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.

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.

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 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.

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.

Synthesis and biochemical characterization of naphthoquinone derivatives targeting bacterial histidine kinases.

Waldiomycin is an inhibitor of histidine kinases (HKs). Although most HK inhibitors target the ATP-binding region, waldiomycin binds to the intracellular dimerization domain (DHp domain) with its naphthoquinone moiety presumed to interact with the conserved H-box region. To further develop inhibitors targeting the H-box, various 2-aminonaphthoquinones with cyclic, aliphatic, or aromatic amino groups and naphtho [2,3-d] isoxazole-4,9-diones were synthesized. These compounds were tested for their inhibitory activity (IC50) against WalK, an essential HK for Bacillus subtilis growth, and their minimum inhibitory concentrations (MIC) against B. subtilis. As a result, 11 novel HK inhibitors were obtained as naphthoquinone derivatives (IC50: 12.6-305 µM, MIC: 0.5-128 µg ml-1). The effect of representative compounds on the expression of WalK/WalR regulated genes in B. subtilis was investigated. Four naphthoquinone derivatives induced the expression of iseA (formerly yoeB), whose expression is negatively regulated by the WalK/WalR system. This suggests that these compounds inhibit WalK in B. subtilis cells, resulting in antibacterial activity. Affinity selection/mass spectrometry analysis was performed to identify whether these naphthoquinone derivatives interact with WalK in a manner similar to waldiomycin. Three compounds were found to competitively inhibit the binding of waldiomycin to WalK, suggesting that they bind to the H-box region conserved in HKs and inhibit HK activity.

Aprosamine Derivatives Active against Multidrug-Resistant Gram-Negative Bacteria.

Novel aprosamine derivatives were synthesized for the development of aminoglycoside antibiotics active against multidrug-resistant Gram-negative bacteria. The synthesis of aprosamine derivatives involved glycosylation at the C-8' position and subsequent modification (epimerization and deoxygenation at the C-5 position and 1-N-acylation) of the 2-deoxystreptamine moiety. All 8'-ß-glycosylated aprosamine derivatives (3a-h) showed excellent antibacterial activity against carbapenem-resistant Enterobacteriaceae and 16S ribosomal RNA methyltransferase-producing multidrug-resistant Gram-negative bacteria compared to the clinical drug, arbekacin. The antibacterial activity of 5-epi (6a-d) and 5-deoxy derivatives (8a,b and 8h) of ß-glycosylated aprosamine was further enhanced. On the other hand, the derivatives (10a,b and 10h) in which the amino group at the C-1 position was acylated with (S)-4-amino-2-hydroxybutyric acid showed excellent activity (MICs 0.25-0.5 µg/mL) against resistant bacteria that produce the aminoglycoside-modifying enzyme, aminoglycoside 3-N-acetyltransferase IV, which induces high resistance against parent apramycin (MIC > 64 µg/mL). In particular, 8b and 8h showed approximately 2- to 8-fold antibacterial activity against carbapenem-resistant Enterobacteriaceae and 8- to 16-fold antibacterial activity against resistant Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, compared to apramycin. Our results showed that aprosamine derivatives have immense potential in the development of therapeutic agents for multidrug-resistant bacteria.

Cycloimidamicins, Novel natural lead compounds for translation inhibition in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most concerning pathogenic bacteria. We screened antibiotics using a highly drug-sensitive P. aeruginosa strain and an oligotrophic medium, and successfully isolated novel antibiotics, namely cycloimidamicins (CIMs), from a rare actinomycete strain, Lentzea sp. MM249-143F7. X-ray and nuclear magnetic resonance analyses revealed that CIMs possess a distinctive and unprecedented molecular structure, containing tetramic acid and an imidazole ring bound directly to indolone. The CIMs exhibited potent antibacterial activity against Gram-negative bacteria, as well as translation inhibition in Escherichia coli in both intact cells and in vitro. Additionally, E. coli strains resistant to known translation inhibitors did not exhibit cross-resistance to CIMs, suggesting that CIMs inhibit bacterial growth by blocking translation through a novel mechanism.

Amycolamicin: a novel broad-spectrum antibiotic inhibiting bacterial topoisomerase.

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Wychimicins, a new class of spirotetronate polyketides from Actinocrispum wychmicini MI503-A4.

