Re-discovery of MS-347a as a fungicide candidate through a new drug discovery platform with a multidrug-sensitive Saccharomyces cerevisiae screening system and the introduction of a global secondary metabolism regulator, laeA gene.

We found that the culture broth of fungi showed anti-fungal activity against multidrug-sensitive budding yeast. However, we could not identify the anti-fungal compound due to the small quantity. Therefore, we attempted to increase the productivity of the target compound by the introduction of a global secondary metabolism regulator, laeA to the strain, which led to the successful isolation of ten-folds greater amount of MS-347a (1) than Aspergillus sp. FKI-5362. Compound 1 was not effective against Candida albicans and the detailed anti-fungal activity of 1 remains unverified. After our anti-fungal activity screening, 1 was found to inhibit the growth of broad plant pathogenic fungal species belonging to the Ascomycota. It is noteworthy that 1 showed little insecticidal activity against silkworms, suggesting its selective biological activity against plant pathogenic fungi. Our study implies that the combination strategy of multidrug-sensitive yeast and the introduction of laeA is useful for new anti-fungal drug discovery.

Design, synthesis, and biological evaluation of new biaryl derivatives of cycloalkyl diacetamide bearing chalcone moiety as type II c-MET kinase inhibitors.

Many human cancers have been associated with the deregulation of the mesenchymal-epithelial transition factor tyrosine kinase (MET) receptor, a promising drug target for anticancer drug discovery. Herein, we report the discovery of a novel structure of potent chalcone-based derivatives type II c-Met inhibitors which are comparable to Foretinib (IC50 = 14 nM) as a potent reference drug. Based on our design strategy, we also expected an anti-tubulin activity for the compounds. However, the weak inhibitory effects on microtubules were confirmed by cell cycle analyses implicated that the observed cytotoxicity against HeLa cells probably was not derived from tubulin inhibition. Compounds 14q and 14k with IC50 values of 24 nM and 45 nM, respectively, demonstrated favorable inhibition of MET kinase activity, and desirable bonding interactions in the ligand-MET enzyme complex stability in molecular docking studies.

Clinical factors associated with the therapeutic efficacy of atezolizumab plus bevacizumab in patients with unresectable hepatocellular carcinoma: A multicenter prospective observational study.

The treatment efficiency and predictors of atezolizumab plus bevacizumab therapy for unresectable hepatocellular carcinoma in real-world practice have not been established. This study aimed to assess the efficacy and safety of atezolizumab plus bevacizumab and to investigate predictors of progression-free survival and overall survival. Patients with unresectable hepatocellular carcinoma treated with atezolizumab plus bevacizumab therapy in 19 hospitals were enrolled before treatment and observed prospectively. The outcomes of 222 patients in this cohort were analyzed. The objective response rate and disease control rate were 22.0% and 70.6%, respectively, whereas the median progression-free survival was 5.7 months. Independent risk factors for shortened progression-free survival were younger age (<75 years; 3.9 months vs. 8.6 months), higher number of intrahepatic tumors (≥5; 4.0 months vs. 7.9 months), macrovascular invasion (2.3 months vs. 6.7 months), and higher neutrophil-to-lymphocyte ratio (≥3.03; 3.0 months vs. 7.8 months). The median overall survival was not reached; however, independent risk factors for shortened overall survival were absence of hyperlipidemia, higher number of intrahepatic tumors (≥5), macrovascular invasion, higher α-fetoprotein level (≥400 ng/mL), worse Child-Pugh score (≥6), and higher neutrophil-to-lymphocyte ratio (≥3.03). Severe adverse events (grade ≥3) were observed in 96 patients (36.0%), with proteinuria being the most frequent. In conclusion, patients with older age, lower number of intrahepatic tumors, absent macrovascular invasion, and lower neutrophil-to-lymphocyte ratio are expected to have better progression-free survival with atezolizumab plus bevacizumab therapy for unresectable hepatocellular carcinoma.

Total Synthesis of Fusaramin, Enabling Stereochemical Elucidation, Structure-Activity Relationship, and Uncovering the Hidden Antimicrobial Activity against Plant Pathogenic Fungi.

