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.

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.

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.

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.

Tetrandrine induces lipid accumulation through blockade of autophagy in a hepatic stellate cell line.

Macroautophagy, or autophagy, is a cellular response in which unnecessary cytoplasmic components, including lipids and organelles, are self-degraded. Recent studies closely related autophagy to activation of hepatic stellate cells (HSCs), a process critical in the pathogenesis of liver fibrosis. During HSC activation, cytoplasmic lipid droplets (LDs) are degraded as autophagic cargo, and then cells express fibrogenic genes. Thus, inhibition of autophagy in HSCs is a potential therapeutic approach for attenuating liver fibrosis. We found that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from Stephania tetrandra, induced lipid accumulation, a phenotype associated with quiescent HSCs, through blockade of autophagy in the rat-derived HSC line HSC-T6. Tetrandrine inhibited autophagic flux without affecting lysosomal function. A phenotypic comparison using siRNA knockdown suggested that tetrandrine may target regulators, involved in fusion between autophagosomes and lysosomes (e.g., syntaxin 17). Moreover, perilipin 1, an LD-coated protein, co-localized specifically with LC3, a marker protein for autophagosomes, in tetrandrine-treated HSC-T6 cells. This suggests a potential role for perilipin 1 in autophagy-mediated LD degradation in HSCs. Our results identified tetrandrine as a potential tool for prevention and treatment of HSC activation.

Eudistomin C, an Antitumor and Antiviral Natural Product, Targets 40S Ribosome and Inhibits Protein Translation.

Eudistomin C (EudiC), a natural product, shows potent antitumor and antiviral activities, but the target molecule and the mechanism of action remain to be revealed. Here, we show that the 40S ribosome is the target in EudiC cytotoxicity. We isolated EudiC-resistant mutants from a multidrug-sensitive yeast strain, and a genetic analysis classified these YER (yeast EudiC resistance) mutants into three complementation groups. A genome-wide study revealed that the YER1-6 mutation is in the uS11 gene (RPS14A). Biotinylated EudiC pulled down Rps14p-containing complexes from 40S and 80S ribosomes, but not from the 60S ribosome. EudiC strongly inhibited translation of the wild-type strain but not of YER1-6 in cells and in vitro. These results indicate that EudiC is a protein synthesis inhibitor targeting the uS11-containing ribosomal subunit, and shows cytotoxicity by inhibiting protein translation.

Vicenistatin induces early endosome-derived vacuole formation in mammalian cells.

Homotypic fusion of early endosomes is important for efficient protein trafficking and sorting. The key controller of this process is Rab5 which regulates several effectors and PtdInsPs levels, but whose mechanisms are largely unknown. Here, we report that vicenistatin, a natural product, enhanced homotypic fusion of early endosomes and induced the formation of large vacuole-like structures in mammalian cells. Unlike YM201636, another early endosome vacuolating compound, vicenistatin did not inhibit PIKfyve activity in vitro but activated Rab5-PAS pathway in cells. Furthermore, vicenistatin increased the membrane surface fluidity of cholesterol-containing liposomes in vitro, and cholesterol deprivation from the plasma membrane stimulated vicenistatin-induced vacuolation in cells. These results suggest that vicenistatin is a novel compound that induces the formation of vacuole-like structures by activating Rab5-PAS pathway and increasing membrane fluidity.

Total Synthesis and Biological Evaluation of Irciniastatin A (a.k.a. Psymberin) and Irciniastatin B.

Irciniastatin A (a.k.a. psymberin) and irciniastatin B are members of the pederin natural product family, which have potent antitumor activity and structural complexity. Herein, we describe a full account of our total synthesis of (+)-irciniastatin A and (-)-irciniastatin B. Our synthesis features the highly regioselective Eu(OTf)3-catalyzed, DTBMP-assisted epoxide ring opening reaction with MeOH, which enabled a concise synthesis of the C1-C6 fragment, extensive use of AZADO (2-azaadamantane N-oxyl) and its related nitroxyl radical/oxoammonium salt-catalyzed alcohol oxidation throughout the synthesis, and a late-stage assembly of C1-C6, C8-C16, and C17-C25 fragments. In addition, for the synthesis of (-)-irciniastatin B, we achieved the C11-selective control of the oxidation stage via regioselective deprotection and AZADO-catalyzed alcohol oxidation. The synthetic irciniastatins showed high levels of cytotoxic activity against mammalian cells. Furthermore, chemical footprinting experiments using synthetic compounds revealed that the binding site of irciniastatins is the E-site of the ribosome.

Protective effects of Nitraria retusa extract and its constituent isorhamnetin against amyloid ß-induced cytotoxicity and amyloid ß aggregation.

Nitraria retusa is a halophyte species that is distributed in North Africa and used as a traditional medicinal plant. In this study, N. retusa ethanol extract and its constituent isorhamnetin (IRA) protected against amyloid ß (Aß)-induced cytotoxicity in human neuroblastoma SH-SY5Y cells. An in vitro Aß aggregation assay suggested that IRA destabilizes Aß fibrils.

Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5.

