Resting phase-administration of lemborexant ameliorates sleep and glucose tolerance in type 2 diabetic mice.

Sleep disorders are associated with increased risk of obesity and type 2 diabetes. Lemborexant, a dual orexin receptor antagonist (DORA), is clinically used to treat insomnia. However, the influence of lemborexant on sleep and glucose metabolism in type 2 diabetic state has remained unknown. In the present study, we investigated the effect of lemborexant in type 2 diabetic db/db mice exhibiting both sleep disruption and glucose intolerance. Single administration of lemborexant at the beginning of the light phase (i.e., resting phase) acutely increased total time spent in non-rapid eye movement (NREM) and REM sleep in db/db mice. Durations of NREM sleep-, REM sleep-, and wake-episodes were also increased by this administration. Daily resting-phase administration of lemborexant for 3-6 weeks improved glucose tolerance without changing body weight and glucose-stimulated insulin secretion in db/db mice. Similar improvement of glucose tolerance was caused by daily resting-phase administration of lemborexant in obese C57BL/6J mice fed high fat diet, whereas no such effect was observed in non-diabetic db/m+ mice. Diabetic db/db mice treated daily with lemborexant exhibited increased locomotor activity in the dark phase (i.e., awake phase), although they did not show any behavioral abnormality in the Y-maze, elevated plus maze, and forced swim tests. These results suggest that timely promotion of sleep by lemborexant improved the quality of wakefulness in association with increased physical activity during the awake phase, and these changes may underlie the amelioration of glucose metabolism under type 2 diabetic conditions.

Design and structural optimization of thiadiazole derivatives with potent GLS1 inhibitory activity.

GLS1 is an attractive target not only as anticancer agents but also as candidates for various potential pharmaceutical applications such as anti-aging and anti-obesity treatments. We performed docking simulations based on the complex crystal structure of GLS1 and its inhibitor CB-839 and found that compound A bearing a thiadiazole skeleton exhibits GLS1 inhibition. Furthermore, we synthesized 27 thiadiazole derivatives in an effort to obtain a more potent GLS1 inhibitor. Among the synthesized derivatives, 4d showed more potent GLS1 inhibitory activity (IC50 of 46.7 µM) than known GLS1 inhibitor DON and A. Therefore, 4d is a very promising novel GLS1 inhibitor.

Rafoxanide, a salicylanilide anthelmintic, interacts with human plasma protein transthyretin.

Transthyretin (TTR) is a carrier protein for thyroid hormone thyroxine (T4 ) in plasma, placental cytosol, and cerebrospinal fluid. While the potential toxicity of small molecules that compete with T4 for binding to TTR should be carefully studied, these small molecules can also serve as anti-ATTR amyloidosis drugs by stabilizing the TTR structure. Here, we demonstrated that rafoxanide, an EU-approved anthelmintic drug for domesticated animals, binds to the T4 -binding site of TTR. An intrinsic fluorescence quenching assay showed that rafoxanide also binds to the thyroid hormone-related proteins, including serum albumin and thyroid hormone receptor ß. Rafoxanide strongly inhibited TTR amyloidogenesis in fibrillization assay, but the binding of rafoxanide to TTR was interfered with in human plasma, probably due to interactions with thyroid hormone-related proteins. Protein crystallography provided clues for the optimization of binding affinity and selectivity. Our findings emphasize the importance of considering rafoxanide as both a possible thyroid-disrupting chemical and a lead compound for the development of new ATTR amyloidosis inhibitors.

Design and Pharmacological Chaperone Effects of N-(4'-Phenylbutyl)-DAB Derivatives Targeting the Lipophilic Pocket of Lysosomal Acid α-Glucosidase.

This study provides the first example of a strategy to design a practical ligand toward lysosomal acid α-glucosidase (GAA) focusing on N-alkyl derivatives of 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). The optimized N-4'-(p-trifluoromethylphenyl)butyl-DAB (5g) showed a Ki value of 0.73 µM, which was 353-fold higher affinity than N-butyl-DAB (3f) without a terminal phenyl group. Docking analysis showed that the phenyl part of 5g was accommodated in a lipophilic pocket. Furthermore, the p-trifluoromethyl group effectively suppresses the fluctuation of the phenyl group, allowing it to produce a stable bonding form with GAA. 5g increased the midpoint of the protein's protein denaturation temperature (Tm) by 6.6 °C above that in the absence of the ligand and acted as a "thermodynamic stabilizer" to improve the thermal stability of rhGAA. 5g dose-dependently increased intracellular GAA activities in Pompe patient's fibroblasts with the M519V mutation; its effect was comparable to that of DNJ, which is under clinical trials.

