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In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.

The mash of sweet potato shochu (Japanese distilled spirit) has a low pH value because the shochu koji mold produces a large amount of citric acid, which prevents germ contamination. In this study, we examined acid protease PepA's role in shochu production. For this purpose, we constructed pepA deletion and overexpression strains, using a black koji mold Aspergillus luchuensis RIB 2604 (NBRC 4314), with the Agrobacterium-mediated transformation method. The rice koji, prepared using a pepA disruptant (ΔpepA) and pepA-overexpressing strain (OEpepA), demonstrated 1/2- and 24-fold acid protease activities compared to that prepared using the parental strain, respectively. A small-scale test of sweet potato shochu brewing indicated the mash of ΔpepA had a lower amino acid concentration, while the mash of OEpepA had a higher concentration than that produced by the parental strain. Therefore, the mash amino acid concentrations were proportional to these strains' acid proteases activities. After distilling these mashes, we examined each shochu's aroma components. Shochu prepared using ΔpepA had relatively higher aroma components, such as alcohol and ester, compared to that prepared using parental strains. Meanwhile, shochu prepared using OEpepA had lower aroma components than that prepared using the parental strains. Based on these results, the amount of shochu aroma components showed an inverse correlation to the acid protease activity in the mash. Thus, the koji mold's acid protease content had a greater influence on the aroma qualities of sweet potato shochu. Accordingly, we have discussed the possibility of the breeding of shochu koji mold with acid protease as an indicator.

Potential combination partners of a CDC7 inhibitor TAK-931 were identified to guide its clinical development strategy., Cell division cycle 7 (CDC7), a serine/threonine kinase, plays important roles in DNA replication. We developed a highly specific CDC7 inhibitor, TAK-931, as a clinical cancer therapeutic agent. This study aimed to identify the potential combination partners of TAK-931 for guiding its clinical development strategies. Unbiased high-throughput chemical screening revealed that the highest synergistic antiproliferative effects observed were the combinations of DNA-damaging agents with TAK-931. Functional phosphoproteomic analysis demonstrated that TAK-931 suppressed homologous recombination repair activity, delayed recovery from double-strand breaks, and led to accumulation of DNA damages in the combination. Whole-genome small interfering RNA library screening identified sensitivity-modulating molecules, which propose the experimentally predicted target cancer types for the combination, including pancreatic, esophageal, ovarian, and breast cancers. The efficacy of combination therapy in these cancer types was preclinically confirmed in the corresponding primary-derived xenograft models. Thus, our findings would be helpful to guide the future clinical strategies for TAK-931.

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.

In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2

A next generation cancer drug candidate, a CDC7 inhibitor, TAK-931, was developed, which is being evaluated in clinical trials., Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931–induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS–wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.

// Akihiro Ohashi 1 , Momoko Ohori 1 , Kenichi Iwai 1 1 Oncology Drug Discovery Unit, Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, Japan Correspondence to: Akihiro Ohashi, email: akihiro.ohashi@takeda.com Keywords: CENP-E, cytokinetic abscission, midbody localization, PRC1, CENP-E inhibitor Received: May 09, 2016 Accepted: October 26, 2016 Published: November 08, 2016 ABSTRACT 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.

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.

The modulation of pre‐mRNA splicing is proposed as an attractive anti‐neoplastic strategy, especially for the cancers that exhibit aberrant pre‐mRNA splicing. Here, we discovered that T‐025 functions as an orally available and potent inhibitor of Cdc2‐like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T‐025 reduced CLK‐dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo . Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive‐associated biomarker of T‐025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T‐025 treatment. MYC activation, which altered pre‐mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti‐tumor efficacy of T‐025 in an allograft model of spontaneous, MYC‐driven breast cancer, at well‐tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC‐driven cancer patients.

