibitory activity and mitotic arrest in prometaphase with formation of an irregular monopolar spindle. The planar tri- and tetracyclic analogs inhibited KSP ATPase in an ATP-competitive manner just like biphenyl-type inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Effect of CF3-STLC, a potent kinesin spindle protein (KSP) inhibitor, on K562 human CML cell line was investigated. Treatment with CF3-STLC induced mitotic arrest of the cell cycle with the appearance of characteristic monoastral spindles, subsequent apoptotic cell death and cleavage of PARP-1, caspase-3, and 4E-BP1. The wide ranging caspase inhibitor z-VAD fmk prevented the cleavage of caspase-3 and 4E-BP1, but failed to attenuate PARP-1 cleavage or cell death triggered by CF3-STLC. These results suggest that CF3-STLC can induce apoptotic cell death in a caspase-independent manner, and may work effectively as an anti-cancer agent for hematological malignancies. [ABSTRACT FROM AUTHOR]

Kinesin spindle protein (KSP) is expressed only in cells undergoing cell division, and hence represents an attractive target for the treatment of cancer. Several KSP inhibitors have been developed and undergone clinical trial, but their clinical use is limited by their toxicity to rapidly proliferating non - cancerous cells. To create new KSP inhibitors that are highly selective for cancer cells, we optimized the amino acid moiety of S -trityl - l - cysteine (STLC) derivative 1 using in silico modeling. Molecular docking and molecular dynamics simulation were performed to investigate the binding mode of 1 with KSP. Consistent with the structure activity relationship studies, we found that a cysteine amino moiety plays an important role in stabilizing the interaction. Based on these findings and the structure of GSH, a substrate of γ - glutamyltransferase (GGT), we designed and synthesized the prodrug N- γ - glutamylated STLC derivative 9 , which could be hydrolyzed by GGT to produce 1. The KSP ATPase inhibitory activity of 9 was lower than that of 1 , and LC - MS analysis indicated that 9 was converted to 1 only in the presence of GGT in vitro. In addition, the cytotoxic activity of 9 was significantly attenuated in GGT - knockdown A549 cells. Since GGT is overexpressed on the cell membrane of various cancer cells, these results suggest that compound 9 could be a promising prodrug that selectively inhibits the proliferation of GGT - expressing cancer cells. [Display omitted] • A series of novel S -trityl- l -cysteine derivatives as KSP inhibitors were synthesized. • Docking studies were performed to understand the pharmacophore and add new functions. • Prodrug 9 , activated by GGT and exhibiting cytotoxicity, was designed. • The correlation between cellular GGT levels and cytotoxicity treatment with 9 was investigated. • A KSP inhibitor activated by GGT was identified as a novel prodrug. [ABSTRACT FROM AUTHOR]