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.

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.

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.

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.

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.

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.

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.

Inhibitory activities of anthraquinone and xanthone derivatives against transthyretin amyloidogenesis.

Transthyretin is a tetrameric protein which functions as a transporter of thyroxine and retinol-binding protein. Misfolding and amyloid aggregation of transthyretin are known to cause wild-type and hereditary transthyretin amyloidosis. Stabilization of the transthyretin tetramer by low molecular weight compounds is an efficacious strategy to inhibit the aggregation pathway in the amyloidosis. Here, we investigated the inhibitory activities of anthraquinone and xanthone derivatives against amyloid aggregation, and found that xanthone-2-carboxylic acid with one chlorine or methyl group has strong inhibitory activity comparable with that of diflunisal, which is one of the best known stabilizers of transthyretin. X-ray crystallographic structures of transthyretin in complex with the compounds revealed that the introduction of chlorine, which is buried in a hydrophobic region, is important for the strong inhibitory effect of the stabilizer against amyloidogenesis. An in vitro absorption, distribution, metabolism and elimination (ADME) study and in vivo pharmacokinetic study demonstrated that the compounds have drug-like features, suggesting that they have potential as therapeutic agents to stabilize transthyretin.

Aldo-keto reductase inhibitors increase the anticancer effects of tyrosine kinase inhibitors in chronic myelogenous leukemia.

Tyrosine kinase inhibitors (TKIs) are widely utilized in clinical practice to treat carcinomas, but secondary tumor resistance during chronic treatment can be problematic. AKR1B1 and AKR1B10 of the aldo-keto reductase (AKR) superfamily are highly expressed in cancer cells and are believed to be involved in drug resistance. The aim of this study was to understand how TKI treatment of chronic myelogenous leukemia (CML) cells changes their glucose metabolism and if inhibition of AKRs can sensitize CML cells to TKIs. K562 cells were treated with the TKIs imatinib, nilotinib, or bosutinib, and the effects on glucose metabolism, cell death, glutathione levels, and AKR levels were assessed. To assess glucose dependence, cells were cultured in normal and low-glucose media. Pretreatment with AKR inhibitors, including epalrestat, were used to determine AKR-dependence. Treatment with TKIs increased intracellular glucose, AKR1B1/10 levels, glutathione oxidation, and nuclear translocation of nuclear factor erythroid 2-related factor 2, but with minimal cell death. These effects were dependent on intracellular glucose accumulation. Pretreatment with epalrestat, or a selective inhibitor of AKR1B10, exacerbated TKI-induced cell death, suggesting that especially AKR1B10 was involved in protection against TKIs. Thus, by disrupting cell protective mechanisms, AKR inhibitors may render CML more susceptible to TKI treatments.

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.

Chemical constituents of Callistemon citrinus from Egypt and their antiausterity activity against PANC-1 human pancreatic cancer cell line.

Human pancreatic cancer is resistant to almost all conventional chemotherapeutic agents. It is known to proliferate aggressively within hypovascular tumor microenvironment by exhibiting remarkable tolerance to nutrition starvation,  a phenomenon termed as "austerity". Search for the new agents that eliminate the tolerance of cancer cells to nutrition starvation is a promising strategy in anticancer drug discovery. In this study, two new meroterpenoids named callistrilones O and P (1 and 2) together with eight known triterpenes (3-10) were isolated from the active dichloromethane extract of Callistemon citrinus leaves. The structure elucidation of the new compounds was achieved by HRFABMS, 1D, 2D NMR, and ECD quantum calculations. All isolated compounds were tested for their preferential cytotoxicity against PANC-1 human pancreatic cancer cells. Among these, callistrilone O (1) exhibited the most potent preferential cytotoxicity with a PC50 value of 0.3 nM, the strongest activity with over 2000 times potent than the positive control arctigenin. Callistrilone O (1) induced dramatic alterations in PANC-1 cell morphology leading to cell death under nutrient-deprived conditions. Compound 1 also inhibited PANC-1 cell migration and -PANC-1 colony formation under the nutrient-rich condition.

Highly Potent Antiausterity Agents from Callistemon citrinus and Their Mechanism of Action against the PANC-1 Human Pancreatic Cancer Cell Line.