Background: Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication. We developed a CDC7-selective inhibitor, TAK-931, designed to inhibit CDC7 activity in a time-dependent and ATP-competitive manner. In this study, we aimed to identify potential combination partners for TAK-931 to guide clinical combination strategies. Methods: In vitro combination studies of various agents with TAK-931 were carried out in COLO205, A549, SW620, SW48, H460, and HCT116 cancer cells using a fully automated system for assay execution and data analysis (Double Agent studies). Agents tested in combination with TAK-931 included: DNA damaging agents, tubulin binders, mitosis inhibitors, cell signaling modulators, proteasome inhibitors, and other targeted agents with significant single-agent activity against these cell lines. Treated stable isotope labeling using amino acids in cell culture (SILAC)-labeled H460 cells were subjected to quantitative mass spectrometry. In vivo combination studies of TAK-931 using DNA damaging agents or IR were conducted in multiple human xenograft models including patient-derived xenograft models (PDXs). Results: The Double Agent studies revealed that DNA damaging agents, such as topoisomerase inhibitors and platinum compounds, had the highest occurrence of synergistic antiproliferative effects in combination with TAK-931. In the phosphoproteomics analysis, gene ontology (GO) enrichment analysis revealed that DNA repair (p Citation Format: Kenichi Iwai, Kurt Eng, Tadahiro Nambu, Jie Yu, Masamitsu Gotou, Sakiyo Tsukamoto, Saomi Murai, Shun-Ichiro Kageyama, Yukie Kashima, Susumu Kobayashi, Akihiro Ohashi. Potential combination partners for a novel CDC7-selective Inhibitor, TAK-931 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A093. doi:10.1158/1535-7163.TARG-19-A093

Inhibitor of apoptosis proteins (IAP), which are key regulators of apoptosis, are inhibited by second mitochondria-derived activator of caspase (SMAC). Small-molecule IAP antagonists have recently been reported as novel therapeutic treatments for cancer. In this study, we showed that the octahydro-pyrrolo[1,2-a]pyrazine derivative, T-3256336, is a novel and orally available small-molecule IAP antagonist. T-3256336 selectively binds to and antagonizes protein interactions involving cellular IAP-1 (cIAP-1), cIAP-2, and X-linked IAP (XIAP). T-3256336 induced the rapid proteasomal degradation of cIAP-1 and activated TNF-α–dependent extrinsic apoptosis signaling in cultured cells. In a MDA-MB-231-Luc breast cancer xenograft model, T-3256336 induced cIAP-1 degradation, TNF-α production, and caspase activation in tumors, which resulted in strong antitumor activities. T-3256336 induced increases in the plasma levels of TNF-α and fragmented cytokeratin-18, which correlated with the antitumor potency in MDA-MB-231-Luc xenograft models. This study provided further insights into biomarkers of IAP antagonists. Furthermore, our data provided evidence that T-3256336 is a promising new anticancer drug worthy of further evaluation and development. Mol Cancer Ther; 12(2); 230–40. ©2012 AACR.

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.

Introduction A new disseminated intravascular coagulation (DIC) scoring system was recently announced by Japanese Association for Acute Medicine (JAAM). We have conducted a prospective external validation study to assess the accuracy of this scoring system. Materials and methods All patients admitted to the ICU in a tertiary academic hospital in 2007 were prospectively observed. All patients younger than 15years of age, those who stayed in the ICU for less than 24hours, had cardiac surgery, hematological diseases, recent chemotherapy or radiotherapy or liver cirrhosis were excluded. The remaining patients were then screened using the JAAM DIC scoring system. Results DIC was diagnosed by the JAAM DIC scoring system in 45 of the 242 patients screened (18.6%). The DIC patients were older, had a higher Acute Physiology and Chronic Health Evaluation (APACHE) II score and stayed in the ICU longer in comparison to the non-DIC patients. However, hospital mortality was similar between the two groups (p=0.98). There was no difference in the JAAM DIC score between the surviving and non-surviving DIC patients (p=0.40). A multivariate logistic regression analysis revealed the DIC diagnosed by JAAM to have a non-significant low odds ratio for hospital mortality (OR 0.29, 95%CI 0.08-1.08, p=0.066). Conclusion We have reported an external validation study of the JAAM DIC scoring system, which was conducted outside of the centers where data for developing the score were collected. DIC diagnosed by this scoring system was not related to hospital mortality.