Fusaramin (1) was isolated as a mitochondrial inhibitor. However, the fungal producer stops producing 1, which necessitates us to supply 1 by total synthesis. We proposed the complete stereochemical structure based on the biosynthetic pathway of sambutoxin. We have established concise and robust total synthesis of 1, enabling us to determine the complete stereochemical structure and to elucidate the structure-activity relationship, and uncover the hidden antiplant pathogenic fungal activity.

Capsaicin indirectly regulates TRPA1 via the arachidonic acid cascade, resulting in TJ opening.

Capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. We previously revealed that TRPA1 is involved in the capsaicin-induced Ca2+ influx and TJ permeability increase, although there are no reports that capsaicin directly activates TRPA1. In this study, we investigated the upstream factors of TRPA1 using RNA-seq analysis, and found that the cyclooxygenase 2 (COX2) gene was upregulated by capsaicin. Cyclooxygenase 2 converts arachidonic acid (AA), a metabolite by phospholipase A2 (PLA2), to prostaglandins. Prostaglandin E2 (PGE2) production was stimulated by capsaicin, and capsaicin-induced Ca2+ influx was effectively inhibited by PLA2 and COX2 inhibitors. The AA-induced TJ permeability increase was inhibited by a TRPA1 antagonist, but the capsaicin- and AA-induced TJ permeability increases were hardly inhibited by a COX2 inhibitor. These results suggest that capsaicin-induced PLA2 activation and AA production are the important steps for the TJ permeability increase.

RSK4 confers paclitaxel resistance to ovarian cancer cells, which is resensitized by its inhibitor BI-D1870.

Many ovarian cancers initially respond well to chemotherapy, but often become drug-resistant after several years. Therefore, analysis of drug resistance mechanisms and overcoming resistance are urgently needed. Paclitaxel is one of the first-choice and widely-used drugs for ovarian cancer, but like most drugs, drug resistance is observed in subsequent use. RSK4 is known as a tumor-suppressor, however, it has increasingly been reported to lead to drug resistance. Here, we found that RSK4 expression was elevated in paclitaxel-resistant ovarian cancer cells using DNA microarray, quantitative real-time PCR, and western blotting analysis. We examined the contribution of RSK4 to paclitaxel resistance and found that paclitaxel sensitivity was restored by RSK inhibitor co-treatment. We analyzed the mechanism by which resistance is developed when RSK4 level is elevated, and accelerated phosphorylation of the downstream translation factor eIF4B was discovered. In the Kaplan-Meier plot, the overall survival time was longer with RSK4 high, supporting its role as a tumor suppressor, as in previous findings, but the tendency was reversed when focusing on paclitaxel treatment. In addition, RSK4 levels were higher in non-responders than in responders in the ROC plotter. Finally, external expression of RSK4 in ovarian cancer cells increased the cell viability under paclitaxel treatment. These findings suggest that RSK4 may contribute to paclitaxel resistance, and that co-treatment with RSK4 inhibitors is effective treatment of paclitaxel-resistant ovarian cancer in which RSK4 is elevated.

X-Ray Conformation and Structure-Activity Relationships of MA026, a Reversible Tight Junction Opener.

MA026, a cyclic lipodepsipeptide, opens the tight junction (TJ) probably via binding to claudin-1. We reported that (1) TJ-opening activity is dependent on the amino acid sequence order at Glu10-Leu11; (2) an epimer at the C3 position of the N-terminal acyl tail decreased the TJ-opening activity; and (3) the epimers D-Leu1/L-Gln6 and L-Leu1/D-Gln6 showed more potent TJ-opening activity than natural MA026, although no systematic structure-activity relationship (SAR) study was conducted. Here, we report the three-dimensional structure and systematic SAR study of MA026. X-Ray crystallography and circular dichroism analysis of MA026 revealed that MA026 forms a left-handed α-helical structure, and hydrophobic amino acids are clustered on one side. Furthermore, the SAR results clearly showed that the hydrophobic region of MA026 is important for TJ-opening activity. These results suggest that MA026 interacts with claudin-1 via the hydrophobic cluster region and provide novel structural insights toward the development of a TJ opener targeting claudin-1.