In the course of our screening program for new anti-methicillin-resistant Staphylococcus aureus antibiotics, four novel antibiotics, termed wychimicins A-D, were isolated from the culture broth of the rare actinomycete Actinocrispum wychmicini strain MI503-AF4. Wychimicins are spirotetronates possessing a macrocyclic 13-membered ring containing trans-decalin and ß-D-xylo-hexopyranose moieties connected to C-17 by an O-glycosidic linkage according to MS, NMR and X-ray analyses. In X-ray crystal structure analysis, the Flack constant was 0.10 (11). The stereochemistry of the spirocarbon C-25 was R. Wychimicins had a minimum inhibitory concentration of 0.125-2 µg ml-1 against methicillin-resistant Staphylococcus aureus.

Rediscovery of 4-Trehalosamine as a Biologically Stable, Mass-Producible, and Chemically Modifiable Trehalose Analog.

Nonreducing disaccharide trehalose is used as a stabilizer and humectant in various products and is a potential medicinal drug, showing curative effects on the animal models of various diseases. However, its use is limited as it is hydrolyzed by trehalase, a widely expressed enzyme in multiple organisms. Several trehalose analogs are prepared, including a microbial metabolite 4-trehalosamine, and their high biological stability is confirmed. For further analysis, 4-trehalosamine is selected as it shows high producibility. Compared with trehalose, 4-trehalosamine exhibits better or comparable protective activities and a high buffer capacity around the neutral pH. Another advantage of 4-trehalosamine is its chemical modifiability: simple reactions produce its various derivatives. Labeled probes and detergents are synthesized in one-pot reactions to exemplify the feasibility of their production, and their utility is confirmed for their respective applications. The labeled probes are used for mycobacterial staining. Although the derivative detergents can be effectively used in membrane protein research, long-chain detergents show 1000-3000-fold stronger autophagy-inducing activity in cultured cells than trehalose and are expected to become a drug lead and research reagent. These results indicate that 4-trehalosamine is a useful trehalose substitute for various purposes and a material to produce new useful derivative substances.

Saccharobipyrimicin, a new antibiotic from the leaf-litter actinomycete Saccharothrix sp. MM696L-181F4.

In the course of screening for new antimicrobial compounds, a new antibiotic substance named saccharobipyrimicin was isolated from the leaf-litter actinomycete Saccharothrix sp. MM696L-181F4. The structure of saccharobipyrimicin was elucidated by various spectral methods, mainly single-crystal X-ray analysis and chemical degradation. It revealed that saccharobipyrimicin contained a 2,2'-bipyridine skeletal structure. Saccharobipyrimicin showed moderate and broad-spectrum antimicrobial activity. Two chemical derivatives of saccharobipyrimicin showed weaker antimicrobial activities than that of saccharobipyrimicin against most test microorganisms except two tolC mutants of Escherichia coli and Neisseria gonorrhoeae.

Sealutomicins, new enediyne antibiotics from the deep-sea actinomycete Nonomuraea sp. MM565M-173N2.

A Nonomuraea sp. strain MM565M-173N2 was isolated from deep-sea sediment off the Sanriku coast, and new antibiotics were evaluated against carbapenem-resistant Enterobacteriaceae (CRE), which is a problematic group of bacteria because of their antimicrobial resistance. From 220 l of fermented broth from strain MM565M-173N2, we isolated four new antibiotics by gel filtration and HPLC, designated as sealutomicins A (1.8 mg), B (1.5 mg), C (0.8 mg), and D (0.8 mg). Their structures were determined from MS, NMR, and CD spectra. Sealutomicin A was found to be a new enediyne antibiotic, while sealutomicins B-D were aromatized products from sealutomicin A. Sealutomicin A showed strong antibacterial activity (MIC 0.05-0.2 µg ml-1) against CRE.

The synthesis of paleic acid, an antimicrobial agent effective against Mannheimia and Pasteurella, and its structurally related derivatives.

A synthetic route to paleic acid 1, antimicrobial agent effective against Mannheimia haemolytica and Pasteurella multocida, has been established. The absolute configuration of the secondary hydroxyl group was controlled by a catalytic asymmetric alkylation of an aldehyde using a chiral titanium sulfonamide complex and the cis double bond was installed using a Wittig reaction. This synthetic route was also applied to the preparation of structurally related analogs, which were used in structure-activity relationship studies for antibacterial activity.

Synthesis and biological activity of analogs of CPZEN-45, a novel antituberculosis drug.

Analogs of CPZEN-45, which is expected to be a promising new antituberculosis drug that overcomes the shortcomings of caprazamycins, were synthesized and their biological activities were evaluated. The biological activity of analogs 1-3, which converted the anilide portion, and analogs 4 and 5, focusing on the seven-membered ring, were lower than that of CPZEN-45. These results suggest that the inhibitory activity of CPZEN-45 against TagO, an ortholog of WecA, has a strict structural limitation, and it was hoped for elucidation of the mode of action of CPZEN-45 using structural biology in the future.