Benziodarone and 6-hydroxybenziodarone are potent and selective inhibitors of transthyretin amyloidogenesis.

Transthyretin amyloidosis is a progressive systemic disorder that is caused by the amyloid deposition of transthyretin in various organs. Stabilization of the native transthyretin is an effective strategy for the treatment of transthyretin amyloidosis. In this study we demonstrate that the clinically used uricosuric agent benziodarone is highly effective to stabilize the tetrameric structure of transthyretin. An acid-induced aggregation assay showed that benziodarone had strong inhibitory activity similar to that of tafamidis, which is currently used as a therapeutic agent for transthyretin amyloidosis. Moreover, a possible metabolite, 6-hydroxybenziodarone, retained the strong amyloid inhibitory activity of benziodarone. An ex vivo competitive binding assay using a fluorogenic probe showed that benziodarone and 6-hydroxybenziodarone were highly potent for selective binding to transthyretin in human plasma. An X-ray crystal structure analysis revealed that the halogenated hydroxyphenyl ring was located at the entrance of the thyroxine binding channel of transthyretin and that the benzofuran ring was located in the inner channel. These studies suggest that benziodarone and 6-hydroxybenziodarone would potentially be effective against transthyretin amyloidosis.

Targeting Pancreatic Cancer with Novel Plumbagin Derivatives: Design, Synthesis, Molecular Mechanism, In Vitro and In Vivo Evaluation.

Pancreatic tumors grow in an "austerity" tumor microenvironment characterized by nutrient deprivation and hypoxia. This leads to the activation of adaptive pathways in pancreatic cancer cells, promoting tolerance to nutrition starvation and aggressive malignancy. Conventional anticancer drugs are often ineffective against tumors that grow in such austerity condition. Plumbagin, a plant-derived naphthoquinone, has shown potent preferential cytotoxicity against pancreatic cancer cells under nutrient-deprived conditions. Therefore, we synthesized a series of plumbagin derivatives and found that 2-(cyclohexylmethyl)-plumbagin (3f) was the most promising compound with a PC50 value of 0.11 µM. Mechanistically, 3f was found to inhibit the PI3K/Akt/mTOR signaling pathways, leading to cancer cell death under nutrient-deprived conditions. In vivo studies using pancreatic cancer xenograft mouse models confirmed the efficacy of 3f, demonstrating significant inhibition of tumor growth in a dose-dependent manner. Compound 3f represents a highly promising lead for anticancer drug development based on an antiausterity strategy.

Design, synthesis, structure-activity relationship studies, and evaluation of novel GLS1 inhibitors.

Glutaminase converts glutamine into glutamic acid and has two isoforms: glutaminase 1 (GLS1) and glutaminase 2 (GLS2). GLS1 is overexpressed in several tumors, and research to develop glutaminase inhibitors as antitumor drugs is currently underway. The present study examined candidate GLS1 inhibitors using in silico screening and attempted to synthesize novel GLS1 inhibitors and assess their GLS1 inhibitory activities in a mouse kidney extract and against recombinant mouse and human GLS1. Novel compounds were synthesized using compound C as the lead compound, and their GLS1 inhibitory activities were evaluated using the mouse kidney extract. Among the derivatives tested, the trans-4-hydroxycyclohexylamide derivative 2j exhibited the strongest inhibitory activity. We also assessed the GLS1 inhibitory activities of the derivatives 2j, 5i, and 8a against recombinant mouse and human GLS1. The derivatives 5i and 8a significantly decreased the production of glutamic acid at 10 mM. In conclusion, we herein identified two compounds that exhibited GLS1 inhibitory activities with equal potencies as known GLS1 inhibitors. These results will contribute to the development of effective novel GLS1 inhibitors with more potent inhibitory activity.

Nicolaioidesin C: An Antiausterity Agent Shows Promising Antitumor Activity in a Pancreatic Cancer Xenograft Mouse Model.

