Naphthyl-Substituted Indole and Pyrrole Carboxylic Acids as Effective Antibiotic Potentiators-Inhibitors of Bacterial Cystathionine γ-Lyase.

Over the past decades, the problem of bacterial resistance to most antibiotics has become a serious threat to patients' survival. Nevertheless, antibiotics of a novel class have not been approved since the 1980s. The development of antibiotic potentiators is an appealing alternative to the challenging process of searching for new antimicrobials. Production of H2S-one of the leading defense mechanisms crucial for bacterial survival-can be influenced by the inhibition of relevant enzymes: bacterial cystathionine γ-lyase (bCSE), bacterial cystathionine ß-synthase (bCBS), or 3-mercaptopyruvate sulfurtransferase (MST). The first one makes the main contribution to H2S generation. Herein, we present data on the synthesis, in silico analyses, and enzymatic and microbiological assays of novel bCSE inhibitors. Combined molecular docking and molecular dynamics analyses revealed a novel binding mode of these ligands to bCSE. Lead compound 2a manifested strong potentiating activity when applied in combination with some commonly used antibiotics against multidrug-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The compound was found to have favorable in vitro absorption, distribution, metabolism, excretion, and toxicity parameters. The high effectiveness and safety of compound 2a makes it a promising candidate for enhancing the activity of antibiotics against high-priority pathogens.

Nybomycin inhibits both types of E. coli DNA gyrase - fluoroquinolone-sensitive and fluoroquinolone-resistant.

Bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are targets of many antibiotics including fluoroquinolones (FQs). Unfortunately, a number of bacterial species easily acquire resistance to FQs by mutations in either DNA gyrase or topoisomerase IV genes. The emergence of resistant pathogenic strains is a global problem in healthcare, therefore, identifying alternative pathways to thwart their persistence is the current frontier in drug discovery. An attractive class of compounds is nybomycins, reported to be "reverse antibiotics" that selectively inhibit growth of some Gram-positive FQ-resistant bacteria by targeting the mutant form of DNA gyrase, while being inactive against wild-type strains with FQ-sensitive gyrases. The strong "reverse" effect was demonstrated only for a few Gram-positive organisms resistant to FQs due to the S83L/I mutation in GyrA subunit of DNA gyrase. However, the activity of nybomycins has not been extensively explored among Gram-negative species. Here, we observed that in Gram-negative E. coli ΔtolC strain with enhanced permeability, wild-type gyrase and GyrA S83L mutant, resistant to fluoroquinolones, are both similarly sensitive to nybomycin.

Characterization of a novel natural tetracenomycin reveals crucial role of 4-hydroxy group in ribosome binding.

The aromatic polyketides tetracenomycins were recently found to be potent inhibitors of protein synthesis. Their binding site is located in a unique locus within the tunnel of the large ribosomal subunit. Here we report the isolation and structure elucidation of a novel natural tetracenomycin congener - O4-Me-tetracenomycin C (O4-Me-TcmC). This compound is isomeric to tetracenomycin X (TcmX), however, in contrast to TcmX, O4-Me-TcmC exhibited no antimicrobial activity and was unable to inhibit protein synthesis in vitro. Structural alignment of tetracenomycins to the binding locus from cryo-EM TcmX-70S ribosome data revealed the crucial role of the 4-hydroxyl group. These findings are important for further development of semi-synthetic tetracenomycins as potential antibacterials.

A New Albomycin-Producing Strain of Streptomyces globisporus subsp. globisporus May Provide Protection for Ants Messor structor.

