Discovery of a novel series of medium-sized cyclic enteropeptidase inhibitors.

Enteropeptidase is located in the duodenum that involved in intestinal protein digestion. We have reported enteropeptidase inhibitors with low systemic exposure. The aim of this study was to discover novel enteropeptidase inhibitors showing more potent in vivo efficacy while retaining low systemic exposure. Inhibitory mechanism-based drug design led us to cyclize ester 2 to medium-sized lactones, showing potent enteropeptidase inhibitory activity and improving the ester stability, thus increasing fecal protein output in vivo. Optimization on the linker between two benzene rings resulted in discovery of ether lactone 6b, exhibiting further enhanced enteropeptidase inhibitory activity and long duration of inhibitory state. Oral administration of 6b in mice significantly elevated fecal protein output compared with the lead 2. In addition, 6b showed low systemic exposure along with low intestinal absorption. Furthermore, we identified the 10-membered lactonization method for scale-up synthesis of 6b, which does not require high-dilution conditions.

Selective Acetyl-CoA Carboxylase 1 Inhibitor Improves Hepatic Steatosis and Hepatic Fibrosis in a Preclinical Nonalcoholic Steatohepatitis Model.

Acetyl-CoA carboxylase (ACC) 1 and ACC2 are essential rate-limiting enzymes that synthesize malonyl-CoA (M-CoA) from acetyl-CoA. ACC1 is predominantly expressed in lipogenic tissues and regulates the de novo lipogenesis flux. It is upregulated in the liver of patients with nonalcoholic fatty liver disease (NAFLD), which ultimately leads to the formation of fatty liver. Therefore, selective ACC1 inhibitors may prevent the pathophysiology of NAFLD and nonalcoholic steatohepatitis (NASH) by reducing hepatic fat, inflammation, and fibrosis. Many studies have suggested ACC1/2 dual inhibitors for treating NAFLD/NASH; however, reports on selective ACC1 inhibitors are lacking. In this study, we investigated the effects of compound-1, a selective ACC1 inhibitor for treating NAFLD/NASH, using preclinical in vitro and in vivo models. Compound-1 reduced M-CoA content and inhibited the incorporation of [14C] acetate into fatty acids in HepG2 cells. Additionally, it reduced hepatic M-CoA content and inhibited de novo lipogenesis in C57BL/6J mice after a single dose. Furthermore, compound-1 treatment of 8 weeks in Western diet-fed melanocortin 4 receptor knockout mice-NAFLD/NASH mouse model-improved liver hypertrophy and reduced hepatic triglyceride content. The reduction of hepatic M-CoA by the selective ACC1 inhibitor was highly correlated with the reduction in hepatic steatosis and fibrosis. These findings support further investigations of the use of this ACC1 inhibitor as a new treatment of NFLD/NASH. SIGNIFICANCE STATEMENT: This is the first study to demonstrate that a novel selective inhibitor of acetyl-CoA carboxylase (ACC) 1 has anti-nonalcoholic fatty liver disease (NAFLD) and anti-nonalcoholic steatohepatitis (NASH) effects in preclinical models. Treatment with this compound significantly improved hepatic steatosis and fibrosis in a mouse model. These findings support the use of this ACC1 inhibitor as a new treatment for NAFLD/NASH.

Design and synthesis of a monocyclic derivative as a selective ACC1 inhibitor by chemical modification of biphenyl ACC1/2 dual inhibitors.

A structure-activity relationship (SAR) study towards novel ACC1-selective inhibitors was carried out by modifying the molecular length of the linker in biaryl derivative 1 g, an ACC1/2 dual inhibitor. Ultimately, this leads us to discover novel phenoxybenzyloxy derivative 1i as a potent ACC1-selective inhibitor. Further chemical modification of this scaffold to improve cellular potency as well as physicochemical and pharmacokinetic (PK) properties produced N-2-(pyridin-2-ylethyl)acetamide derivative 1n, which showed highly potent ACC1-selective inhibition as well as sufficient PK profile for further in vivo evaluations. Oral administration of 1n significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of 100 mg/kg. Accordingly, our novel series of potent ACC1-selective inhibitors represents a set of useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid-related diseases.

The identification and pharmacological evaluation of potent, selective and orally available ACC1 inhibitor.