A Combination Strategy of Multidrug-Sensitive Budding Yeast and Chemical Modifications Enabling to Find a New Overlooked Antifungal Compound, Sakurafusariene, from In-House Fractionated Library.

In this report, we disclose our discovery of a new antifungal natural product, sakurafusariene (1), from an in-house fractionated library of the culture broth of Fusarium sp. FKI-7550 strain by using a combination strategy of multidrug-sensitive yeast and chemical modification. Throughout our investigation, we encountered challenges in the isolation of natural product 1. A chemical modification strategy via alkylation of 1 allowed for removal of the impurities enabling us to elucidate the structure of 1. Furthermore, we synthesized ester derivatives using a method inspired by the isolation study of 1, which gave us valuable information to understand a preliminary structure-activity relationship against Pyricularia oryzae growth inhibitory activity.

Pretreatment with antibiotics is associated with reduced therapeutic response to atezolizumab plus bevacizumab in patients with hepatocellular carcinoma.

AIM: Alterations in microbial composition of gut microbiota due to antibiotics (ATB) may lead to resistance to immune checkpoint inhibitors (ICIs). This study aimed to assess the impact of ATB use on therapeutic response in patients with hepatocellular carcinoma (HCC) receiving atezolizumab plus bevacizumab. METHODS: This study retrospectively analyzed 105 patients with HCC treated with atezolizumab plus bevacizumab as a primary systemic therapy from prospectively-registered, multicenter, cohorts. Nineteen patients who received prior ATB were included in the ATB (+) group; 86 patients who did not receive prior ATB were included in the ATB (-) group. The therapeutic outcomes were compared between the two groups. RESULTS: Most of the patients' baseline characteristics were not significantly different between the two groups. The objective response rates according to the Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) (30.1% vs. 11.1%; p = 0.143) and modified RECIST (mRECIST) (44.6% vs. 27.8%; p = 0.190) were not significantly different between the ATB (-) and ATB (+) groups. The disease control rates were higher in the ATB (-) group than in the ATB (+) group according to RECIST v1.1 (74.7% vs. 44.4%; p = 0.012) and mRECIST (78.3% vs. 50.0%; p = 0.020). Prior ATB use was found to be independently associated with radiological progressive disease of the first therapeutic assessment. The median progression-free survival according to RECIST v1.1 (9.1 months vs. 3.0 months; p = 0.049) and mRECIST (9.1 months vs. 3.0 months; p = 0.036), and overall survival (not reached vs. 11.4 months; p = 0.015) were longer in the ATB (-) group than in the ATB (+) group. CONCLUSIONS: Prior ATB use was associated with reduced therapeutic responses in patients with HCC receiving atezolizumab plus bevacizumab.

Acyltransferase Domain Exchange between Two Independent Type†I Polyketide Synthases in the Same Producer Strain of Macrolide Antibiotics.

Streptomyces graminofaciens A-8890 produces two macrolide antibiotics, FD-891 and virustomycin A, both of which show significant biological activity. In this study, we identified the virustomycin A biosynthetic gene cluster, which encodes type I polyketide synthases (PKSs), ethylmalonyl-CoA biosynthetic enzymes, methoxymalony-acyl carrier protein biosynthetic enzymes, and post-PKS modification enzymes. Next, we demonstrated that the acyltransferase domain can be exchanged between the Vsm PKSs and the PKSs involved in FD-891 biosynthesis (Gfs PKSs), without any supply problems of the unique extender units. We exchanged the malonyltransferase domain in the loading module of Gfs PKS with the ethylmalonyltransferase domain and the methoxymalonyltransferase domain of Vsm PKSs. Consequently, the expected two-carbon-elongated analog 26-ethyl-FD-891 was successfully produced with a titer comparable to FD-891 production by the wild type; however, exchange with the methoxymalonyltransferase domain did not produce any FD-891 analogs. Furthermore, 26-ethyl-FD-891 showed potent cytotoxic activity against HeLa cells, like natural FD-891.

High-throughput platform for yeast morphological profiling predicts the targets of bioactive compounds.

