Antiproliferative activities through accelerating autophagic flux by basidalin and its analogs in human cancer cells.

Basidalin, isolated from the basidiomycete Leucoagaricus naucina, has previously demonstrated antibacterial and antitumor properties against murine cancer cells in vivo, but its effects on human cancer cells remain unknown. In this study, we found that basidalin possesses antiproliferative activity against human cancer cell lines. To elucidate the antiproliferative mechanism of basidalin, we focused on autophagy. Treatment with basidalin led to an increase in LC3-II expression level, and accelerated autophagic flux through an mTOR-independent pathway. Moreover, according to the structure-activity relationship analysis-including newly synthesized basidalin analogs-the formyl group, not the amino group, contributes to the antiproliferative activities of basidalin against human cancer cells. Additionally, the antiproliferative activity of basidalin analogs was strongly correlated with autophagy-inducing activity, indicating that basidalin exhibits antiproliferative activity through autophagy induction. These data suggest that basidalin, characterized by its ability to upregulate autophagic flux, emerges as a novel anticancer drug.

Antiplasmodial Activity Evaluation of a Bestatin-Related Aminopeptidase Inhibitor, Phebestin.

The increasing burden and spread of resistant malaria parasites remains an immense burden to public health. These factors have driven the demand to search for a new therapeutic agent. From our screening, phebestin stood out with nanomolar efficacy against Plasmodium falciparum 3D7. Phebestin was initially identified as an aminopeptidase N inhibitor. Phebestin inhibited the in vitro multiplication of the P. falciparum 3D7 (chloroquine-sensitive) and K1 (chloroquine-resistant) strains at IC50 values of 157.90 ± 6.26 nM and 268.17 ± 67.59 nM, respectively. Furthermore, phebestin exhibited no cytotoxic against human foreskin fibroblast cells at 2.5 mM. In the stage-specific assay, phebestin inhibited all parasite stages at 100 and 10-fold its IC50 concentration. Using 72-h in vitro exposure of phebestin at concentrations of 1 µM on P. falciparum 3D7 distorted the parasite morphology, showed dying signs, shrank, and prevented reinvasion of RBCs, even after the compound was washed from the culture. An in silico study found that phebestin binds to P. falciparum M1 alanyl aminopeptidase (PfM1AAP) and M17 leucyl aminopeptidase (PfM17LAP), as observed for bestatin. In vivo evaluation using P. yoelii 17XNL-infected mice with administrations of 20 mg/kg phebestin, once daily for 7 days, resulted in significantly lower parasitemia peaks in the phebestin-treated group (19.53%) than in the untreated group (29.55%). At the same dose and treatment, P. berghei ANKA-infected mice showed reduced parasitemia levels and improved survival compared to untreated mice. These results indicate that phebestin is a promising candidate for development as a potential therapeutic agent against malaria.

Synthesis of novobiocin derivatives and evaluation of their antigonococcal activity and pharmacokinetics.

Gonorrhea has become a serious problem because the number of infected people is increasing and the multi-drug resistance of the causative bacteria, Neisseria gonorrhoeae, is progressing. To develop novel drugs against resistant N. gonorrhoeae, we focused on the antibiotic novobiocin (1). This natural product has a different mechanism of action from existing drugs for gonorrhea, which may make it effective against resistant strains. Actually, it was applied to resistant N. gonorrhoeae, and moderate antibacterial activity was confirmed. Based on this result, we investigated the development of an antigonococcal drug with 1 as the lead compound. The pharmacophore is thought to be the noviose sugar moiety, especially around the 3'-position, so we derivatized this part in order to improve antibacterial activity. As a result, we found that 5 with an methylpyrrole ester structure have a very potent antibacterial activity. This derivative also showed excellent antigonococcal activity against resistant strains in vitro, however it has poor water solubility and pharmacokinetics because it is the acidic lipid-soluble compound. Therefore, we considered introduction of a basic substituent into the molecule would result in an amphoteric compound with improved water solubility, and we investigated further derivatization. As a result of synthesizing various derivatives, we found 47 containing imidazole with strong antigonococcal activity and greatly improved water solubility. This derivative has also improved metabolism and blood concentration in vivo, and is expected to be orally absorbed. Based on these results, we believe that 47 is a very promising anti-gonococcal lead compound and has great potential for further development.