Background: Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. We developed the CDC7-selective small molecule inhibitor TAK-931. Treatment with TAK-931 results in delayed S phase progression, DNA replication stress (RS), centrosome disorganization, mitotic aberrations, and potent growth suppression in cancer cells. In this study, we conducted the time-dependent phospho-proteomics analysis to determine the signaling pathways affected by TAK-931. Materials and Methods: TAK-931 was synthesized by Takeda Pharmaceutical Company, Ltd. The SILAC-labeled COLO205 cells were treated with TAK-931 at 100 nM for 0, 4, 24 hours, applying for the phospho-proteomics analysis in the PhosphoScout® Service at Evotec AG (Munich, Germany). Results: The SILAC-labeled COLO205 cells with TAK-931 treatment were subjected to quantitative mass spectrometry, detecting 15,128 phosphorylation sites of the 4,426 phosphoproteins. At 4 h of treatment, only 2 phosphorylation sites were detected as significantly downregulated: MAX gene-associated protein (MGA) and alpha-enolase (ENO1). At 24 h of treatment, however, 51 phosphorylation sites were identified as being significantly altered, including 44 upregulation and 7 downregulation sites. The gene ontology (GO) enrichment analysis in the categories of biological process (BP) and cellular component (CC) revealed that 10 GO terms from BP and 2 GO terms from CC were significantly enriched in the treated cells after 24 h: cell cycle, cell cycle phase, cell cycle process, mitosis, nuclear division, organelle fusion, organelle organization, mitotic cell cycle, M phase of mitotic cell cycle, M phase, chromosomal part, and spindles. Network analysis on the changed phosphorylation prolife also revealed that significant network modules existed at 24 h of treatment, which included DNA replication/damage, mitotic progression/spindle assembly, nucleus assembly, GTPase activity, cell migration, cell adhesion, TP53, and protein translation. Conclusion: The time-dependent phospho-proteomics analysis revealed the dynamics of phospho-signal transduction affected by TAK-931 in the cancer cell, which may help to provide a deeper understanding of the molecular mechanisms of TAK-931. Citation Format: Kenichi Iwai, Masamitsu Gotou, Kazunori Yamanaka, Akihiro Ohashi. Phospho-proteomics analysis to determine the signaling pathways affected by a novel CDC7-selective inhibitor TAK-931 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2312.

Arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are endocannabinoids involved in septic shock, and 8-epi prostaglandin F2alpha (F2-isoprostane) is a biomarker of oxidative stress in biological systems. Because the antibiotic polymyxin B absorbs endocannabinoids as well as endotoxins, direct hemoperfusion therapy with polymyxin B-immobilized fibers (PMX-DHP) decreases serum levels of endocannabinoids. To investigate the features of sepsis and determine the proper use of PMX-DHP, we measured the changes in levels of endocannabinoids and F2-isoprostane in patients with septic shock. Twenty-six patients with septic shock, including those with septic shock induced by peritonitis, underwent laparotomy for drainage. Endocannabinoids absorption with PMX-DHP was examined in two groups of patients: patients whose mean arterial blood pressure (mABP) had increased more than 20 mm Hg (responder group; N = 13); and patients iwhose mABP did not increase or had increased no more than 20 mm Hg (non-responder group; N = 13). Levels of AEA did not change after PMX-DHP in either the non-responder or responder groups, whereas levels of 2-AG decreased significantly after PMX-DHP in the responder group, but not in the non-responder group. F2-isoprostane gradually increased after PMX-DHP treatment; on the other hand, levels of F2-isoprostane remained constant in the responder group. Patients with septic shock are under considerable oxidative stress, and 2-AG plays an important role in the cardiovascular status of these patients. The removal of 2-AG by PMX-DHP benefits patients with septic shock by stabilizing cardiovascular status and decreasing long-term oxidative stress.

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.