In our effort to explore the potential of ACC1-selective inhibitor as in vivo probe molecule, a series of 1,3-benzoxazole derivatives was synthesized. Previously, we reported a series of novel bicyclic and monocyclic ACC1-selective inhibitors. Among them, compound 1a exhibited highly potent cellular activity (acetate uptake IC50 = 0.76 nM) as well as promising in vivo PD efficacy. However, compound 1a caused severe body weight reduction in repeated dose administration in the mouse model. Since 1a showed potent inhibitory activity against mouse ACC1 as well as strong inhibition of mouse ACC2, we further examined a series of 1a analogues in order to reduce undesirable body weight change. The replacement of acetamide moiety with ureido moiety dramatically improved selectivity of mouse ACC1 against ACC2. In addition, analogue 1b displayed favorable bioavailability in mouse cassette dosing PK study, hence in vivo PD studies were also carried out. Oral administration of 1b significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at doses of more than 30 mg/kg. Furthermore, compound 1b showed significant antitumor efficacy in 786-O xenograft mice at an oral dose of 30 mg/kg (T/C = 0.5%). Accordingly, our novel potent ACC1-selective inhibitor represents a set of useful orally-available research tools, as well as potential therapeutic agents particularly in terms of new cancer therapies.

Design and synthesis of a novel 1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative as an orally available ACC1 inhibitor.

We initiated our structure-activity relationship (SAR) studies for novel ACC1 inhibitors from 1a as a lead compound. Our initial SAR studies of 1H-Pyrrolo[3,2-b]pyridine-3-carboxamide scaffold revealed the participation of HBD and HBA for ACC1 inhibitory potency and identified 1-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1c as a potent ACC1 inhibitor. Although compound 1c had physicochemical and pharmacokinetic (PK) issues, we investigated the 1H-pyrrolo[3,2-b]pyridine core scaffold to address these issues. Accordingly, this led us to discover a novel 1-isopropyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide derivative 1k as a promising ACC1 inhibitor, which showed potent ACC1 inhibition as well as sufficient cellular potency. Since compound 1k displayed favorable bioavailability in mouse cassette dosing PK study, we conducted in vivo Pharmacodynamics (PD) studies of this compound. Oral administration of 1k significantly reduced the concentration of malonyl-CoA in HCT-116 xenograft tumors at a dose of 100 mg/kg. Accordingly, our novel series of potent ACC1 inhibitors represent useful orally-available research tools, as well as potential therapeutic agents for cancer and fatty acid related diseases.

Nutrition intake among the Japanese elderly: an intergenerational comparison based on national health and nutrition survey scores.

Context: The average Japanese lifespan became the longest in the world in 1986. What factors give the Japanese this longevity? Washoku, or the traditional Japanese diet, is respected globally for its nutritionally-balanced and healthy eating habits. This uniquely Japanese way of eating may be one factor that helps extend the Japanese lifespan. Objective: To explain the nutrition intake characteristics of today's Japanese elderly compared with their children's generation and to discuss the association between nutrition intake and various diseases and health issues in the general adult population. Methods: This study compared the characteristics of nutrition status and nutrition intake among today's elderly and their children's generation by using National Health and Nutrition Survey scores. Results: Japanese elderly had high adequacy in all nutrients as well as a high intake of potatoes, pulses, vegetables, fruits, algae, and fish and shellfish compared with their children's generation. Conclusion: Nutrition intake among the Japanese elderly had the characteristics of washoku, but these characteristics were not passed on to the next generation. Extension of the average lifespan and improved health could be achieved by modifying nutrition intake, even after reaching the age of onset of lifestyle- and age-related diseases, typically the 50s.

Correlation between habitual dietary fibre intake and postprandial plasma glucose levels in early adulthood.

Background: Increasing dietary fibre (DF) intake through a habitual diet is recommended for preventing diabetes. Aim: To investigate a stepwise approach to nutrition education on DF intake among young adults in Japan with the largest deficit in habitual DF intake from the recommended value. Subjects and methods: Plasma glucose levels were measured in 54 adults in their 20s and 30s (29 men, 25 women) during fasting and at 30, 60, 90, and 120 minutes. Habitual DF intake and postprandial plasma glucose level were analysed. Results: DF intake was low (men = 5.7 ± 1.5 g/1000 kcal; women = 6.3 ± 1.2 g/1000 kcal) with no sex difference. Comparison between low- and high-DF groups based on the cut-off point (7.0 g/1000 kcal) showed that the pattern of changes in postprandial plasma glucose levels was significant, and a significant increase was observed at 30 minutes in the low-DF group. Comparison by food group demonstrated that habitual intake of potatoes, green/yellow vegetables, other vegetables, seaweed, peas and beans, and fruits was significantly higher in the high-DF group. Conclusion: This study provides valuable information regarding food selection for preventing diabetes and suggests that 7.0 g/1000 kcal of DF is an effective target value for a stepwise approach to nutrition education in Japan.