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5-microtubule interaction and in several Eg5 mutants. 11-Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule-stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S-trityl-L-cysteine (STLC) and GSK-1, both terpendole E and 11-ketopaspaline only partially inhibited Eg5-microtubule interaction. Furthermore, terpendole E and 11-ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5(D130A), Eg5(L214A)) or GSK-1 (Eg5(I299F), Eg5(A356T)), but with the same extent of inhibition against wild-type Eg5. Because Eg5(D130A) and Eg5(L214A) show cross-resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop-binding type Eg5 inhibitors.

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.

Construction of multidrug-sensitive yeast with high sporulation efficiency.

Budding yeast is often used in chemical genetics for screening, target identification, and compound verification, but its high-level drug resistance has made the analysis of compounds difficult. Here we report the construction of 12geneΔ0HSR, a strain that lacks eight efflux pumps located on the plasma membrane and four transcription factors involved in expression of efflux pumps, and contains the RME1(ins-308A) mutation. This strain retained sufficient transformation, mating, and sporulation efficiency for genetic analysis in addition to hypersensitivity against several compounds. 12geneΔ0HSR is a useful tool for chemical biology, not only in chemical screening but in target identification and verification of bioactive compounds.

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.

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.

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.

Transient receptor potential V4 channel stimulation induces reversible epithelial cell permeability in MDCK cell monolayers.

The transient receptor potential V4 channel (TRPV4) is responsive to a variety of physical and chemical stimuli, including a synthetic agonist GSK1016790A (GSK). Here, we show that TRPV4 is functionally expressed in, and that GSK induces the reversible opening of tight junctions (TJs) in epithelial Madin-Darby canine kidney II monolayers. Stimulation of TRPV4 by GSK induces an increase in fluorescein isothiocyanate-conjugated dextran (4 kDa) permeability and a reduction in transepithelial resistance, and these responses are blocked by pretreatment with the specific TRPV4 antagonist. Small conductance, but not large conductance Ca2+ -activated K+ channels, TRPA1 channel, and cofilin activation are involved in TRPV4-mediated reversible opening of TJs. These results suggest that a novel mechanism underlies TRPV4-mediated regulation of the tightness of epithelial barriers.

Cofilin mediates tight-junction opening by redistributing actin and tight-junction proteins.

We previously found that capsaicin induces tight-junction (TJ) opening accompanied with cofilin dephosphorylation/activation in intestinal Caco-2 cells. Here, we examined the role of cofilin in TJ regulation, and analyzed the structural events that lead to TJ opening. We transfected Caco-2 cells with wild-type cofilin [cofilin(wt)] or its constitutively active mutant cofilin(S3A). We found that the decreases in transepithelial electrical resistance (TER) was slower in cofilin(wt) transfectants and faster in cofilin(S3A) mutants than in vector controls. Moreover, cofilin dephosphorylation corresponded to the rate of TER decrease. Capsaicin treatment changed the localization of TJ proteins and altered the F-actin structure, but in a manner different from those depend on myosin light chain kinase (MLCK). These results strongly support the importance of cofilin in TJ opening, suggesting cofilin as a target for TJ permeability regulation in epithelial cells.

TRPA1-dependent reversible opening of tight junction by natural compounds with an α,ß-unsaturated moiety and capsaicin.

The delivery of hydrophilic macromolecules runs into difficulties such as penetration of the cell membrane lipid bilayer. Our prior experiment demonstrated that capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. Herein, we screened paracellular permeability enhancers other than capsaicin. As TJ opening by capsaicin is associated with Ca2+ influx, we first screened the compounds that induce Ca2+ influx in layered MDCK II cells, and then we determined the compounds' abilities to open TJs. Our results identified several natural compounds with α,ß-unsaturated moiety. A structure-activity relationship (SAR) analysis and the results of pretreatment with reducing reagent DTT suggested the importance of α,ß-unsaturated moiety. We also examined the underlying mechanisms, and our findings suggest that the actin reorganization seen in capsaicin treatment is important for the reversibility of TJ opening. Furthermore, our analyses revealed that TRPA1 is involved in the Ca2+ influx and TJ permeability increase not only by an α,ß-unsaturated compound but also by capsaicin. Our results indicate that the α,ß-unsaturated moiety can be a potent pharmacophore for TJ opening.

Brasilicardin A, a natural immunosuppressant, targets amino Acid transport system L.

Lymphocytes in T cell activation require extracellular nutrients to provide energy for cellular proliferation and effector functions. Therefore, inhibitors of nutrient transporters are expected to be a new class of immunosuppressant. Here, we report that the molecular target of brasilicardin A (BraA), an immunosuppressive compound, is the amino acid transporter system L. BraA inhibited the cell-cycle progression of murine T cell lymphocyte CTLL-2 cells in G1 phase, and potently inhibited the uptake of amino acids that are substrates for amino acid transport system L. Moreover, BraA stimulated the GCN2 activation and, subsequently, the phosphorylation of eIF2alpha. These results suggest that the immunosuppressive activity of BraA is induced by amino acid deprivation via the inhibition of system L and that the amino acid transporter is a target for immunosuppressant.