Tyrosinase has a role in melanin synthesis and several defects of the tyrosinase gene lead to albinism. Here, we cloned and characterized rainbow trout tyrosinase cDNAs and carried out the molecular and biochemical characterization of albino mutants. Two types of cDNA were cloned: tyrosinase-1 (Tyr-1) and tyrosinase-2 (Tyr-2). Both contained regions predicted to encode structural features of tyrosinase, and phylogenetic analysis confirmed that Tyr-1 and Tyr-2 were members of the tyrosinase family. Tyr-1 transcripts were first detected in embryos at 5 d post-fertilization (dpf) and Tyr-2 transcripts at 15 dpf. 3,4-dihydroxyphenylalanine assays revealed significantly reduced tyrosinase activities in dominant and recessive albino mutants compared with wild-type embryos. However, reverse-transcription PCR showed no differences in the amounts or lengths of the coding regions of Tyr-1 and Tyr-2 transcripts between wild-type embryos and albino mutants. Antisense morpholino oligonucleotides (AMOs) designed to knockdown tyrosinase gene expression in wild-type embryos led to reduced pigmentation in the retina and skin of embryos at 25 and 35 dpf, respectively. Furthermore, the tyrosinase activities of AMO-treated embryos were significantly reduced. We conclude that both Tyr-1 and Tyr-2 are crucial for melanin synthesis in rainbow trout embryos. Furthermore, we describe a potential application of AMOs in the treatment of hyperpigmentation.

Proper control of gene expression at the level of transcription and RNA processing is a fundamental process of tumor cell growth. Here we showed that a novel pre-mRNA splicing modulator, Cdc2-like kinase (CLK) inhibitor T-025, suppressed tumor cell growth in vitro and in vivo. To reveal the mechanisms linking aberrant splicing and tumor growth suppression by T-025, we combined transcriptomic RNA-seq and proteomic p-SILAC data. We identified MDM4 as a mediator for triggering p53-dependent apoptosis in response to T-025. CLK inhibition led to exon 6 skipping of MDM4 mRNA, a potential target for nonsense-mediated decay, decreasing MDM4 protein. Consistent with these results, overexpression of MDM4 or knockdown of p53 attenuated growth-suppressive effects of T-025. We then investigated the relationship between genomic features and sensitivity to T-025 by evaluating the antiproliferative effect of T-025 against 240 cancer cell lines. Strikingly, expression of MDM4 was correlated with that of CLK2 and cell sensitivity to T-025. In addition, we found that copy number variation and mRNA expression of MYC were correlated with cell sensitivity to T-025. T-025 exhibited greater sensitivity in MYC-expressing cells and MYC-driven spontaneous breast cancer models. Together, we demonstrated that MDM4-p53 signaling and MYC-activation have key roles in antitumor activity of CLK inhibitor. Citation Format: Kazuho Nishimura, Masahiro Yaguchi, Yukiko Yamamoto, Shunsuke Ebara, Kawakita Yoichi, Ryo Mizojiri, Yusuke Nakayama, Kozo Hayashi, Shuichi Miyakawa, Kenichi Iwai, Toshiyuki Nomura. Small molecule inhibitor of pre-mRNA splicing evokes antitumor activity via MDM4-p53 pathway [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B060.

Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays important roles in initiation of DNA replication by phosphorylating MCM2. Kinase activity of CDC7 is controlled by its binding protein DBF4 in a cell-cycle dependent manner. Here we developed a potent CDC7 inhibitor TAK-931 (IC50< 0.3 nM) as a cancer therapeutic drug candidate, which exhibits a time-dependent ATP-competitive kinetics to its ATP-binding pocket. The selectivity studies using the 308 kinases revealed >120-fold selectivity of TAK-931 for CDC7 kinase inhibition compared to other kinase inhibitions. Treatment with TAK-931 suppressed the cellular MCM2 phosphorylation at Ser40, resulting in a delayed S phase progression, checkpoint activation, apoptosis, and potent growth suppression in various cancer cell lines. Furthermore, oral administration of TAK-931 as a single agent caused a significant antitumor activity in multiple xenograft models which include both cell line-based xenografts and patient-derived xenograft (PDX) models. These results demonstrate that TAK-931 is a highly potent and selective inhibitor of CDC7 kinase, and causes a potent antiproliferation both in vitro and in vivo studies using various cancer cells. Next, to identify potential predictive biomarkers to guide patient-selection strategies, in vitro cell panel screening of TAK-931 using was tested for its ability to antiproliferation in 246 cell lines, which includes both solid and hematological cancer cells. TAK-931 inhibited proliferation of multiple cancer cell lines, with mean concentration producing a half-maximal response (EC50) values ranging from 29.1 nM to > 30 μM (median = 554.5 nM). While the wide range of TAK-931 antiproliferative spectrum was observed, neither doubling speed nor CDC7 expression profile did predict tTAK-931 sensitivity in cancer cell lines. A correlative study of the tumor genetic mutations in relation to antiproliferative activity that KRAS mutant cancer cells were more sensitive to TAK-931 compared to KRAS non-mutant cell lines (p60% TGI. Our findings suggest that the KRAS-mutant pancreatic tumors could be the potential candidate for the TAK-931 target indication. Citation Format: Kenichi Iwai, Tadahiro Nambu, Osamu Kurasawa, Noriko Uchiyama, Ryo Dairiki, Yukiko Yamamoto, Satoru Nishizawa, Mengkun Zhang, Yuko Ishii, Huifeng Niu, Akihiro Ohashi. Potential predictive biomarkers of clinical responses for a novel CDC7-selective inhibitor TAK-931 [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 3073. doi:10.1158/1538-7445.AM2017-3073

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

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., CENP-E regulates chromosome alignment during mitosis to distribute chromosomes equally into daughter cells. Here, the authors show that CENP-E inhibition causes p53-mediated post-mitotic apoptosis in tumours where the spindle assembly checkpoint is compromised, suggesting that CENP-E is a therapeutic target for these cancers.

Antithrombin III (ATIII) of low doses (1500-3000 units per day for 3-5 days) has been used for treatment of disseminated intravascular coagulation (DIC) for decades in Japan. In this study, we have examined the impact of ATIII practice change on outcome in critically ill patients with sepsis and DIC. From April 2005 to September 2008, all septic patients admitted to our ICU were divided into two groups: before withdrawing ATIII (period 1) and after withdrawing ATIII (period 2). Patients treated with ATIII in the period 1 and those not treated with ATIII in the period 2 were then matched according to the similar Acute Physiology and Chronic Health Evaluation II scores (± 3) and the same diagnosis grouping. Sensitivity analysis was also conducted for patients with DIC. Forty-one out of 98 patients (41.8%) in the period 1 and only one out of 80 patients (1.3%) in the period 2 were treated with ATIII. Thirty pairs of the patients were matched. There was no difference between the two groups regarding the platelet counts and Sepsis-related Organ Failure Assessment scores at day 1 and day 4. A subgroup analysis was conducted with 12 patients diagnosed with DIC out of the 30 pairs. There was no difference between the two DIC groups for platelet counts, Sepsis-related organ failure assessment scores and DIC score at day 1 and also day 4. Although not significant, hospital mortality tended lower in the period 2. This study found that withdrawing ATIII administration from management of septic patients with or without DIC did not influence outcome.

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.

To develop novel inhibitor of apoptosis (IAP) proteins antagonists, we designed a bicyclic octahydropyrrolo[1,2-a]pyrazine scaffold as a novel proline bioisostere. This design was based on the X-ray co-crystal structure of four N-terminal amino acid residues (AVPI) of the second mitochondria-derived activator of caspase (Smac) with the X-chromosome-linked IAP (XIAP) protein. Lead optimization of this scaffold to improve oral absorption yielded compound 45, which showed potent cellular IAP1 (cIAP1 IC(50): 1.3 nM) and XIAP (IC(50): 200 nM) inhibitory activity, in addition to potent tumor growth inhibitory activity (GI(50): 1.8 nM) in MDA-MB-231 breast cancer cells. X-ray crystallographic analysis of compound 45 bound to XIAP and to cIAP1 was achieved, revealing the various key interactions that contribute to the higher cIAPI affinity of compound 45 over XIAP. Because of its potent IAP inhibitory activities, compound 45 (T-3256336) caused tumor regression in a MDA-MB-231 tumor xenograft model (T/C: -53% at 30 mg/kg).