An efficient CRISPR interference-based prediction method for synergistic/additive effects of novel combinations of anti-tuberculosis drugs.

Tuberculosis (TB) is treated by chemotherapy with multiple anti-TB drugs for a long period, spanning 6 months even in a standard course. In perspective, to prevent the emergence of antimicrobial resistance, novel drugs that act synergistically or additively in combination with major anti-TB drugs and, if possible, shorten the duration of TB therapy are needed. However, their combinatorial effect cannot be predicted until the lead identification phase of the drug development. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool that enables high-throughput screening of novel drug targets. The development of anti-TB drugs promises to be accelerated by CRISPRi. This study determined whether CRISPRi could be applicable for predictive screening of the combinatorial effect between major anti-TB drugs and an inhibitor of a novel target. In the checkerboard assay, isoniazid killed Mycobacterium smegmatis synergistically or additively in combinations with rifampicin or ethambutol, respectively. The susceptibility to rifampicin and ethambutol was increased by knockdown of inhA, which encodes a target molecule of isoniazid. Additionally, knockdown of rpoB, which encodes a target molecule of rifampicin, increased the susceptibility to isoniazid and ethambutol, which act synergistically with rifampicin in the checkerboard assay. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A, a novel TB drug candidate, with isoniazid or rifampicin. These results demonstrate that CRISPRi is a useful tool not only for drug target exploration but also for screening the combinatorial effects of novel combinations of anti-TB drugs. This study provides a rationale for anti-TB drug development using CRISPRi.

Synthesis of Mannosylerythritol Lipid Analogues and their Self-Assembling Properties, Recovery Effects on Damaged Skin Cells, and Antibacterial Activity.

Synthesis of three types of purpose-designed mannosylerythritol lipid (MEL)-D analogues with decanoyl groups, ß-GlcEL-D, α-GlcEL-D, and α-MEL-D, was accomplished utilizing our boron-mediated aglycon delivery (BMAD) methods. Their self-assembling properties, recovery effects on damaged skin cells, and antibacterial activity were evaluated. It was revealed, for the first time, that α-GlcEL-D and α-MEL-D only generated giant vesicles, indicating that slight differences in the steric configuration of an erythritol moiety and fatty acyl chains affect the ability to form vesicles. Analogue α-MEL-D exhibited significant recovery effects on damaged skin cells. Furthermore, α-MEL-D exhibited antibacterial activity as high as that for MEL-D, indicating that α-MEL-D is a promising artificial sugar-based material candidate for enhancing the barrier function of the stratum corneum, superior to a known cosmetic ingredient, and possesses antibacterial activity.

Synthesis and antiviral activity of formycin derivatives with anti-influenza virus activity.

In a screening using our unique natural product library, the C-nucleoside antibiotic formycin A, which exerts strong anti-influenza virus activity, was rediscovered. Aiming to develop a new type of anti-influenza virus drug, we synthesized new derivatives of formycin and evaluated its anti-influenza virus activity. Structural modifications were focused on the base moiety and sugar portion, respectively, and >40 novel formycin derivatives were synthesized. Modification of the C-7 position of the pyrazolopyrimidine ring strongly contributed to improve the activity. In particular, excellent anti-influenza virus activity was observed in the NHMe (10), SMe (12), and SeMe (15) derivatives, in which heteroatoms were introduced. In addition, in the modification of the sugar moiety, the presence of a hydroxyl group and its stereochemistry greatly affected both the expression and intensity of the activity. Furthermore, the evaluation results of the 7-SEt derivative (29) and the 2'-modified derivative (59) suggested that structural modifications may reduce cytotoxicity.

An immuno-dot blot assay for screening histidine kinase inhibitors.

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.

Total Synthesis of Caprazamycin A: Practical and Scalable Synthesis of syn-ß-Hydroxyamino Acids and Introduction of a Fatty Acid Side Chain to 1,4-Diazepanone.