Human pancreatic cancer cells display remarkable tolerance to nutrition starvation that help them to survive in a hypovascular tumor microenvironment, a phenomenon known as "austerity". The elucidation of agents countering this tolerance is an established antiausterity strategy in anticancer drug discovery. In this study, a Callistemon citrinus leaf extract inhibited the viability of PANC-1 human pancreatic cancer cells preferentially under nutrient-deprived medium (NDM) with a PC50 value of 7.4 µg/mL. Workup of this extract resulted in the isolation of three new meroterpenoids, callistrilones L-N (1-3), together with 14 known compounds (4-17). The structure elucidation of the new compounds was achieved by HRFABMS and by NMR and ECD spectroscopic analysis. The new compounds showed highly potent preferential cytotoxicity against PANC-1 cells with PC50 values ranging from 10 to 65 nM in NDM. Of these, callistrilone L (1) inhibited PANC-1 cell migration and colony formation in a normal nutrient-rich condition. Callistrilone L (1) also strongly suppressed the migration of PANC-1 cells in real time. Mechanistically, 1 was found to inhibit the Akt/mTOR and autophagy activation pathway. Callistrilone L (1) and related meroterpenoids are promising leads for anticancer drug development based on the antiausterity strategy used in this work.

Formal Syntheses of (-)-Lepadiformines A, C, and (-)-Fasicularin.

Marine tricyclic alkaloids lepadiformine and fasicularin, with a unique perhydropyrrolo[2,1- j]quinoline or perhydropyrido[2,1- j]quinoline framework, were synthesized starting from the B ring of the tricyclic system. This approach includes a highly stereocontrolled diallylation of a cyclic enaminoester and subsequent ring-closing metathesis to construct the A/B ring system, which was transformed into key lactams 32 and 33, and amino alcohol 37. Thus, we achieved formal syntheses of (-)-lepadiformines A, C, and (-)-fasicularin in a divergent manner.

Design and synthesis of functionalized coumarins as potential anti-austerity agents that eliminates cancer cells' tolerance to nutrition starvation.

Human pancreatic tumor cells have inherent ability to tolerate nutrition starvation which enables them to survive in the hypovascular tumor microenvironment. Discovery of agents that selectively inhibit the cancer cells' tolerance to nutrition starvation leading to cancer cell death is a new anti-austerity approach in anti-cancer drug discovery. A series of coumarins derivatives were synthesized and evaluated for their anti-austerity activity against PANC-1 human pancreatic cancer cell line. The compound 7-Hydroxy-2-oxo-2H-chromene-3-carboxylic acid (3-phenylpropyl)amide (2c) showed highly potent selective cytotoxicity against PANC-1 cells under nutrient-deprived conditions, with a PC50 value of 0.44 µM, without exhibiting toxicity in normal, nutrient-rich medium. Compound 2c caused dramatic alterations in PANC-1 cell morphology, leading to cell death. The compound 2c was found to inhibit PANC-1 cell migration and colony formation in a concentration-dependent manner. The compound 2c is a lead structure for the anti-austerity drug development against pancreatic cancer.

The novel small-molecule antagonist of PAC1 receptor attenuates formalin-induced inflammatory pain behaviors in mice.

We recently developed PA-8, a novel small-molecule antagonist of PACAP type 1 (PAC1) receptor. In the present study, we examined whether PA-8 was effective against formalin-induced inflammatory pain in mice. Both intrathecal and oral administration of PA-8 resulted in the dose-dependent attenuation of the second phase of formalin-induced nociceptive responses. PA-8 also inhibited c-fos upregulation in the ipsilateral dorsal horn of the spinal cord. The results suggested that PACAP-PAC1 receptor signaling system in the spinal cord were primarily involved in the transmission of inflammatory pain, and PA-8 could be useful for the development of novel analgesics for treating inflammatory pain.

Identification and Total Synthesis of Two Previously Unreported Odd-Chain Bis-Methylene-Interrupted Fatty Acids with a Terminal Olefin that Activate Protein Phosphatase, Mg2+/Mn2+-Dependent 1A (PPM1A) in Ovaries of the Limpet Cellana toreuma.

