Akihiro Ohashi

Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells

The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations—misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours.

Synthetic studies of centromere-associated protein-E (CENP-E) inhibitors: 1.Exploration of fused bicyclic core scaffolds using electrostatic potential map.

Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine 7, which showed potent CENP-E enzyme inhibition (IC50: 50nM) and cellular activity with accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM analysis proved to be useful tools for the rational design of CENP-E inhibitors.

Synthetic Studies on Centromere-Associated Protein-E (CENP-E) Inhibitors: 2. Application of Electrostatic Potential Map (EPM) and Structure-Based Modeling to Imidazo[1,2-a]pyridine Derivatives as Anti-Tumor Agents

To develop centromere-associated protein-E (CENP-E) inhibitors for use as anticancer therapeutics, we designed novel imidazo[1,2-a]pyridines, utilizing previously discovered 5-bromo derivative 1a. By site-directed mutagenesis analysis, we confirmed the ligand binding site. A docking model revealed the structurally important molecular features for effective interaction with CENP-E and could explain the superiority of the inhibitor (S)-isomer in CENP-E inhibition vs the (R)-isomer based on the ligand conformation in the L5 loop region. Additionally, electrostatic potential map (EPM) analysis was employed as a ligand-based approach to optimize functional groups on the imidazo[1,2-a]pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo[1,2-a]pyridine derivative (+)-(S)-12, which showed potent CENP-E inhibition (IC50: 3.6 nM), cellular phosphorylated histone H3 (p-HH3) elevation (EC50: 180 nM), and growth inhibition (GI50: 130 nM) in HeLa cells. Furthermore, (+)-(S)-12 demonstrated antitumor activity (T/C: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice.

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

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.

Identification of a new class of potent Cdc7 inhibitors designed by putative pharmacophore model: Synthesis and biological evaluation of 2,3-dihydrothieno[3,2-d]pyrimidin-4(1H)-ones

Cell division cycle 7 (Cdc7) is a serine/threonine kinase that plays important roles in the regulation of DNA replication process. A genetic study indicates that Cdc7 inhibition can induce selective tumor-cell death in a p53-dependent manner, suggesting that Cdc7 is an attractive target for the treatment of cancers. In order to identify a new class of potent Cdc7 inhibitors, we generated a putative pharmacophore model based on in silico docking analysis of a known inhibitor with Cdc7 homology model. The pharmacophore model provided a minimum structural motif of Cdc7 inhibitor, by which preliminary medicinal chemistry efforts identified a dihydrothieno[3,2-d]-pyrimidin-4(1H)-one scaffold having a heteroaromatic hinge-binding moiety. The structure-activity relationship (SAR) studies resulted in the discovery of new, potent, and selective Cdc7 inhibitors 14a, c, e. Furthermore, the high selectivity of 14c, e for Cdc7 over Rho-associated protein kinase 1 (ROCK1) is discussed by utilizing a docking study with Cdc7 and ROCK2 crystal structures.

2-Aminomethylthieno[3,2-d]pyrimidin-4(3H)-ones bearing 3-methylpyrazole hinge binding moiety: Highly potent, selective, and time-dependent inhibitors of Cdc7 kinase

In order to increase the success rate for developing new Cdc7 inhibitors for cancer therapy, we explored a new chemotype which can comply with the previously-constructed pharmacophore model. Substitution of a pyridine ring of a serendipitously-identified Cdc7 inhibitor 2b with a 3-methylpyrazole resulted in a 4-fold increase in potency and acceptable kinase selectivity, leading to the identification of thieno[3,2-d]pyrimidin-4(3H)-one as an alternative scaffold. Structure-activity relationship (SAR) study revealed that incorporation of a substituted aminomethyl group into the 2-position improved kinase selectivity. Indeed, a pyrrolidinylmethyl derivative 10c was a potent Cdc7 inhibitor (IC50=0.70nM) with high selectivity (Cdk2/Cdc7≥14,000, ROCK1/Cdc7=200). It should be noted that 10c exhibited significant time-dependent Cdc7 inhibition with slow dissociation kinetics, cellular pharmacodynamic (PD) effects, and COLO205 growth inhibition. Additionally, molecular basis of high kinase selectivity of 10c is discussed by using the protein structures of Cdc7 and Cdk2.