The first total synthesis of caprazamycin A (1), a representative liponucleoside antibiotic, is described. Diastereoselective aldol reactions of aldehydes 12 and 25-27, derived from uridine, with diethyl isocyanomalonate 13 and phenylcarbamate 21 were investigated using thiourea catalysts 14 or bases to synthesize syn-ß-hydroxyamino acid derivatives. The 1,4-diazepanone core of 1 was constructed using a Mitsunobu reaction, and the fatty acid side chain was introduced using a stepwise sequence based on model studies. Notably, global deprotection was realized using palladium black and formic acid without hydrogenating the olefin in the uridine unit.

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes.

In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1 min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.

Leucinostatin Y: A Peptaibiotic Produced by the Entomoparasitic Fungus Purpureocillium lilacinum 40-H-28.

Leucinostatin Y, a new peptaibiotic, was isolated from the culture broth of the entomoparasitic fungus Purpureocillium lilacinum 40-H-28. The planar structure was elucidated by detailed analysis of its NMR and MS/MS data. The absolute configurations of the amino acids were partially determined by an advanced Marfey's method. The biological activities of leucinostatin Y were assessed using human pancreatic cancer cells, revealing the importance of the C-terminus of leucinostatins for preferential cytotoxicity to cancer cells under glucose-deprived conditions and inhibition of mitochondrial function.

Systematic and Stereoselective Total Synthesis of Mannosylerythritol Lipids and Evaluation of Their Antibacterial Activity.

The total synthesis of the 20 homogeneous members of mannosylerythritol lipids (MELs) with different alkyl chain lengths was effectively and systematically accomplished from a strategically designed common key intermediate that was stereoselectively constructed by the borinic acid catalyzed ß-mannosylation reaction. In addition, their antibacterial activities against Gram-positive bacteria were evaluated. Our results demonstrated that not only the length of the alkyl chains but also the pattern of Ac groups on the mannose moiety were important factors for antibacterial activity.

Monotherapy with a novel intervenolin derivative, AS-1934, is an effective treatment for Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori (H. pylori) infection causes various gastrointestinal diseases including gastric cancer. Hence, eradication of this infection could prevent these diseases. The most popular first-line treatment protocol to eradicate H. pylori is termed "triple therapy" and consists of a proton pump inhibitor (PPI), clarithromycin, and amoxicillin or metronidazole. However, the antibiotics used to treat H. pylori infection are hindered by the antibiotics-resistant bacteria and by their antimicrobial activity against intestinal bacteria, leading to side effects. Therefore, an alternative treatment with fewer adverse side effects is urgently required to improve the overall eradication rate of H. pylori. OBJECTIVE: The aim of this study was to assess the effectiveness and mechanism of action of an antitumor agent, intervenolin, and its derivatives as an agent for the treatment of H. pylori infection. RESULTS: We demonstrate that intervenolin, and its derivatives showed selective anti-H. pylori activity, including antibiotic-resistant strains, without any effect on intestinal bacteria. We showed that dihydroorotate dehydrogenase, a key enzyme for de novo pyrimidine biosynthesis, is a target and treatment with intervenolin or its derivatives decreased the protein and mRNA levels of H. pylori urease, which protects H. pylori against acidic conditions in the stomach. Using a mouse model of H. pylori infection, oral monotherapy with the intervenolin derivative AS-1934 had a stronger anti-H. pylori effect than the triple therapy commonly used worldwide to eradicate H. pylori. CONCLUSION: AS-1934 has potential advantages over current treatment options for H. pylori infection.

Identification of the Three Genes Involved in Controlling Production of a Phytotoxin Tropolone in Burkholderia plantarii.

UNLABELLED: Tropolone, a phytotoxin produced by Burkholderia plantarii, causes rice seedling blight. To identify genes involved in tropolone synthesis, we systematically constructed mutations in the genes encoding 55 histidine kinases and 72 response regulators. From the resulting defective strains, we isolated three mutants, KE1, KE2, and KE3, in which tropolone production was repressed. The deleted genes of these mutants were named troR1, troK, and troR2, respectively. The mutant strains did not cause rice seedling blight, and complementation experiments indicated that TroR1, TroK, and TroR2 were involved in the synthesis of tropolone in B. plantarii However, tropolone synthesis was repressed in the TroR1 D52A, TroK H253A, and TroR2 D46A site-directed mutants. These results suggest that the putative sensor kinase (TroK) and two response regulators (TroR1 and TroR2) control the production of tropolone in B. plantarii IMPORTANCE: A two-component system is normally composed of a sensor histidine kinase (HK) and a cognate response regulator (RR) pair. In this study, HK (TroK) and two RRs (TroR1 and TroR2) were found to be involved in controlling tropolone production in B. plantarii These three genes may be part of a bacterial signal transduction network. Such networks are thought to exist in other bacteria to regulate phytotoxin production, as well as environmental adaptation and signal transduction.