Diverse non-methylene-interrupted (NMI) fatty acids (FAs) with odd-chain lengths have been recognized in triacylglycerols and polar lipids from the ovaries of the limpet Cellana toreuma, however their biological properties remain unclear. In this study, two previously unreported odd-chain NMI FAs, (12Z)-12,16-heptadecadienoic (1) and (14Z)-14,18-nonadecadienoic (2) acids, from the ovary lipids of C. toreuma were identified by a combination of equivalent chain length (ECL) values of their methyl esters and capillary gas chromatography-mass spectrometry (GC-MS) of their 3-pyridylcarbinol derivatives. On the basis of the experimental results, both 1 and 2 were synthesized to prove their structural assignments and to test their biological activity. The ECL values and electron impact-mass (EI-MS) spectra of naturally occurring 1 and 2 were in agreement with those of the synthesized 1 and 2. In an in vitro assay, both 1 and 2 activated protein phosphatase, Mg2+/Mn2+-dependent 1A (PPM1A) up to 100 µM in a dose-dependent manner.

In Silico Screening Identified Novel Small-molecule Antagonists of PAC1 Receptor.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors are present in the spinal dorsal horn and dorsal root ganglia, suggesting an important role of PACAP signaling systems in the modulation of spinal nociceptive transmission. Previously, we found that intrathecal injection of PACAP or maxadilan, a selective PACAP type I (PAC1) receptor agonist, induced transient aversive responses followed by a long-lasting mechanical allodynia in mice, suggesting that PACAP-PAC1 receptor systems are involved in chronic pain and that selective PAC1 antagonists may become a new class of analgesics. Although several PAC1 antagonists, such as PACAP 6-38, have been reported, all of them are peptide compounds. In the present study, we identified new small-molecule antagonists of the PAC1 receptor using in silico screening and in vitro/vivo pharmacological assays. The identified small-molecule compounds, named PA-8 and PA-9, dose dependently inhibited the phosphorylation of CREB induced by PACAP in PAC1-, but not VPAC1- or VPAC2-receptor-expressing CHO cells. PA-8 and PA-9 also dose dependently inhibited PACAP-induced cAMP elevation with an IC50 of 2.0 and 5.6 nM, respectively. In vivo pharmacological assays showed that intrathecal injection of these compounds blocked the induction of PACAP-induced aversive responses and mechanical allodynia in mice. In contrast, the compounds when administered alone exerted neither agonistic nor algesic actions in the in vitro/vivo assays. The compounds identified in the present study are new and the first small-molecule antagonists of the PAC1 receptor; they may become seed compounds for developing novel analgesics.

Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase.

Most of the endogenous free d-serine (about 90%) in the brain is produced by serine racemase (SR). d-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico screening using the structural information. As a result, we identified two hit compounds by their in vitro evaluations using wild-type SR. Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent inhibitor than compound 24, which was synthesized by our group based upon the structural information of the mutant-type SR.

Design and synthesis of novel anti-hyperalgesic agents based on 6-prenylnaringenin as the T-type calcium channel blockers.

Since 6-prenylnaringenin (6-PNG) was recently identified as a novel T-type calcium channel blocker with the IC50 value around 1 µM, a series of flavanone derivatives were designed, synthesized and subsequently evaluated for T-channel-blocking activity in HEK293 cells transfected with Cav3.2 T-type channels using a patch-clamp technique. As a result, several new flavanones blocked Cav3.2-dependent T-currents more potently than 6-PNG. In the synthesized compounds, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(2-hydroxyphenyl)chroman-4-one 8j, 6-(3-ethylpent-2-enyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11b, 6-(2-cyclopentylideneethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 11d, and 6-(2-Cyclopentylethyl)-5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one 12c were more potent blocker than 6-PNG with the IC50 value of 0.39, 0.26, 0.46, and 0.50 µM, respectively. Among the above four derivatives, the compound 8j provided the best result in the in vivo experiments; i.e. systemic administration of 8j at the minimum dose completely restored neuropathic pain induced by partial sciatic nerve ligation in mice.