Morphological profiling is an omics-based approach for predicting intracellular targets of chemical compounds in which the dose-dependent morphological changes induced by the compound are systematically compared to the morphological changes in gene-deleted cells. In this study, we developed a reliable high-throughput (HT) platform for yeast morphological profiling using drug-hypersensitive strains to minimize compound use, HT microscopy to speed up data generation and analysis, and a generalized linear model to predict targets with high reliability. We first conducted a proof-of-concept study using six compounds with known targets: bortezomib, hydroxyurea, methyl methanesulfonate, benomyl, tunicamycin, and echinocandin B. Then we applied our platform to predict the mechanism of action of a novel diferulate-derived compound, poacidiene. Morphological profiling of poacidiene implied that it affects the DNA damage response, which genetic analysis confirmed. Furthermore, we found that poacidiene inhibits the growth of phytopathogenic fungi, implying applications as an effective antifungal agent. Thus, our platform is a new whole-cell target prediction tool for drug discovery.

Perineal stapled prolapse resection in combination with Thiersch operation for relapsed rectal prolapse: a case report.

BACKGROUND: Treatment options for complete rectal prolapse include over 100 procedures. In previous reports, operative rectal prolapse repair, regardless of the technique by perineal approach, was associated with high recurrence rates. However, there is no consensus on the optimal surgical procedure for relapsed rectal prolapse. CASE PRESENTATION: A 97-year-old woman was admitted to our hospital with a chief complaint of complete rectal prolapse measuring > 5 cm. The patient had a history of laparoscopic anterior suture rectopexy without sigmoid resection under general anesthesia for complete rectal prolapse one year prior. The patient's postoperative course was uneventful. However, her dementia worsened (Hasegawa's dementia scale: 5/30 points) after the first operation. Further, moderate-to-severe aortic valve stenosis was first diagnosed with heart failure 6 months after the operation. Nine months after the initial surgery, she experienced a recurrence of complete rectal prolapse measuring approximately 5 cm. Considering the coexistence of advanced age, severe dementia, and aortic valve stenosis, surgery under general anesthesia was not indicated. Perineal stapled prolapse resection in combination with the t operation was planned because of its minimal invasiveness and shortened hospital stay. The procedure was performed by a team of two surgeons in the jack knife position, under spinal anesthesia. The prolapse was cut along the long-axis direction with three linear staplers and resected along the short-axis direction with four linear staplers. The cross-section of the linear stapler was reinforced with 3-0 Vicryl sutures. After rectal resection, the Thiersch operation using 1-0 nylon thread 1 cm away from the anal verge was additionally performed. The operative time was 24 min, and intraoperative blood loss was 1 mL. The postoperative course was uneventful. Three months after the operation, no recurrence was observed, and defecation function was good with improvements of Wexner score. CONCLUSIONS: Perineal stapled prolapse resection in combination with the Thiersch operation could be a useful option for patients with relapsed rectal prolapse and with poor general condition, who are not indicated for other surgical procedures.

Traminines A and B, produced by Fusarium concentricum, inhibit oxidative phosphorylation in Saccharomyces cerevisiae mitochondria.

Two new tetramic acid derivatives, traminines A (1) and B (2), were isolated from a culture broth of Fusarium concentricum FKI-7550 by bioassay-guided fractionation using multidrug-sensitive Saccharomyces cerevisiae 12geneΔ0HSR-iERG6. The chemical structures of 1 and 2 were elucidated by NMR studies. Compounds 1 and 2 inhibited the growth of the multidrug-sensitive yeast strain on nonfermentable medium containing glycerol, but not on fermentable medium containing glucose. These results strongly suggest that they target mitochondrial machineries presiding over ATP production via oxidative phosphorylation. Throughout the assay monitoring overall ADP-uptake/ATP-release in yeast mitochondria, 1 and 2 were shown to inhibit one or more enzymes involving oxidative phosphorylation. Based on biochemical characterization, we found that the interference with oxidative phosphorylation by 1 is attributable to the dual inhibition of complex III and FoF1-ATPase, whereas that by 2 is solely due to the inhibition of complex III.

Structural Revision of Natural Cyclic Depsipeptide MA026 Established by Total Synthesis and Biosynthetic Gene Cluster Analysis.

