Takaharu Hirayama

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.

Discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (TAK-593), a highly potent VEGFR2 kinase inhibitor

Vascular endothelial growth factor (VEGF) plays important roles in tumor angiogenesis, and the inhibition of its signaling pathway is considered an effective therapeutic option for the treatment of cancer. In this study, we describe the design, synthesis, and biological evaluation of 2-acylamino-6-phenoxy-imidazo[1,2-b]pyridazine derivatives. Hybridization of two distinct imidazo[1,2-b]pyridazines 1 and 2, followed by optimization led to the discovery of N-[5-({2-[(cyclopropylcarbonyl)amino]imidazo[1,2-b]pyridazin-6-yl}oxy)-2-methylphenyl]-1,3-dimethyl-1H-pyrazole-5-carboxamide (23a, TAK-593) as a highly potent VEGF receptor 2 kinase inhibitor with an IC50 value of 0.95 nM. The compound 23a strongly suppressed proliferation of VEGF-stimulated human umbilical vein endothelial cells with an IC50 of 0.30 nM. Kinase selectivity profiling revealed that 23a inhibited platelet-derived growth factor receptor kinases as well as VEGF receptor kinases. Oral administration of 23a at 1 mg/kg bid potently inhibited tumor growth in a mouse xenograft model using human lung adenocarcinoma A549 cells (T/C=8%).

Structure-based design, synthesis, and evaluation of imidazo[1,2-b]pyridazine and imidazo[1,2-a]pyridine derivatives as novel dual c-Met and VEGFR2 kinase inhibitors.

To identify compounds with potent antitumor efficacy for various human cancers, we aimed to synthesize compounds that could inhibit c-mesenchymal epithelial transition factor (c-Met) and vascular endothelial growth factor receptor 2 (VEGFR2) kinases. We designed para-substituted inhibitors by using co-crystal structural information from c-Met and VEGFR2 in complex with known inhibitors. This led to the identification of compounds 3a and 3b, which were capable of suppressing both c-Met and VEGFR2 kinase activities. Further optimization resulted in pyrazolone and pyridone derivatives, which could form intramolecular hydrogen bonds to enforce a rigid conformation, thereby producing potent inhibition. One compound of particular note was the imidazo[1,2-a]pyridine derivative (26) bearing a 6-methylpyridone ring, which strongly inhibited both c-Met and VEGFR2 enzyme activities (IC50=1.9, 2.2 nM), as well as proliferation of c-Met-addicted MKN45 cells and VEGF-stimulated human umbilical vein endothelial cells (IC50=5.0, 1.8 nM). Compound 26 exhibited dose-dependent antitumor efficacy in vivo in MKN45 (treated/control ratio [T/C]=4%, po, 5mg/kg, once-daily) and COLO205 (T/C=13%, po, 15 mg/kg, once-daily) mouse xenograft models.

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.

Prolyl-tRNA synthetase inhibition promotes cell death in SK-MEL-2 cells through GCN2-ATF4 pathway activation.

Protein translation is highly activated in cancer tissues through oncogenic mutations and amplifications, and this can support survival and aberrant proliferation. Therefore, blocking translation could be a promising way to block cancer progression. The process of charging a cognate amino acid to tRNA, a crucial step in protein synthesis, is mediated by tRNA synthetases such as prolyl tRNA synthetase (PRS). Interestingly, unlike pan-translation inhibitors, we demonstrated that a novel small molecule PRS inhibitor (T-3861174) induced cell death in several tumor cell lines including SK-MEL-2 without complete suppression of translation. Additionally, our findings indicated that T-3861174-induced cell death was caused by activation of the GCN2-ATF4 pathway. Furthermore, the PRS inhibitor exhibited significant anti-tumor activity in several xenograft models without severe body weight losses. These results indicate that PRS is a druggable target, and suggest that T-3861174 is a potential therapeutic agent for cancer therapy.