Actinocrispum wychmicini gen. nov., sp. nov., a novel member of the family Pseudonocardiaceae, isolated from soil.

A novel actinomycete, designated MI503-A4T, was isolated from soil. Comparative analysis of 16S rRNA gene sequences indicated that MI503-A4T was phylogenetically related to members of the family Pseudonocardiaceae. The most closely related genus was Kibdelosporangium (95.7-96.2 % sequence similarity). Substrate mycelia were branched and pale yellow to pale yellowish-brown. Straight- to zigzag-shaped aerial mycelia were observed, but Sporangium-like structures were absent. The whole-cell hydrolysate contained meso-diaminopimelic acid. The muramic acid residues in the peptidoglycan were N-acetylated. Whole-cell sugars were rhamnose, ribose, arabinose and galactose (cell wall chemotype IV). The predominant menaquinone was MK-9(H4). A small amount of MK-8(H4) was also detected. The DNA G+C content was 70.3-71.1 mol%. Polar lipids contained diphosphatidylglycerol, phosphatidylethanolamine and hydroxyl-phosphatidylethanolamine. Cellular fatty acid analysis of MI503-A4T detected predominantly iso-C14 : 0 (11.5 %), iso-C15 : 0 (13.3 %) and iso-C16 : 0 (35.7 %). Phenotypic and phylogenetic characteristics differentiated MI503-A4T from members of all genera within the family Pseudonocardiaceae with validly published names. Therefore, MI503-A4T is proposed to be a representative of a novel species in a novel genus, Actinocrispum wychmicini gen. nov., sp. nov. The type strain of the type species is MI503-A4T (=NBRC 109632T=DSM 45934T).

Identification of self-growth-inhibiting compounds lauric acid and 7-(Z)-tetradecenoic acid from Helicobacter pylori.

Helicobacter pylori growth medium is usually supplemented with horse serum (HS) or FCS. However, cyclodextrin derivatives or activated charcoal can replace serum. In this study, we purified self-growth-inhibiting (SGI) compounds from H. pylori growth medium. The compounds were recovered from porous resin, Diaion HP-20, which was added to the H. pylori growth medium instead of known supplements. These SGI compounds were also identified from 2,6-di-O-methyl-ß-cyclodextrin, which was supplemented in a pleuropneumonia-like organisms broth. The growth-inhibiting compounds were identified as lauric acid (LA) and 7-(Z)-tetradecenoic acid [7-(Z)-TDA]. Although several fatty acids had been identified in H. pylori, these specific compounds were not previously found in this species. However, we confirmed that these fatty acids were universally present in the cultivation medium of the H. pylori strains examined in this study. A live/dead assay carried out without HS indicated that these compounds were bacteriostatic; however, no significant growth-inhibiting effect was observed against other tested bacterial species that constituted the indigenous bacterial flora. These findings suggested that LA and 7-(Z)-TDA might play important roles in the survival of H. pylori in human stomach epithelial cells.

Total Synthesis and Antimicrobial Activity of Chlorocatechelin A.

Chlorocatechelin A (1) is a structurally unique microbial siderophore containing two units of 4-chloro-2,3-dihydroxybenzoic acid (CDB) and a characteristic acylguanidine structure. Purification from the microbe culture is not an easy task due to the lability of the acylguanidine and its chelating nature. Here we report the first convergent total synthesis and antimicrobial activity of chlorocatechelin A (1). The bis-acylated arginine was constructed using a Schotten-Baumann reaction whereas the CDB component was synthesized from o-vanillin (8). Condensation with an ornithine derivative synthesized from 1-benzyl d-glutamate was followed by deprotection in basic and neutral conditions to complete the total synthesis. We examined the antimicrobial activity of chlorocatechelin A (1) and found that this siderophore was active against desferrioxamine B (DFB)-sensitive microbes including the fish pathogen Pasteurella piscicida.