There are several well-studied examples of protective symbiosis between insect host and symbiotic actinobacteria, producing antimicrobial metabolites to inhibit host pathogens. These mutualistic relationships are best described for some wasps and leaf-cutting ants, while a huge variety of insect species still remain poorly explored. For the first time, we isolated actinobacteria from the harvester ant Messor structor and evaluated the isolates' potential as antimicrobial producers. All isolates could be divided into two morphotypes of single and mycelial cells. We found that the most common mycelial morphotype was observed among soldiers and least common among larvae in the studied laboratory colony. The representative of this morphotype was identified as Streptomyces globisporus subsp. globisporus 4-3 by a polyphasic approach. It was established using a E. coli JW5503 pDualRep2 system that crude broths of mycelial isolates inhibited protein synthesis in reporter strains, but it did not disrupt the in vitro synthesis of proteins in cell-free extracts. An active compound was extracted, purified and identified as albomycin δ2. The pronounced ability of albomycin to inhibit the growth of entomopathogens suggests that Streptomyces globisporus subsp. globisporus may be involved in defensive symbiosis with the Messor structor ant against infections.

Mechanism-Based Approach to New Antibiotic Producers Screening among Actinomycetes in the Course of the Citizen Science Project.

Since the discovery of streptomycin, actinomycetes have been a useful source for new antibiotics, but there have been diminishing rates of new finds since the 1960s. The decreasing probability of identifying new active agents led to reduced interest in soil bacteria as a source for new antibiotics. At the same time, actinomycetes remain a promising reservoir for new active molecules. In this work, we present several reporter plasmids encoding visible fluorescent protein genes. These plasmids provide primary information about the action mechanism of antimicrobial agents at an early stage of screening. The reporters and the pipeline described have been optimized and designed to employ citizen scientists without specialized skills or equipment with the aim of essentially crowdsourcing the search for new antibiotic producers in the vast natural reservoir of soil bacteria. The combination of mechanism-based approaches and citizen science has proved its effectiveness in practice, revealing a significant increase in the screening rate. As a proof of concept, two new strains, Streptomyces sp. KB-1 and BV113, were found to produce the antibiotics pikromycin and chartreusin, respectively, demonstrating the efficiency of the pipeline.

Amycolatopsis camponoti sp. nov., new tetracenomycin-producing actinomycete isolated from carpenter ant Camponotus vagus.

An actinobacterial strain A23T, isolated from adult ant Camponotus vagus collected in Ryazan region (Russia) and established as tetracenomycin X producer, was subjected to a polyphasic taxonomic study. Morphological characteristics of this strain included well-branched substrate mycelium and aerial hyphae fragmented into rod-shaped elements. Phylogenetic analyses based on 16S rRNA gene and genome sequences showed that strain A23T was most closely related to Amycolatopsis pretoriensis DSM 44654T. Average nucleotide identity and digital DNA-DNA hybridization values between the genome sequences of isolate A23T and its closest relative, Amycolatopsis pretoriensis DSM 44654T, were 39.5% and 88.6%, which were below the 70% and 95-96% cut-off point recommended for bacterial species demarcation, respectively. The genome size of the isolate A23T was 10,560,374 bp with a DNA G + C content of 71.2%. The whole-cell hydrolysate contained meso-diaminopimelic acid and arabinose and galactose as main diagnostic sugars as well as ribose and rhamnose. It contained MK-9(H4) as the predominant menaquinone and iso-C16:0, iso-C15:0, anteiso-C17:0 and C16:0 as the major cellular fatty acids. Diphosphatidylglycerol and phosphatidylethanolamine prevailed among phospholipids. Mycolic acids were not detected. Based on the phenotypic, genomic and phylogenetic data, isolate A23T represents a novel species of the genus Amycolatopsis, for which the name Amycolatopsis camponoti sp. nov. is proposed, and the type strain is A23T (= DSM 111725T = VKM 2882T).

Biological evaluation and spectral characterization of a novel tetracenomycin X congener.

