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.

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.

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.

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.

A CDC7 inhibitor sensitizes DNA-damaging chemotherapies by suppressing homologous recombination repair to delay DNA damage recovery.

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.

Discovery of a Novel, Highly Potent, and Selective Thieno[3,2-d]pyrimidinone-Based Cdc7 Inhibitor with a Quinuclidine Moiety (TAK-931) as an Orally Active Investigational Antitumor Agent.

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.

Effect of pepA deletion and overexpression in Aspergillus luchuensis on sweet potato shochu brewing.

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.

Dietary fermented products using koji mold and sweet potato-shochu distillery by-product promotes hepatic and serum cholesterol levels and modulates gut microbiota in mice fed a high-cholesterol diet.

It has been reported that fermented products (FPs) prepared from sweet potato-shochu distillery by-product suppressed weight gain and decreased serum cholesterol levels in mice under normal dietary conditions. Furthermore, from the information gained from the above data regarding health benefits of the FPs, the aim of this study was evaluating the effects of dietary FPs on lipid accumulation and gut microbiota in mice with or without cholesterol-load in the diet. C57BL/6N mice were fed normal (CO) diet, CO with 10% FPs (CO + FPs) diet, cholesterol loaded (HC) diet, or HC with 10% FPs (HC + FPs) diet for 8 weeks. The mice were then euthanized, and blood samples, tissue samples, and feces were collected. The adipose tissue weight and liver triglyceride levels in the HC + FPs diet groups were significantly reduced compared to that in the HC diet groups. However, FPs significantly increased the serum non-high-density lipoprotein cholesterol (HDL-C) levels, the ratio of non-HDL-C to HDL-C and hepatic total cholesterol levels in mice fed cholesterol-loaded diet compared with that of the HC diet group. Since dietary FPs significantly decreased the protein expression levels of cholesterol 7 alpha-hydroxylase 1 in the HC + FPs diet groups, the cholesterol accumulation in FPs group may be explained by insufficient catabolism from cholesterol to bile acid. In addition, the dietary FPs tended to increase Clostridium cluster IV and XIVa, which are butyrate-producing bacteria. Related to the result, n-butyrate was significantly increased in the CO + FPs and the HC + FPs diet groups compared to their respective control groups. These findings suggested that dietary FPs modulated the lipid pool and gut microbiota.

Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor.

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.

Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability.

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.

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.

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.

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.

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.

Impact of withdrawing antithrombin III administration from management of septic patients with or without disseminated intravascular coagulation.

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.

Antitumor activity and pharmacodynamic biomarkers of a novel and orally available small-molecule antagonist of inhibitor of apoptosis proteins.

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.

Design and synthesis of potent inhibitor of apoptosis (IAP) proteins antagonists bearing an octahydropyrrolo[1,2-a]pyrazine scaffold as a novel proline mimetic.

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).

Prospective external validation of the new scoring system for disseminated intravascular coagulation by Japanese Association for Acute Medicine (JAAM).

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 15 years of age, those who stayed in the ICU for less than 24 hours, 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.

Removal of 2-arachidonylglycerol by direct hemoperfusion therapy with polymyxin B immobilized fibers benefits patients with septic shock.

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.