Total synthesis of (-)-caprazamycin A.

Caprazamycin A has significant antibacterial activity against Mycobacterium tuberculosis (TB). The first total synthesis is herein reported and features a) the scalable preparation of the syn-ß-hydroxy amino acid with a thiourea-catalyzed diastereoselective aldol reaction, b) construction of a diazepanone with an unstable fatty-acid side chain, and c) global deprotection with hydrogenation. This report provides a route for the synthesis of related liponucleoside antibiotics with fatty-acid side chains.

An unusual terpene cyclization mechanism involving a carbon-carbon bond rearrangement.

Terpene cyclization reactions are fascinating owing to the precise control of connectivity and stereochemistry during the catalytic process. Cyclooctat-9-en-7-ol synthase (CotB2) synthesizes an unusual 5-8-5 fused-ring structure with six chiral centers from the universal diterpene precursor, the achiral C20 geranylgeranyl diphosphate substrate. An unusual new mechanism for the exquisite CotB2-catalyzed cyclization that involves a carbon-carbon backbone rearrangement and three long-range hydride shifts is proposed, based on a powerful combination of in vivo studies using uniformly (13)C-labeled glucose and in vitro reactions of regiospecifically deuterium-substituted geranylgeranyl diphosphate substrates. This study shows that CotB2 elegantly demonstrates the synthetic virtuosity and stereochemical control that evolution has conferred on terpene synthases.

Inhibition of the first step in synthesis of the mycobacterial cell wall core, catalyzed by the GlcNAc-1-phosphate transferase WecA, by the novel caprazamycin derivative CPZEN-45.

Because tuberculosis is one of the most prevalent and serious infections, countermeasures against it are urgently required. We isolated the antitubercular agents caprazamycins from the culture of an actinomycete strain and created CPZEN-45 as the most promising derivative of the caprazamycins. Herein, we describe the mode of action of CPZEN-45 first against Bacillus subtilis. Unlike the caprazamycins, CPZEN-45 strongly inhibited incorporation of radiolabeled glycerol into growing cultures and showed antibacterial activity against caprazamycin-resistant strains, including a strain overexpressing translocase-I (MraY, involved in the biosynthesis of peptidoglycan), the target of the caprazamycins. By contrast, CPZEN-45 was not effective against a strain overexpressing undecaprenyl-phosphate-GlcNAc-1-phosphate transferase (TagO, involved in the biosynthesis of teichoic acid), and a mutation was found in the tagO gene of the spontaneous CPZEN-45-resistant strain. This suggested that the primary target of CPZEN-45 in B. subtilis is TagO, which is a different target from that of the parent caprazamycins. This suggestion was confirmed by evaluation of the activities of these enzymes. Finally, we showed that CPZEN-45 was effective against WecA (Rv1302, also called Rfe) of Mycobacterium tuberculosis, the ortholog of TagO and involved in the biosynthesis of the mycolylarabinogalactan of the cell wall of M. tuberculosis. The outlook for WecA as a promising target for the development of antituberculous drugs as a countermeasure of drug resistant tuberculosis is discussed.

Identification of a chemical inhibitor for nuclear speckle formation: implications for the function of nuclear speckles in regulation of alternative pre-mRNA splicing.

Nuclear speckles are subnuclear structures enriched with RNA processing factors and poly (A)(+) RNAs comprising mRNAs and poly (A)(+) non-coding RNAs (ncRNAs). Nuclear speckles are thought to be involved in post-transcriptional regulation of gene expression, such as pre-mRNA splicing. By screening 3585 culture extracts of actinomycetes with in situ hybridization using an oligo dT probe, we identified tubercidin, an analogue of adenosine, as an inhibitor of speckle formation, which induces the delocalization of poly (A)(+) RNA and dispersion of splicing factor SRSF1/SF2 from nuclear speckles in HeLa cells. Treatment with tubercidin also decreased steady-state MALAT1 long ncRNA, thought to be involved in the retention of SRSF1/SF2 in nuclear speckles. In addition, we found that tubercidin treatment promoted exon skipping in the alternative splicing of Clk1 pre-mRNA. These results suggest that nuclear speckles play a role in modulating the concentration of splicing factors in the nucleoplasm to regulate alternative pre-mRNA splicing.