Radiation therapy, as a primary therapy or as a combination partner, is used in half of all worldwide cancer treatments. Research is ongoing to identify agents which potentiate the effects of ionizing radiation (IR) in tumor cells. Because IR causes DNA double strand breaks (DSBs), inhibition of DNA damage repair mechanisms could enhance the effects of radiation. DNA repair at DSBs is mediated by the non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways, both of which rely on the post-translational modification of proteins by ubiquitin (Ub). A phosphorylation and ubiqutination cascasde at DSBs results in Ub-dependent recruitment of 53BP1 and BRCA1 complexes. We have identified a first in class investigational drug, TAK-243 (MLN7243), which targets the ubiquitin activating enzyme, UAE (UBA1), the enzyme responsible for activating > 99% of all cellular Ub. Previously, TAK-243 was shown to inhibit mono-Ub of PCNA and FANCD2, key proteins within the translesion synthesis (TLS) and Fanconi Anemia (FA) DNA repair pathways, and also to inhibit Ub transfer to UBC13, an E2 ubiquitin-conjugating enzyme utilized in DSB repair. We hypothesized that TAK-243 would prevent repair of DSBs and thereby potentiate IR-induced cell death. Here we show that TAK-243 pre-treatment potentiates the effect of IR on HCT-116 cells in a colony formation assay in vitro. To link this combination benefit to the disruption of DNA damage repair, we demonstrate that TAK-243 pre-treatment blocks the IR-induced recruitment of 53BP1 to sites of DNA damage both in vitro and in vivo. In a patient-derived xenograft (PDX) model of non-small cell lung cancer, formation of IR-induced 53BP1 foci is inhibited when TAK-243 is dosed 1 hour before beam-focused radiation exposure. In contrast, levels of IR-induced pH2Ax are not significantly changed by TAK-243 treatment, suggesting that TAK-243 does not prevent formation or detection of DSBs, but rather acts downstream to prevent DNA damage repair. Additive-to-synergistic effects on tumor growth inhibition were observed in several xenograft models treated with the combination of TAK-243 and beam-focused IR, with persistent tumor regressions noted in some NSCLC and breast cancer models. The results of our experiments provide a mechanistic rationale for combining radiation with TAK-243 in the clinical setting. Currently, TAK-243 is being evaluated in a solid tumor phase I clinical trial evaluating safety, tolerability, pharmacokinetics, pharmacodynamics and anti-tumor activity (NCT02045095). Citation Format: Michael A. Milhollen, Judy Qiuju Shi, Tary Traore, Jessica Huck, Darshan Sappal, Kenichi Iwai, Akihiro Ohashi, Claudia Rabino, Jennifer A. Duffy, Eric Lightcap, Yuko Ishii, Jeffrey Ciavarri, Neil Bence, Allison J. Berger, Marc L. Hyer. TAK-243, a small molecule inhibitor of the ubiquitin activating enzyme (UAE), disrupts DNA damage repair and sensitizes tumor cells and xenografts to ionizing radiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3719.