Discovery of Novel and Potent Stearoyl Coenzyme A Desaturase 1 (SCD1) Inhibitors as Anticancer Agents.

A lead compound A was identified previously as an stearoyl coenzyme A desaturase (SCD) inhibitor during research on potential treatments for obesity. This compound showed high SCD1 binding affinity, but a poor pharmacokinetic (PK) profile and limited chemical accessibility, making it suboptimal for use in anticancer research. To identify potent SCD1 inhibitors with more promising PK profiles, we newly designed a series of 'non-spiro' 4, 4-disubstituted piperidine derivatives based on molecular modeling studies. As a result, we discovered compound 1a, which retained moderate SCD1 binding affinity. Optimization around 1a was accelerated by analyzing Hansch-Fujita and Hammett constants to obtain 4-phenyl-4-(trifluoromethyl)piperidine derivative 1n. Fine-tuning of the azole moiety of 1n led to compound 1o (T-3764518), which retained nanomolar affinity and exhibited an excellent PK profile. Reflecting the good potency and PK profile, orally administrated compound 1o showed significant pharmacodynamic (PD) marker reduction (at 0.3mg/kg, bid) in HCT116 mouse xenograft model and tumor growth suppression (at 1mg/kg, bid) in 786-O mouse xenograft model. In conclusion, we identified a new series of SCD1 inhibitors, represented by compound 1o, which represents a promising new chemical tool suitable for the study of SCD1 biology as well as the potential development of novel anticancer therapies.

CD151 expression marks atrial- and ventricular- differentiation from human induced pluripotent stem cells.

Current differentiation protocols for human induced pluripotent stem cells (hiPSCs) produce heterogeneous cardiomyocytes (CMs). Although chamber-specific CM selection using cell surface antigens enhances biomedical applications, a cell surface marker that accurately distinguishes between hiPSC-derived atrial CMs (ACMs) and ventricular CMs (VCMs) has not yet been identified. We have developed an approach for obtaining functional hiPSC-ACMs and -VCMs based on CD151 expression. For ACM differentiation, we found that ACMs are enriched in the CD151low population and that CD151 expression is correlated with the expression of Notch4 and its ligands. Furthermore, Notch signaling inhibition followed by selecting the CD151low population during atrial differentiation leads to the highly efficient generation of ACMs as evidenced by gene expression and electrophysiology. In contrast, for VCM differentiation, VCMs exhibiting a ventricular-related gene signature and uniform action potentials are enriched in the CD151high population. Our findings enable the production of high-quality ACMs and VCMs appropriate for hiPSC-derived chamber-specific disease models and other applications.

ERRγ agonist under mechanical stretching manifests hypertrophic cardiomyopathy phenotypes of engineered cardiac tissue through maturation.

Engineered cardiac tissue (ECT) using human induced pluripotent stem cell-derived cardiomyocytes is a promising tool for modeling heart disease. However, tissue immaturity makes robust disease modeling difficult. Here, we established a method for modeling hypertrophic cardiomyopathy (HCM) malignant (MYH7 R719Q) and nonmalignant (MYBPC3 G115∗) pathogenic sarcomere gene mutations by accelerating ECT maturation using an ERRγ agonist, T112, and mechanical stretching. ECTs treated with T112 under 10% elongation stimulation exhibited more organized and mature characteristics. Whereas matured ECTs with the MYH7 R719Q mutation showed broad HCM phenotypes, including hypertrophy, hypercontraction, diastolic dysfunction, myofibril misalignment, fibrotic change, and glycolytic activation, matured MYBPC3 G115∗ ECTs displayed limited phenotypes, which were primarily observed only under our new maturation protocol (i.e., hypertrophy). Altogether, ERRγ activation combined with mechanical stimulation enhanced ECT maturation, leading to a more accurate manifestation of HCM phenotypes, including non-cardiomyocyte activation, consistent with clinical observations.

CCR7 Mediates Cell Invasion and Migration in Extrahepatic Cholangiocarcinoma by Inducing Epithelial-Mesenchymal Transition.

The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.

Design, synthesis, and structure-activity relationships of novel spiro-piperidines as acetyl-CoA carboxylase inhibitors.

Spiro-lactone (S)-1 is a potent acetyl-CoA carboxylase (ACC) inhibitor and was found to be metabolically liable in human hepatic microsomes. To remove one of the risk factors in human study by improving the metabolic stability, we focused on modifying the spiro-lactone ring and the benzothiophene portion of the molecule. Spiro-imide derivative 8c containing a 6-methylthieno[2,3-b]pyridine core exhibited potent ACC inhibitory activity and favorable pharmacokinetic profiles in rats.