Motor activity of centromere-associated protein-E contributes to its localization at the center of the midbody to regulate cytokinetic abscission

Accurate control of cytokinesis is critical for genomic stability to complete high-fidelity transmission of genetic material to the next generation. A number of proteins accumulate in the intercellular bridge (midbody) during cytokinesis, and the dynamics of these proteins are temporally and spatially orchestrated to complete the process. In this study, we demonstrated that localization of centromere-associated protein-E (CENP-E) at the midbody is involved in cytokinetic abscission. The motor activity of CENP-E and the C-terminal midbody localization domain, which includes amino acids 2659–2666 (RYFDNSSL), are involved in the anchoring of CENP-E to the center of the midbody. Furthermore, CENP-E motor activity contributes to the accumulation of protein regulator of cytokinesis 1 (PRC1) in the midbody during cytokinesis. Midbody localization of PRC1 is critical to the antiparallel microtubule structure and recruitment of other midbody-associated proteins. Therefore, CENP-E motor activity appears to play important roles in the organization of these proteins to complete cytokinetic abscission. Our findings will be helpful for understanding how each step of cytokinesis is regulated to complete cytokinetic abscission.

Different cell fates after mitotic slippage: From aneuploidy to polyploidy

ABSTRACT The molecular mechanism responsible for cell fate after mitotic slippage remains unclear. We investigated the different postmitotic effects of aneuploidy versus polyploidy using chemical inhibitors of centromere-associated protein-E (CENP-E) and kinesin family member 11 (KIF11, also known as Eg5). Aneuploidy caused substantial proteotoxic stress and DNA damage accompanied by p53-mediated postmitotic apoptosis, whereas polyploidy did not induce these antiproliferative effects.

Design and synthesis of fused bicyclic inhibitors targeting the L5 loop site of centromere-associated protein E.

Centromere-associated protein-E (CENP-E) is a mitotic kinesin which plays roles in cell division, and is regarded as a promising therapeutic target for the next generation of anti-mitotic agents. We designed novel fused bicyclic CENP-E inhibitors starting from previous reported dihydrobenzofuran derivative (S)-(+)-1. Our design concept was to adjust the electron density distribution on the benzene ring of the dihydrobenzofuran moiety to increase the positive charge for targeting the negatively charged L5 loop of CENP-E, using predictions from electrostatic potential map (EPM) analysis. For the efficient synthesis of our 2,3-dihydro-1-benzothiophene 1,1-dioxide derivatives, a new synthetic method was developed. As a result, we discovered 6-cyano-7-trifluoromethyl-2,3-dihydro-1-benzothiophene 1,1-dioxide derivative (+)-5d (Compound A) as a potent CENP-E inhibitor with promising potential for in vivo activity. In this Letter, we discuss the design and synthetic strategy used in the discovery of (+)-5d and structure-activity relationships for its analogs possessing various fused bicyclic L5 binding moieties.

Abstract 5041: Translational pharmacokinetic-pharmacodynamic xenograft model for TAK-931, a small molecule cell division cycle 7 (CDC7) kinase inhibitor

TAK-931 is a small molecule inhibitor of the cell division cycle 7 (CDC7) kinase. As a serine/threonine kinase that contributes to DNA replication and the DNA damage response, CDC7 is hypothesized to be a promising cancer drug target. CDC7 inhibition with TAK-931 has demonstrated antiproliferative activity with cancer cell lines and tumor growth inhibition (TGI) in murine ectopic xenograft models. Herein, the analysis of multiple models to characterize pharmacokinetic (PK) and pharmacodynamic (PD) relationships with xenograft TGI is described. TAK-931 treatment-induced TGI was dose schedule-independent and could be described using plasma drug concentrations or tumor PD inhibition. However, the efficacious doses were at least 10-fold higher for the PK-TGI relationship than for the PD-TGI relationship. This discrepancy was used to select a dynamic PK-PD-TGI modeling approach to project the minimal efficacious dose (MED) and minimal biological active dose (MBAD) for TAK-931 due to the large differences in time-concentration profiles predicted for humans versus mice. The Phase I human trial is on-going and will be used to verify the dynamic PK-PD-driven modeling approach for the CDC7 inhibitor. Citation Format: Charles Locuson, Mayank Patel, Akihiro Ohashi, Kenichi Iwai, Tadahiro Nambu, Toshiyuki Takeuchi, Akifumi Kogame, Douglas Bowman, Stephen Tirrell, Huifeng Niu, Cindy Xia. Translational pharmacokinetic-pharmacodynamic xenograft model for TAK-931, a small molecule cell division cycle 7 (CDC7) kinase inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5041. doi:10.1158/1538-7445.AM2017-5041