Arachidonylethanolamide (AEA) and 2-arachidonylglycerol (2-AG) are endocannabinoids involved in septic shock, and 8-epi prostaglandin F2alpha (F2-isoprostane) is a biomarker of oxidative stress in biological systems. Because the antibiotic polymyxin B absorbs endocannabinoids as well as endotoxins, direct hemoperfusion therapy with polymyxin B-immobilized fibers (PMX-DHP) decreases serum levels of endocannabinoids. To investigate the features of sepsis and determine the proper use of PMX-DHP, we measured the changes in levels of endocannabinoids and F2-isoprostane in patients with septic shock. Twenty-six patients with septic shock, including those with septic shock induced by peritonitis, underwent laparotomy for drainage. Endocannabinoids absorption with PMX-DHP was examined in two groups of patients: patients whose mean arterial blood pressure (mABP) had increased more than 20 mm Hg (responder group; N = 13); and patients iwhose mABP did not increase or had increased no more than 20 mm Hg (non-responder group; N = 13). Levels of AEA did not change after PMX-DHP in either the non-responder or responder groups, whereas levels of 2-AG decreased significantly after PMX-DHP in the responder group, but not in the non-responder group. F2-isoprostane gradually increased after PMX-DHP treatment; on the other hand, levels of F2-isoprostane remained constant in the responder group. Patients with septic shock are under considerable oxidative stress, and 2-AG plays an important role in the cardiovascular status of these patients. The removal of 2-AG by PMX-DHP benefits patients with septic shock by stabilizing cardiovascular status and decreasing long-term oxidative stress.

To the Editor: The pulmonary artery catheter (PAC) and PiCCO monitor (Pulsion Medical Systems, Munich, Germany) use the thermodilution technique to measure cardiac output [1]. Because the indicator injection site (central vein) and temperature measurement site (femoral artery) are more distant in PiCCO than in PAC (right atrium and pulmonary artery), PiCCO can potentially detect a wider variety of left-to-right shunts (LRS) than PAC. A 68-year-old man was admitted to the ICU for presumed diagnoses of septic shock and acute kidney injury resulting from an infected right common iliac artery (CIA) aneurysm. He was treated with continuous veno-venous hemodialysis (CVVHD) and vasopressin infusion with massive fluid resuscitation and noradrenaline infusion for septic shock. Ten days after ICU admission, he still required a small amount of noradrenaline and CVVHD. To evaluate his cardiac function and manage his shock state more meticulously, a 5-Fr. catheter (Pulsioncath; Pulsion Medical Systems, Munich, Germany) was inserted in his left femoral artery and was connected to the PiCCO Plus monitor. Despite the absence of hypoxemia and lung infiltrates in the chest X-ray, hemodynamic variables showed low cardiac index (CI, 1.37 l/min/m) and very low intrathoracic blood volume index (ITBVI, 336 ml), with massively increased extravascular lung water index (EVLWI, 43.7 ml/kg) for an average value of two measurements. The thermodilution curve showed a significantly prolonged flattening of the descending portion of the curve, suggesting an LRS. A dynamic computed tomography (CT) scan of the abdomen was subsequently conducted, which revealed an arteriovenous fistula between the right CIA aneurysm and the inferior vena cava. To repair the fistula, endovascular stenting was planned. However, on the day of surgery, the patient suddenly developed cardiac arrest and could not be resuscitated. Although an autopsy was not performed, the cause of cardiac arrest was thought to be rupture of the aneurysm. The dilution curve of LRS is characterized by an early recirculation of the cold indicator, which results in premature flattening of the descending portion of the curve. Previously, only two cases of LRS detected by PiCCO have been reported [2]. One patient had a ventricular septal defect and the other had an aorto-vena caval fistula. One patient had an ITBVI of 857 ml/m, EVLWI of 31.7 ml/kg, and CI of 2.66 l/min/m. Our case showed even more abnormal values of ITBVI and EVLWI than the previous cases. Retrospectively, our patient had probably developed the LRS before ICU admission, when his condition deteriorated. The persistent LRS could have contributed to the sustained shock state and acute kidney injury and might have deteriorated gradually during his stay in the ICU. We think that this is the reason why we found exceptionally low ITBVI and high EVLWI values compared with previous cases. Electronic supplementary material The online version of this article (doi:10.1007/s00540-014-1788-8) contains supplementary material, which is available to authorized users.

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

Numerous studies on cerebral angiography have been conducted since Egas Moniz's study in 1927.It is known that complications due to cerebral angiography are mainly resulted from the contrast medium. We have selected organic tri-iodide solution as a contrast medium because it is safe and offers a high contrast. Urografin (60% and 76%) were used in 1869 cerebral angiographies. Present studies were conducted to observe complications due to this medium using two concentrations of Urografin.