PLoS One. 2015 Dec 9;10(12):e0144675.

A Novel Time-Dependent CENP-E Inhibitor with Potent Antitumor Activity.

Ohashi A1, Ohori M1, Iwai K1, Nambu T1, Miyamoto M2, Kawamoto T3, Okaniwa M1.
  • 1Oncology Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Japan.
  • 2DMPK Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Japan.
  • 3Biomolecular Research Laboratories, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Fujisawa, Japan.



Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.

PMID: 26649895



Antimitotic drugs targeting microtubule dynamics, such as taxanes and vinca alkaloids, are widely used in the clinical treatment of cancer [1]. However, peripheral neuropathy is a major adverse effect of these drugs, presumably because they directly inhibit the assembly of microtubule structures even in non-dividing neural cells [2]. To reduce the incidence of this debilitating side effect, the components of mitotic spindles that are non-structural but essential for mitosis have recently attracted attention as target molecules for next-generation anticancer drugs. A mitotic kinesin, centromere-associated protein E (CENP-E), is emerging as promising target molecules for anticancer drugs [3]. CENP-E is a mitotic spindle motor protein of the kinesin superfamily [4], which is localized at the kinetochore of chromosomes [4, 5] and controls chromosome alignment during metaphase by capturing the microtubule plus end at the kinetochore [6-8]. More recently, CENP-E has been reported to transport the pole-proximal chromosomes toward the metaphase plate, and CENP-E-driven chromosome congression is guided by tubulin post-translational modification [9].

We have developed a novel time-dependent CENP-E inhibitor, Compound-A (Cmpd-A), based on a biochemical screening of the ATPase activity of the CENP-E motor domain [10]. In our recent study published in PLOS ONE [11], we report the characterization of Cmpd-A on the enzymatic mode of action, cellular morphology, pharmacokinetics (PK), and pharmacodynamics (PD) and demonstrate its antiproliferative activities both in vitro and in vivo. The cell-free enzymatic assays using the CENP-E motor domains revealed that Cmpd-A is a highly-potent CENP-E inhibitor with time-dependent enzymatic action, and has the potential to overcome high ATP concentrations in cellular settings when it occupies the allosteric site of CENP-E. Because of this unique enzymatic mechanism, Cmpd-A exhibits potent cellular activity and potential growth inhibition of cancer cells (GI50 = 80 nM in HeLa cells [10]). In addition, Cmpd-A potently suppresses cellular proliferation in multiple cancer cell lines [11].

The in vivo antitumor activity of CENP-E was investigated in a COLO205-xenograft nude mouse model. Cmpd-A was intraperitoneally administered to the COLO205 xenograft nude mouse model at a dose of 100 mg/kg three times (0, 8, and 24 h) on the first day. The administration of Cmpd-A on this schedule significantly enhanced antitumor efficacy in the COLO205 model [11]. The antitumor activity (T/C %) was 11% on Day 8 (p < 0.01), but there was no significant bodyweight loss compared with control COLO205 xenograft nude mice treated with vehicle. Although it is difficult to directly monitor CENP-E cellular activity modulated by Cmpd-A, the inhibitory effects of Cmpd-A were determined in cells with pole-proximal misaligned chromosomes, in which the inter-kinetochore tension was significantly weakened [11]. In parallel with the mitotic aberration induced by Cmpd-A, the mitotic marker pHH3 was also elevated in vitro and in vivo. Furthermore, pHH3 levels and the antiproliferative activity of Cmpd-A were correlated [11]. Although pHH3 is not a biomarker to monitor CENP-E motor activity directly, this correlation indicates that pHH3 could potentially be used as a surrogate for the PD biomarker as well as an efficacy marker. Further preclinical studies in multiple tumor models are needed to develop the utility of pHH3 as a biomarker for CENP-E inhibitors. Our investigations contribute to an increased understanding of the linking chromosome instability and antiproliferative activity in cancer cells and confirm that small-molecule inhibitors of CENP-E motor activity have important potential as anticancer drugs.



Figure 1 Check items of Cmpd-A pharmacological profile


Figure 2 Mechanism of action of Cmpd-A to induce antiproliferation in cancer cells



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This research was supported by Takeda Pharmaceutical Company, Ltd.



*Corresponding author

Akihiro Ohashi, Ph.D.

Project lead

Oncology Drug Discovery Unit, Pharmaceutical Research Division

Takeda Pharmaceutical Company Limited

26-1 Muraoka Higashi 2-chome, Fujisawa, Kanagawa 251-8555, Japan


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