A revised structure of natural 14-mer cyclic depsipeptide MA026, isolated from Pseudomonas sp. RtlB026 in 2002 was established by physicochemical analysis with HPLC, MS/MS, and NMR and confirmed by total solid-phase synthesis. The revised structure differs from that previously reported in that two amino acid residues, assigned in error, have been replaced. Synthesized MA026 with the revised structure showed a tight junction (TJ) opening activity like that of the natural one in a cell-based TJ opening assay. Bioinformatic analysis of the putative MA026 biosynthetic gene cluster (BGC) of RtIB026 demonstrated that the stereochemistry of each amino acid residue in the revised structure can be reasonably explained. Phylogenetic analysis with xantholysin BGC indicates an exceptionally high homology (ca. 90 %) between xantholysin and MA026. The TJ opening activity of MA026 when binding to claudin-1 is a key to new avenues for transdermal administration of large hydrophilic biologics.

Mutational Biosynthesis of Hitachimycin Analogs Controlled by the ß-Amino Acid-Selective Adenylation Enzyme HitB.

Hitachimycin is a macrolactam antibiotic with an (S)-ß-phenylalanine (ß-Phe) at the starter position of its polyketide skeleton. (S)-ß-Phe is formed from l-α-phenylalanine by the phenylananine-2,3-aminomutase HitA in the hitachimycin biosynthetic pathway. In this study, we produced new hitachimycin analogs via mutasynthesis by feeding various (S)-ß-Phe analogs to a ΔhitA strain. We obtained six hitachimycin analogs with F at the ortho, meta, or para position and Cl, Br, or a CH3 group at the meta position of the phenyl moiety, as well as two hitachimycin analogs with thienyl substitutions. Furthermore, we carried out a biochemical and structural analysis of HitB, a ß-amino acid-selective adenylation enzyme that introduces (S)-ß-Phe into the hitachimycin biosynthetic pathway. The KM values of the incorporated (S)-ß-Phe analogs and natural (S)-ß-Phe were similar. However, the KM values of unincorporated (S)-ß-Phe analogs with Br and a CH3 group at the ortho or para position of the phenyl moiety were high, indicating that HitB functions as a gatekeeper to select macrolactam starter units during mutasynthesis. The crystal structure of HitB in complex with (S)-ß-3-Br-phenylalanine sulfamoyladenosine (ß-m-Br-Phe-SA) revealed that the bulky meta-Br group is accommodated by the conformational flexibility around Phe328, whose side chain is close to the meta position. The aromatic group of ß-m-Br-Phe-SA is surrounded by hydrophobic and aromatic residues, which appears to confer the conformational flexibility that enables HitB to accommodate the meta-substituted (S)-ß-Phe. The new hitachimycin analogs exhibited different levels of biological activity in HeLa cells and multidrug-sensitive budding yeast, suggesting that they may target different molecules.

A novel translation inhibitor, mersicarpine, inhibits S-phase progression and induces apoptosis in HL60 cells.

Mersicarpine is an aspidosperma alkaloid isolated from the Kopsia genus of plants. Its intriguing structural features have attracted much attention in synthetic organic chemistry, but no biological activity has been reported. Here, we report the effects of mersicarpine on human leukemia cell line HL60. At concentrations above 30 µm, mersicarpine reversibly arrested cell cycle progression in S-phase. At higher concentrations, it induced not only production of reactive oxygen species, but also apoptosis. Macromolecular synthesis assay revealed that mersicarpine specifically inhibits protein synthesis. These results suggest that mersicarpine is a novel translation inhibitor that induces apoptosis.

Structure Optimization of Gatastatin for the Development of γ-Tubulin-Specific Inhibitor.

Gatastatin (O 7-benzyl glaziovianin A) is a γ-tubulin-specific inhibitor that is used to investigate γ-tubulin function in cells. We have previously reported that the unsubstituted phenyl ring of the O 7-benzyl group in gatastatin is important for γ-tubulin inhibition. To obtain further structural information regarding γ-tubulin inhibition, we synthesized several gatastatin derivatives containing a fixed O 7-benzyl moiety. Modifications of the B-ring resulted in drastic decrease in cytotoxicity, abnormal spindle formation activity, and inhibition of microtubule (MT) nucleation. In contrast, various O 6-alkylated gatastatin derivatives showed potent cytotoxicity, induced abnormal spindle formation, and inhibited MT nucleation. We had previously reported that O 6-benzyl glaziovianin A is a potent α/ß-tubulin inhibitor; thus, these new results suggest that the O 6-position restricts affinity for α/ß- and γ-tubulin. Considering that an O 7-benzyl group increases specificity for γ-tubulin, more potent and specific γ-tubulin inhibitors can be generated through O 6-modifications of gatastatin.