Instability of the 16S rRNA methyltransferase-encoding npmA gene: why have bacterial cells possessing npmA not spread despite their high and broad resistance to aminoglycosides?

The NpmA bacterial 16S rRNA methyltransferase, which is identified from Escherichia coli strains, confers high resistance to many types of aminoglycoside upon its host cells. But despite its resistance-conferring ability, only two cases of its isolation from E. coli (14 years apart) have been reported to date. Here, we investigated the effect of the npmA gene on aminoglycoside resistance in Pseudomonas aeruginosa and Klebsiella pneumoniae and its stability in E. coli cells by comparing it with armA, another 16S rRNA methyltransferase gene currently spreading globally. As a result, we found that npmA conferred resistance to all types of aminoglycoside antibiotics we tested (except streptomycin) in both P. aeruginosa and K. pneumoniae, as well in E. coli. In addition, co-expression of armA and npmA resulted in an additive effect for the resistance. However, in return for the resistance, we also observed that the growth rates and the cell survivability of the strains transformed with the npmA-harboring plasmids were inferior than those of the control strains and that these plasmids were easily disrupted by IS10, IS1, and IS5 insertion sequences. We discuss these data in the context of the threat posed by pathogenic strains possessing npmA.

Quadoctomycin, a 48-membered macrolide antibiotic from Streptomyces sp. MM168-141F8.

Drug-resistant bacteria are still emerging, and screening of new skeletal antibiotics is important. During our continuous screening for antimicrobial agents, we discovered a new antimicrobial, named quadoctomycin, from solid culture of Streptomyces sp. MM168-141F8. The substance was purified by solvent extraction, silica gel chromatography and HPLC. Structural elucidation of quadoctomycin was performed by MS and NMR analyses and chemical degradation. Quadoctomycin possesses a 48-membered polyol macrolide skeleton in which an α-D-mannoside is connected to C-22 by an O-glycosidic linkage. The structure of quadoctomycin was found to be related to that of monazomycin A based on the analyses of NMR spectra in the same solvent (pyridine-d5). Quadoctomycin showed potent antibacterial activity against Staphylococcus aureus, including methicillin-resistant S. aureus, and other Gram-positive pathogenic bacteria such as Enterococcus faecalis and E. faecium (including drug-resistant strains), but did not show activity toward Gram-negative bacteria or Candida albicans.The Journal of Antibiotics advance online publication 15 November 2017; doi:10.1038/ja.2017.140.

Kribellosides, novel RNA 5'-triphosphatase inhibitors from the rare actinomycete Kribbella sp. MI481-42F6.

Yeast capping enzymes differ greatly from those of mammalian, both structurally and mechanistically. Yeast-type capping enzyme repressors are therefore candidate antifungal drugs. The 5'-guanine-N7 cap structure of mRNAs are an essential feature of all eukaryotic organisms examined to date and is the first co-transcriptional modification of cellular pre-messenger RNA. Inhibitors of the RNA 5'-triphosphatase in yeast are likely to show fungicidal effects against pathogenic yeast such as Candida. We discovered a new RNA 5'-triphosphatase inhibitor, designated as the kribellosides, by screening metabolites from actinomycetes. Kribellosides belong to the alkyl glyceryl ethers. These novel compounds inhibit the activity of Cet1p (RNA 5'-triphosphatase) from Saccharomyces cerevisiae in vitro with IC50s of 5-8 µM and show antifungal activity with MICs ranging from 3.12 to 100 µg ml-1 against S. cerevisiae.

Biological activity of intervenolin analogs with a phenyl substituent.

Intervenolin analogs with a phenyl substituent at the 2- or 3-position were synthesized. The compounds (3-11) showed weak or no inhibitory activity toward the growth of MKN-74 gastric adenocarcinoma cells, even in the presence or absence of the corresponding Hs738 stromal cells, whereas 2-substituted analogs exhibited selective anti-Helicobacter pylori activity. Introduction of a pendant side chain on the nitrogen alleviated their acute toxicity in mice. The 2-phenyl-substituted analogs are reasonable structural templates for structure-activity relationship studies toward the development of anti-H. pylori agents that do not affect human cells.The Journal of Antibiotics advance online publication, 11 October 2017; doi:10.1038/ja.2017.123.