Symmetrical approach of spiro-pyrazolidinediones as acetyl-CoA carboxylase inhibitors.

Spiro-pyrazolidinedione derivatives without quaternary chiral center were discovered by structure-based drug design and characterized as potent acetyl-CoA carboxylase (ACC) inhibitors. The high metabolic stability of the spiro-pyrazolo[1,2-a]pyridazine scaffold and enhancement of the activity by incorporation of a 7-methoxy group on the benzothiophene core successfully led to the identification of compound 4c as an orally bioavailable and highly potent ACC inhibitor. Oral administration of 4c significantly decreased the values of the respiratory quotient in rats, indicating the stimulation of fatty acid oxidation.

Time resolved DCE-MRI of the kidneys: Evaluation of the renal vasculatures and tumors using F-DISCO with and without compressed sensing in normal and wide-bore 3T systems.

Dynamic contrast-enhanced MR imaging (DCE-MRI) has been widely used for the evaluation of renal arteries. This method is also useful for tumor and renal parenchyma characterization. The very fast MRI may provide stable and precise information regarding vasculature and soft tissues. The purpose of this study was to evaluate the ability of DCE-MRI to assess renal vasculatures and tumor perfusions using Differential subsampling with Cartesian ordering with spectrally selected inversion recovery with adiabatic pulses (F-DISCO) with and without compressed sensing (CS) in normal and wide-bore 3T systems. Fifty-one patients who underwent DCE-MRI using F-DISCO with or without CS for evaluation of renal or adrenal regions were included. Image quality, artifacts, fat saturation, and selective visual recognition of renal vasculatures were assessed by using a 5-point scale. Tumor recognition was verified by using a 5-point scale of confidence level. Signal intensities of each structure were also measured. In all cases, the temporal resolution of each phase for DCE-MRI was 1.9 to 2.0 seconds. Image quality, artifacts, fat saturation, and selective visual recognition of vasculatures were all acceptable (mean score 4.2-4.9). The selective visualization of renal arteries and veins was successfully accomplished (mean score 4.0-4.9). Contrast media perfusion for renal vasculature, renal parenchyma, and tumors was also recognized. DCE-MRI for the evaluation of renal vasculatures and tumors using F-DISCO with or without CS can be performed with high temporal and spatial resolutions in normal and wide-bore 3T systems. This information can be obtained in a stable fashion throughout the dynamic contrast study. CS can additionally provide benefits that the total imaging time may be shorter than without CS.

Design, Synthesis, and Biological Evaluation of a Novel Series of 4-Guanidinobenzoate Derivatives as Enteropeptidase Inhibitors with Low Systemic Exposure for the Treatment of Obesity.

To discover a novel series of potent inhibitors of enteropeptidase, a membrane-bound serine protease localized to the duodenal brush border, 4-guanidinobenzoate derivatives were evaluated with minimal systemic exposure. The 1c docking model enabled the installation of an additional carboxylic acid moiety to obtain an extra interaction with enteropeptidase, yielding 2a. The oral administration of 2a significantly elevated the fecal protein output, a pharmacodynamic marker, in diet-induced obese (DIO) mice, whereas subcutaneous administration did not change this parameter. Thus, systemic exposure of 2a was not required for its pharmacological effects. Further optimization focusing on the in vitro IC50 value and T1/2, an indicator of dissociation time, followed by enhanced in vivo pharmacological activity based on the ester stability of the compounds, revealed two series of potent enteropeptidase inhibitors, a dihydrobenzofuran analogue ((S)-5b, SCO-792) and phenylisoxazoline (6b), which exhibited potent anti-obesity effects despite their low systemic exposure following their oral administration to DIO rats.

Discovery of potent, selective, and orally bioavailable quinoline-based dipeptidyl peptidase IV inhibitors targeting Lys554.

Dipeptidyl peptidase IV (DPP-4) inhibition is a validated therapeutic option for type 2 diabetes, exhibiting multiple antidiabetic effects with little or no risk of hypoglycemia. In our studies involving non-covalent DPP-4 inhibitors, a novel series of quinoline-based inhibitors were designed based on the co-crystal structure of isoquinolone 2 in complex with DPP-4 to target the side chain of Lys554. Synthesis and evaluation of designed compounds revealed 1-[3-(aminomethyl)-4-(4-methylphenyl)-2-(2-methylpropyl)quinolin-6-yl]piperazine-2,5-dione (1) as a potent, selective, and orally active DPP-4 inhibitor (IC50=1.3 nM) with long-lasting ex vivo activity in dogs and excellent antihyperglycemic effects in rats. A docking study of compound 1 revealed a hydrogen-bonding interaction with the side chain of Lys554, suggesting this residue as a potential target site useful for enhancing DPP-4 inhibition.

Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl Moiety.

The therapeutic potential of monoacylglycerol lipase (MAGL) inhibitors in central nervous system-related diseases has attracted attention worldwide. However, the availability of reversible-type inhibitor is still limited to clarify the pharmacological effect. Herein, we report the discovery of novel spiro chemical series as potent and reversible MAGL inhibitors with a different binding mode to MAGL using Arg57 and His121. Starting from hit compound 1 and its co-crystal structure with MAGL, structure-based drug discovery (SBDD) approach enabled us to generate various spiro scaffolds like 2a (azetidine-lactam), 2b (cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl moiety in 1 with higher lipophilic ligand efficiency (LLE). Optimization of the left hand side afforded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL inhibitory activity (IC50 6.2 nM), good oral absorption, blood-brain barrier penetration, and significant pharmacodynamic changes (2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3-10 mg/kg, po. in mice.

ERRγ enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes.

One of the earliest maturation steps in cardiomyocytes (CMs) is the sarcomere protein isoform switch between TNNI1 and TNNI3 (fetal and neonatal/adult troponin I). Here, we generate human induced pluripotent stem cells (hiPSCs) carrying a TNNI1EmGFP and TNNI3mCherry double reporter to monitor and isolate mature sub-populations during cardiac differentiation. Extensive drug screening identifies two compounds, an estrogen-related receptor gamma (ERRγ) agonist and an S-phase kinase-associated protein 2 inhibitor, that enhances cardiac maturation and a significant change to TNNI3 expression. Expression, morphological, functional, and molecular analyses indicate that hiPSC-CMs treated with the ERRγ agonist show a larger cell size, longer sarcomere length, the presence of transverse tubules, and enhanced metabolic function and contractile and electrical properties. Here, we show that ERRγ-treated hiPSC-CMs have a mature cellular property consistent with neonatal CMs and are useful for disease modeling and regenerative medicine.

Microsurgical observation of the posterior vitreous in patients with vitreous hemorrhage caused by Terson syndrome.

PURPOSE: To describe features characteristic of vitreous hemorrhage in patients with Terson syndrome observed through a microsurgical scope. METHODS: Between May 2015 and February 2019, 12 eyes of 10 patients with vitreous hemorrhage occurring after subarachnoid hemorrhage (SAH) underwent pars plana vitrectomy. RESULTS: During vitreous surgery, we found 10 of 12 eyes did not have posterior vitreous detachment (PVD). Furthermore, we observed in 9 of the 10 eyes without PVD (90.0%) that there was no hemorrhage in the posterior vitreous cavity at the posterior pole while we removed vitreous hemorrhage. We confirmed that this clean space could be the posterior precortical vitreous pocket (PPVP). CONCLUSION AND IMPORTANCE: Terson syndrome may have no hemorrhage in the PPVP regardless of the presence of severe vitreous hemorrhage. The cases presented in our study may suggest one of the mechanisms of Terson syndrome.

Thromboxane A2-induced signal transduction is negatively regulated by KIAA1005 that directly interacts with thromboxane A2 receptor.

Thromboxane A(2) (TXA(2)), a potent inducer of platelet aggregation and smooth muscle contraction, exerts its action through TXA(2) receptor (TP). There are two alternative splicing variants of TP, TP alpha and TP beta. To clarify the signal transduction of TP pathway, we searched for putative TP binding proteins using a yeast two-hybrid system with the C-terminal region of TP alpha or TP beta as bait. We found KIAA1005 as a novel interacting protein of the TP alpha and TP beta C-terminal region (TP interacting protein, TPIP). KIAA1005/TPIP was co-immunoprecipitated with TP alpha or TP beta in HEK293 cells expressing myc-KIAA1005/TPIP and FLAG-TP isoforms. Expression analysis showed a ubiquitous expression pattern of KIAA1005/TPIP mRNA, including prominent expression in the thymus. Furthermore, TP-mediated phosphoinositide hydrolysis, phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and interleukin-6 production were reduced by the expression of KIAA1005/TPIP. The expression of KIAA1005/TPIP decreased cell-surface TP alpha and TP beta levels. Thus, we show for the first time that KIAA1005/TPIP is a novel TP interacting protein that regulates TP-mediated signal transduction negatively.