Non-electrophilic TRPA1 agonists, menthol, carvacrol and clotrimazole, open epithelial tight junctions via TRPA1 activation.

Activation of the transient receptor potential A1 channel (TRPA1) by electrophilic agonists was reported to induce the opening of tight junctions (TJs). Because compounds that increase TJ permeability can be paracellular permeability enhancers, we investigated the effect of non-electrophilic TRPA1 activators, including food ingredients (menthol and carvacrol) and medication (clotrimazole), on epithelial permeability. We show that all three compounds induced increase of the permeability of fluorescein isothiocyanate-conjugated dextran (4 kDa) and decrease of transepithelial electrical resistance, accompanied by Ca2+ influx and cofilin activation in epithelial MDCK II monolayers. These phenotypes were attenuated by pretreatment of a TRPA1 antagonist, suggesting TRPA1-mediated opening of TJs. These results suggest that non-electrophilic TRPA1 activators with established safety can be utilized to regulate epithelial barriers.

Dual Inhibition of γ-Tubulin and Plk1 Induces Mitotic Cell Death.

α/ß-Tubulin inhibitors that alter microtubule (MT) dynamics are commonly used in cancer therapy, however, these inhibitors also cause severe side effects such as peripheral neuropathy. γ-Tubulin is a possible target as antitumor drugs with low side effects, but the antitumor effect of γ-tubulin inhibitors has not been reported yet. In this study, we verified the antitumor activity of gatastatin, a γ-tubulin specific inhibitor. The cytotoxicity of gatastatin was relatively weak compared with that of the conventional MT inhibitors, paclitaxel and vinblastine. To improve the cytotoxicity, we screened the chemicals that improve the effects of gatastatin and found that BI 2536, a Plk1 inhibitor, greatly increases the cytotoxicity of gatastatin. Co-treatment with gatastatin and BI 2536 arrested cell cycle progression at mitosis with abnormal spindles. Moreover, mitotic cell death induced by the combined treatment was suppressed by the Mps1 inhibitor, reversine. These findings suggest that co-treatment with Plk1 and γ-tubulin inhibitors causes spindle assembly checkpoint-dependent mitotic cell death by impairing centrosome functions. These results raise the possibility of Plk1 and γ-tubulin inhibitor co-treatment as a novel cancer chemotherapy.

Pyrenocine A induces monopolar spindle formation and suppresses proliferation of cancer cells.

Pyrenocine A, a phytotoxin, was found to exhibit cytotoxicity against cancer cells with an IC50 value of 2.6-12.9 µM. Live cell imaging analysis revealed that pyrenocine A arrested HeLa cells at the M phase with characteristic ring-shaped chromosomes. Furthermore, as a result of immunofluorescence staining analysis, we found that pyrenocine A resulted in the formation of monopolar spindles in HeLa cells. Monopolar spindles are known to be induced by inhibitors of the kinesin motor protein Eg5 such as monastrol and STLC. Monastrol and STLC induce monopolar spindle formation and M phase arrest via inhibition of the ATPase activity of Eg5. Interestingly, our data revealed that pyrenocine A had no effect on the ATPase activity of Eg5 in vitro, which suggested the compound induces a monopolar spindle by an unknown mechanism. Structure-activity relationship analysis indicates that the enone structure of pyrenocine A is likely to be important for its cytotoxicity. An alkyne-tagged analog of pyrenocine A was synthesized and suppressed proliferation of HeLa cells with an IC50 value of 2.3 µM. We concluded that pyrenocine A induced monopolar spindle formation by a novel mechanism other than direct inhibition of Eg5 motor activity, and the activity of pyrenocine A may suggest a new anticancer mechanism.