Studies of CDK 8/19 inhibitors: Discovery of novel and selective CDK8/19 dual inhibitors and elimination of their CYP3A4 time-dependent inhibition potential

In this article, synthetic studies around a pyridylacrylamide-based hit compound (1), utilizing structure-based drug design guided by CDK8 docking models, is discussed. Modification of the pendant 4-fluorophenyl group to various heteroaromatic rings was conducted aiming an interaction with the proximal amino acids, and then replacement of the morpholine ring was targeted for decreasing potential of time-dependent CYP3A4 inhibition. These efforts led to the compound 4k, with enhanced CDK8 inhibitory activity and no apparent potential for time-dependent CYP3A4 inhibition (CDK8 IC50: 2.5nM; CYP3A4 TDI: 99% compound remaining). Compound 4k was found to possess a highly selective kinase inhibition profile, and also showed favorable pharmacokinetic profile. Oral administration of 4k (15mg/kg, bid. for 2weeks) suppressed tumor growth (T/C 29%) in an RPMI8226 mouse xenograft model.

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 3407: A novel CENP-E-selective inhibitor exhibits potent anti-tumor efficacy by two distinct mechanisms of action dependent on spindle assembly checkpoint activity.

Centromere-associated protein-E (CENP-E) is a mitotic spindle motor protein belonging to the kinesin superfamily and controls chromosome alignment during metaphase by capturing the microtubule plus end at the kinetochore. Loss of CENP-E function has been reported to result in misaligned chromosomes at metaphase leading to spindle assembly checkpoint (SAC) activation. Here, we developed a novel small-molecule inhibitor of CENP-E (Compound-A), which targets an ATPase domain at the CENP-E N-terminal motor region. Enzymatic kinetics reveals that Compound-A is a slow-off rate type of ATP-competitive CENP-E inhibitor. Treatment of Hela cells with Compound-A induced chromosome misalignment during the SAC-dependent mitotic arrest, resulting in potent growth suppression by apoptosis. Furthermore, intraperitoneal administration of Compound-A displayed potent anti-tumor efficacy in colo205-xenograft mouse models (T/C=11 % at day 8). Given that SAC activation by CENP-E inhibition induced mitotic death, we next investigated whether SAC attenuation by BubR1 knockdown recovers cell viability in these cells. However, CENP-E inhibition was able to cause apoptosis after mitotic slippage in the BubR1-knockdown Hela cells, while inhibition of another mitotic kinesin Eg5, which controls centrosome separation during mitosis, induced polyploidy instead of apoptosis in these cells. Our data suggest that asymmetric chromosome segregation accelerated by CENP-E inhibition, but not polyploidy by Eg5 inhibition, is responsible for apoptosis after mitotic slippage. A comprehensive gene expression analysis of microarray comparisons revealed that p53 pathways are activated by CENP-E inhibition after mitotic slippage, and p53 knockdown suppressed post-mitotic caspase-3/7 activation by CENP-E inhibition. Furthermore, we found that both phosphorylation of at Ser-15 by ATM and ATR kinases and accumulation of p53 protein by unfolding protein response (UPR) were involved in post-mitotic p53 activation after mitotic slippage. In conclusion, we developed the novel CENP-E inhibitor, Compound-A, and CENP-E inhibition by Compound-A induced potent growth inhibition in both SAC-intact and SAC-defective cancer cells. In the latter case, p53 pathways play important roles in the induction of apoptosis. Our data demonstrate that SAC and p53 pathways complementally function to eliminate aberrant chromosome segregation accelerated by CENP-E inhibition. Thus, SAC activation and p53 protein accumulation are available for complementary PD biomarkers of the CENP-E inhibitors, and several molecules in the SAC and p53 pathways could be potential biomarkers to select sensitive tumors to the CENP-E inhibitors. Citation Format: Akihiro Ohashi, Momoko Ohori, Kenichi Iwai, Yusuke Nakayama, Tadahiro Nambu, Daisuke Morishita, Tomohiro Kawamoto, Maki Miyamoto, Takaharu Hirayama, Masanori Okaniwa, Hiroshi Banno, Tomoyasu Ishikawa, Hitoshi Kandori, Kentaro Iwata. A novel CENP-E-selective inhibitor exhibits potent anti-tumor efficacy by two distinct mechanisms of action dependent on spindle assembly checkpoint activity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3407. doi:10.1158/1538-7445.AM2013-3407