Human pancreatic tumors are hypovascular in nature, and their tumor microenvironment is often characterized by hypoxia and severe nutrient deprivation due to uncontrolled heterogeneous growth, a phenomenon known as "austerity". However, pancreatic tumor cells have the inherent ability to adapt and thrive even in such low nutrient and hypoxic microenvironments. Anticancer drugs such as gemcitabine and paclitaxel, which target rapidly proliferating cells, are often ineffective against nutrient-deprived pancreatic cancer cells. In order to overcome this limitation, the search for novel agents that can eliminate cancer cells' adaptations to nutrition starvation, also known as "antiausterity" agents, represents a promising strategy to make the cancer cells susceptible to treatment. The natural product (+)-nicolaioidesin C (Nic-C) was found to have potent antiausterity activity against the PANC-1 human pancreatic cancer cell line in a nutrient-deprived condition. However, its efficacy in vivo remained untested. To address this, we synthesized Nic-C in its racemic form and evaluated its antitumor potential in a human pancreatic cancer xenograft model. Nic-C inhibited pancreatic cancer cell migration and colony formation and significantly inhibited tumor growth in MIA PaCa-2 xenografts in a dose-dependent manner. Furthermore, Nic-C inhibited the Akt/mTOR and autophagy signaling pathways in both in vitro and in vivo studies. Metabolomic profiling of in vivo tumor samples suggests that Nic-C downregulates amino acid metabolism while upregulating sphingolipid metabolism.

Chlorinated Naringenin Analogues as Potential Inhibitors of Transthyretin Amyloidogenesis.

Misfolding and aggregation of transthyretin are implicated in the fatal systemic disease known as transthyretin amyloidosis. Here, we report the development of a naringenin derivative bearing two chlorine atoms that will be efficacious for preventing aggregation of transthyretin in the eye. The amyloid inhibitory activity of the naringenin derivative was as strong as that of tafamidis, which is the first therapeutic agent targeting transthyretin in the plasma. X-ray crystal structures of the compounds in complex with transthyretin demonstrated that the naringenin derivative with one chlorine bound to the thyroxine-binding site of transthyretin in the forward mode and that the derivative with two chlorines bound to it in the reverse mode. An ex vivo competitive binding assay showed that naringenin derivatives exhibited more potent binding than tafamidis in the plasma. Furthermore, an in vivo pharmacokinetic study demonstrated that the dichlorinated derivative was significantly delivered to the eye.

Small-molecule non-peptide antagonists of the PACAP receptor attenuate acute restraint stress-induced anxiety-like behaviors in mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved pleiotropic neuropeptide, implicated in emotional stress responses and anxiety-related disorders. Here, we examined whether our recently developed small-molecule non-peptide PACAP receptor antagonists could ameliorate anxiety-like behaviors induced by acute restraint stress in mice. The antagonists PA-9 and its derivative PA-915 improved anxiety-like behaviors in mice subjected to restraint stress. An anxiolytic effect was observed with single acute dose, suggesting their fast-acting properties. PA-915 demonstrated a statistically significant anxiolytic effect whereas fluoxetine did not. These results indicate the potential of PAC1 antagonists as a novel treatment for anxiety.

Discovery of pimozide derivatives as novel T-type calcium channel inhibitors with little binding affinity to dopamine D2 receptors for treatment of somatic and visceral pain.

T-type Ca2+ channels (T-channels), particularly Cav3.2 and Cav3.1 isoforms, are promising targets for treating various diseases including intractable pain. Given the potent inhibitory activity of pimozide, an antipsychotic, against T-channels, we conducted structure-activity relationship studies of pimozide derivatives, and identified several compounds including 3a, 3s, and 4 that had potency comparable to that of pimozide in inhibiting T-channels, but little binding affinity to dopamine D2 receptors. The introduction of a phenylbutyl group on the benzoimidazole nuclei of pimozide was considered a key structural modification to reduce the binding affinity to D2 receptors. Those pimozide derivatives potently suppressed T-channel-dependent somatic and visceral pain in mice, without causing any motor dysfunctions attributable to D2 receptor blockade, including catalepsy. The present study thus provides an avenue to develop novel selective T-channel inhibitors available for pain management via the structural modification of existing medicines.