The aromatic polyketide tetracenomycin X (TcmX) was recently found to be a potent inhibitor of protein synthesis; its binding site is located in a unique locus within the tunnel of the large ribosomal subunit. The distinct mode of action makes this relatively narrow class of aromatic polyketides promising for drug development in the quest to prevent the spread of drug-resistant pathogens. Here we report the isolation and structure elucidation of a novel natural tetracenomycin X congener - 6-hydroxytetraceonomycin X (6-OH-TcmX). In contrast to TcmX, 6-OH-TcmX exhibited lower antimicrobial and cytotoxic activity, but comparable in vitro protein synthesis inhibition ability. A survey on spectral properties of tetracenomycins revealed profound differences in both UV-absorption and fluorescence spectra between TcmX and 6-OH-TcmX, suggesting a significant influence of 6-hydroxylation on the tetracenomycin X chromophore. Nonetheless, characteristic spectral properties of tetracenomycins make them suitable candidates for semi-synthetic drug development (e.g., for targeted delivery, chemical biology, or cell imaging).

Gausemycins†A,B: Cyclic Lipoglycopeptides from Streptomyces sp.*.

We report a novel family of natural lipoglycopeptides produced by Streptomyces sp. INA-Ac-5812. Two major components of the mixture, named gausemycins A and B, were isolated, and their structures were elucidated. The compounds are cyclic peptides with a unique peptide core and several remarkable structural features, including unusual positions of d-amino acids, lack of the Ca2+ -binding Asp-X-Asp-Gly (DXDG) motif, tyrosine glycosylation with arabinose, presence of 2-amino-4-hydroxy-4-phenylbutyric acid (Ahpb) and chlorinated kynurenine (ClKyn), and N-acylation of the ornithine side chain. Gausemycins have pronounced activity against Gram-positive bacteria. Mechanistic studies highlight significant differences compared to known glyco- and lipopeptides. Gausemycins exhibit only slight Ca2+ -dependence of activity and induce no pore formation at low concentrations. Moreover, there is no detectable accumulation of cell wall biosynthesis precursors under treatment with gausemycins.

Identification of isocyclosporins by collision-induced dissociation of doubly protonated species.

Nonribosomal cyclopeptide cyclosporin A (CsA), produced by fungus Tolypocladium inflatum, is an extremely important immunosuppressive drug used in organ transplantations and for therapy of autoimmune diseases. Here we report for the first time production of CsA, along with related cyclosporins B and C, by Tolypocladium inflatum strains of marine origin (White Sea). Cyclosporins A-C contain an unusual amino acid, (4R)-4-((E)-2-butenyl)-4,N-dimethyl-l-threonine (MeBmt), and are prone to isomerization to non-active isocyclosporin by N→O acyl shift of valine connected to MeBmt in acidic conditions. CsA and isoCsA are not distinguishable in MS analysis of [M+H]+ ions due to rapid [CsA + H]+→[isoCsA + H]+ conversion. We found that the N→O acyl shift is completely suppressed in cyclosporine [M+2H]2+ ions, and their collision-induced dissociation (CID) can be used for rapid and unambiguous analysis of cyclosporins and isocylosporins. Fragmentation patterns of [CsA+2H]2+ and [isoCsA+2H]2+ ions were analyzed and explained. The developed approach could be useful for MS analysis of other peptides containing ß-hydroxy-α-amino acids.

Chemical Ecology of Streptomyces albidoflavus Strain A10 Associated with Carpenter Ant Camponotus vagus.

Antibiotics produced by symbiotic microorganisms were previously shown to be of crucial importance for ecological communities, including ants. Previous works on ant-actinobacteria symbiosis are mainly focused on farming ants, which use antifungal microbial secondary metabolites to control pathogens in their fungal gardens. In this work, we studied microorganisms associated with carpenter ant Camponotus vagus. Pronounced antifungal activity of isolated actinobacteria strain A10 was found to be facilitated by biosynthesis of the antimycin A complex, consisting of small hydrophobic depsipeptides with high antimicrobial and cytotoxic activity. The actinomycete strain A10 was identified as Streptomyces albidoflavus. We studied the antagonistic activity of strain A10 against several entomopathogenic microorganisms. The antifungal activity of this strain potentially indicates a defensive symbiosis with the host ant, producing antimycins to protect carpenter ants against infections. The nature of this ant-microbe association however remains to be established.

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.