Design and synthesis of novel pipernonaline derivatives as anti-austerity agents against human pancreatic cancer PANC-1 cells.

Pipernonaline (1), one of the components of the spice pepper, preferentially reduced the survival of human pancreatic cancer PANC-1 cells under nutrient-deprived conditions witha PC50 value of 7.2 µM, suggesting that1couldpotentially lead to the development ofnew anticanceragents basedon theanti-austerity strategy. We have synthesized a total of 31 pipernonaline derivatives, revealing clear structure-activity relationships. Compound 9, which showed the strongest preferential cytotoxicity among synthesized derivatives, inhibited Akt activation and cancer cell migration, making it an extremely promising candidate compound for new pancreatic cancer agents based on the anti-austerity strategy.

A New Method for Albuminuria Measurement Using a Specific Reaction between Albumin and the Luciferin of the Firefly Squid Watasenia scintillans.

This study demonstrates that the luciferin of the firefly squid Watasenia scintillans, which generally reacts with Watasenia luciferase, reacted with human albumin to emit light in proportion to the albumin concentration. The luminescence showed a peak wavelength at 540 nm and was eliminated by heat or protease treatment. We used urine samples collected from patients with diabetes to quantify urinary albumin concentration, which is essential for the early diagnosis of diabetic nephropathy. Consequently, we were able to measure urinary albumin concentrations by precipitating urinary proteins with acetone before the reaction with luciferin. A correlation was found with the result of the immunoturbidimetric method; however, the Watasenia luciferin method tended to produce lower albumin concentrations. This may be because the Watasenia luciferin reacts with only intact albumin. Therefore, the quantification method using Watasenia luciferin is a new principle of urinary albumin measurement that differs from already established methods such as immunoturbidimetry and high-performance liquid chromatography.

Coupling of acceptor-substituted diazo compounds and tertiary thioamides: synthesis of enamino carbonyl compounds and their pharmacological evaluation.

This paper describes the synthesis of enamino carbonyl compounds by the copper(i)-catalyzed coupling of acceptor-substituted diazo compounds and tertiary thioamides. We plan to use this method to synthesize indolizidine (-)-237D analogs to find α6-selective antismoking agents. Therefore, we also performed in silico α6-nAchRs binding studies of selected products. Compounds with low root-mean-square deviation values showed more favorable binding free energies. We also report preliminary pharmacokinetic data on indolizidine (-)-237D and found it to have weak activity at CYP3A4. In addition, as enamino carbonyl compounds are also known for antimicrobial properties, we screened previously reported and new enamino carbonyl compounds for antibacterial, antimicrobial, and antifungal properties. Eleven compounds showed significant antimicrobial activities.

Abietane diterpenes from Abies spectabilis and their anti-pancreatic cancer activity against the MIA PaCa-2 cell line.

An ethanolic extract of the stem of Abies spectabilis exhibited strong cytotoxicity against MIA PaCa-2 human pancreatic cancer cells preferentially under nutrient-deprived conditions. Therefore, phytochemical investigation of this bioactive extract was carried out, and that led the isolation of ten compounds (1-10) including a new abietane-type diterpene (1). The structure of the new compound (1) was elucidated by combined spectroscopic techniques, including HRFABMS, NMR and quantum ECD calculation. All the isolated compounds were evaluated for their efficacy against MIA PaCa-2 human pancreatic cancer cell line by employing an anti-austerity strategy. Among the tested compounds, dehydroabietinol (5) displayed the most potent activity with a PC50 value of 6.6 µM. Dehydroabietinol (5) was also found to retard the MIA PaCa-2 cell migration under normal nutrient-rich conditions displaying its anti-metastatic potential. Investigation on the mechanism suggested that dehydroabietinol (5) is an inhibitor of the key cancer cell survival Akt/mTOR/autophagy signaling pathway.

Discovery and Structure-Based Optimization of Novel Atg4B Inhibitors for the Treatment of Castration-Resistant Prostate Cancer.

Autophagy inhibition is an attractive target for cancer therapy. In this study, we discovered inhibitors of Atg4B essential for autophagosome formation and evaluated their potential as therapeutics for prostate cancer. Seventeen compounds were identified as candidates after in silico screening and a thermal shift assay. Among them, compound 17 showed the most potent Atg4B inhibitory activity, inhibited autophagy induced by anti-castration-resistant prostate cancer (CRPC) drugs, and significantly enhanced apoptosis. Although 17 has been known as a phospholipase A2 (PLA2) inhibitor, other PLA2 inhibitors had no effect on Atg4B and autophagy. We then performed structural optimization based on molecular modeling and succeeded in developing 21f (by shortening the alkyl chain of 17), which was a potent competitive inhibitor for Atg4B (Ki = 3.1 µM) with declining PLA2 inhibitory potency. Compound 21f enhanced the anticancer activity of anti-CRPC drugs via autophagy inhibition. These findings suggest that 21f can be used as an adjuvant drug for therapy with anti-CRPC drugs.

Design and synthesis of pyrido[2,3-d]pyrimidine derivatives for a novel PAC1 receptor antagonist.

Since PA-8 (5-(4-(Allyloxy)-3-methoxyphenyl)-2-amino-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione) was recently identified as a novel small-molecule antagonist of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC1) receptor, a series of pyrido[2,3-d]pyrimidine derivatives have been designed, synthesized and subsequently evaluated for antagonistic activity on the PAC1 receptor. In this study, we synthesized 21 derivatives based on the PA-8 structure. Among them, the compound 2o (2-Amino-5-(3-trifluoromethoxy-phenyl)-5,8-dihydro-3H,6H-pyrido[2,3-d]pyrimidine-4,7-dione) showed more potent antagonistic activities than PA-8. Intrathecal (i.t.) injection of 2o blocked the induction of PACAP-induced aversive behaviors and mechanical allodynia in mice, and the effects were more potent than those of PA-8. A single i.t. injection of 2o also inhibited spinal nerve ligation (SNL)-induced mechanical allodynia. Repeated intraperitoneal administration of 2o gradually reduced the SNL-induced mechanical allodynia, and this effect appeared earlier than for PA-8. In addition, 2o exhibited a favorable ADME and pharmacokinetics profiles. These results suggest that 2o may become an analgesic for the treatment of neuropathic pain.

Suvorexant and mirtazapine improve chronic pain-related changes in parameters of sleep and voluntary physical performance in mice with sciatic nerve ligation.

Both chronic pain and sleep disorders are associated with a reduction in the quality of life. They can be both a cause and a consequence of each other, and should therefore be simultaneously treated. However, optimal treatments for chronic pain-related sleep disorders are not well established. Here, we aimed to investigate the effects of suvorexant, a novel sleep drug, and mirtazapine, a noradrenergic and specific serotonergic antidepressant, on pain-related changes in sleep parameters in a preclinical chronic pain mice model, by partial sciatic nerve ligation. We evaluated the quantity, duration, and depth of sleep by analyzing the electroencephalogram and voluntary activity by counting the number of wheel rotations to determine various symptoms of sleep disorders, including reduced total sleep time, fragmentation, low quality, and impaired activity in the daytime. Suvorexant and mirtazapine normalized the reduction in sleep time and fragmented sleep, further regaining the sleep depth at sleep onset in the chronic pain state in nerve-ligated mice. Mirtazapine also increased the percentage of rapid eye movement sleep in mice. Suvorexant decreased voluntary activity, which was prolonged after administration; however, mirtazapine did not decrease it. Although the effects of suvorexant and mirtazapine on sleep and activity are different, both suvorexant and mirtazapine could be potential therapeutic agents for chronic pain-related sleep disorders.

Total Synthesis of Decahydroquinoline Poison Frog Alkaloids ent-cis-195A and cis-211A.

The total synthesis of two decahydroquinoline poison frog alkaloids ent-cis-195A and cis-211A were achieved in 16 steps (38% overall yield) and 19 steps (31% overall yield), respectively, starting from known compound 1. Both alkaloids were synthesized from the common key intermediate 11 in a divergent fashion, and the absolute stereochemistry of natural cis-211A was determined to be 2R, 4aR, 5R, 6S, and 8aS. Interestingly, the absolute configuration of the parent decahydroquinoline nuclei of cis-211A was the mirror image of that of cis-195A, although both alkaloids were isolated from the same poison frog species, Oophaga (Dendrobates) pumilio, from Panama.