ent-Verticilide B1 Inhibits Type 2 Ryanodine Receptor Channels and is Antiarrhythmic in Casq2 -/- Mice.

Intracellular Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; "ent-B1") in RyR2 single channel and [3H]ryanodine binding assays, and in Casq2 -/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single channels and RyR2-mediated spontaneous Ca2+ release in Casq2 -/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 minutes and half-life of 45 minutes after intraperitoneal administration of 3 mg/kg in mice. In vivo, ent-B1 significantly reduced catecholamine-induced ventricular arrhythmias in Casq2 -/- mice in a dose-dependent manner. Hence, we have identified a novel chemical entity - ent-B1 - that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics. SIGNIFICANCE STATEMENT: The cardiac ryanodine receptor (RyR2) is an untapped target in the stagnant field of antiarrhythmic drug development. We have confirmed RyR2 as an antiarrhythmic target in a mouse model of sudden cardiac death and shown the therapeutic efficacy of a second enantiomeric natural product.

Toxicological evaluation of microbial secondary metabolites in the context of European active substance approval for plant protection products.

Risk assessment (RA) of microbial secondary metabolites (SM) is part of the EU approval process for microbial active substances (AS) used in plant protection products (PPP). As the number of potentially produced microbial SM may be high for a certain microbial strain and existing information on the metabolites often are low, data gaps are frequently identified during the RA. Often, RA cannot conclusively clarify the toxicological relevance of the individual substances. This work presents data and RA conclusions on four metabolites, Beauvericin, 2,3-deepoxy-2,3-didehydro-rhizoxin (DDR), Leucinostatin A and Swainsonin in detail as examples for the challenging process of RA. To overcome the problem of incomplete assessment reports, RA of microbial AS for PPP is in need of new approaches. In view of the Next Generation Risk Assessment (NGRA), the combination of literature data, omic-methods, in vitro and in silico methods combined in adverse outcome pathways (AOPs) can be used for an efficient and targeted identification and assessment of metabolites of concern (MoC).

Unearthing a Cryptic Biosynthetic Gene Cluster for the Piperazic Acid-Bearing Depsipeptide Diperamycin in the Ant-Dweller Streptomyces sp. CS113.

Piperazic acid is a cyclic nonproteinogenic amino acid that contains a hydrazine N-N bond formed by a piperazate synthase (KtzT-like). This amino acid, found in bioactive natural products synthesized by non-ribosomal peptide synthetases (NRPSs), confers conformational constraint to peptides, an important feature for their biological activities. Genome mining of Streptomyces strains has been revealed as a strategy to identify biosynthetic gene clusters (BGCs) for potentially active compounds. Moreover, the isolation of new strains from underexplored habitats or associated with other organisms has allowed to uncover new BGCs for unknown compounds. The in-house "Carlos Sialer (CS)" strain collection consists of seventy-one Streptomyces strains isolated from the cuticle of leaf-cutting ants of the tribe Attini. Genomes from twelve of these strains have been sequenced and mined using bioinformatics tools, highlighting their potential to encode secondary metabolites. In this work, we have screened in silico those genomes, using KtzT as a hook to identify BGCs encoding piperazic acid-containing compounds. This resulted in uncovering the new BGC dpn in Streptomyces sp. CS113, which encodes the biosynthesis of the hybrid polyketide-depsipeptide diperamycin. Analysis of the diperamycin polyketide synthase (PKS) and NRPS reveals their functional similarity to those from the aurantimycin A biosynthetic pathway. Experimental proof linking the dpn BGC to its encoded compound was achieved by determining the growth conditions for the expression of the cluster and by inactivating the NRPS encoding gene dpnS2 and the piperazate synthase gene dpnZ. The identity of diperamycin was confirmed by High-Resolution Mass Spectrometry (HRMS) and Nuclear Magnetic Resonance (NMR) and by analysis of the domain composition of modules from the DpnP PKS and DpnS NRPS. The identification of the dpn BGC expands the number of BGCs that have been confirmed to encode the relatively scarcely represented BGCs for depsipeptides of the azinothricin family of compounds and will facilitate the generation of new-to-nature analogues by combinatorial biosynthesis.

Binding proteins of destruxin A from Metarhizium against insect cell.

Destruxin A (DA) is a cyclo-hexadepsipeptidic insecticidal mycotoxin isolated from the entomopathogenic fungi, Metarhizium spp. However, its mode of action is unknown. In this study, we isolated 149 candidate DA-binding proteins by drug affinity response target stability, and determined the interactions of 80 canditates with DA in vitro by surface plasmon resonance. The affinity coefficients (KD) ranged from 24 to 469 µM. Binding proteins were functionally diverse and included cytoskeletal components and cell motility, protein transcription and translation pathways, ubiquitin dependent protein metabolic processes, nucleus pore entry and exit, and endoplasmic reticulum vesicle transport and etc. Electron microscopy revealed that DA damaged the cytoskeleton and multiple organelles, disrupted cell adhesion and motility, and led to cell death. DA appeared to have a multi-targeted approach to cellular structures and multiple life processes, leading to cell death. The results of this study could provide molecular evidence for the analysis of the insecticidal toxicology of DA and further improve the study of the pathogenic insect mechanism of Metarhizium.

Helper bacteria halt and disarm mushroom pathogens by linearizing structurally diverse cyclolipopeptides.

The bacterial pathogen Pseudomonas tolaasii severely damages white button mushrooms by secretion of the pore-forming toxin tolaasin, the main virulence factor of brown blotch disease. Yet, fungus-associated helper bacteria of the genus Mycetocola (Mycetocola tolaasinivorans and Mycetocola lacteus) may protect their host by an unknown detoxification mechanism. By a combination of metabolic profiling, imaging mass spectrometry, structure elucidation, and bioassays, we found that the helper bacteria inactivate tolaasin by linearizing the lipocyclopeptide. Furthermore, we found that Mycetocola spp. impair the dissemination of the pathogen by cleavage of the lactone ring of pseudodesmin. The role of pseudodesmin as a major swarming factor was corroborated by identification and inactivation of the corresponding biosynthetic gene cluster. Activity-guided fractionation of the Mycetocola proteome, matrix-assisted laser desorption/ionization (MALDI) analyses, and heterologous enzyme production identified the lactonase responsible for toxin cleavage. We revealed an antivirulence strategy in the context of a tripartite interaction that has high ecological and agricultural relevance.

Hunting the main protease of SARS-CoV-2 by plitidepsin: Molecular docking and temperature-dependent molecular dynamics simulations.

COVID-19 has shaken all the countries across the globe and researchers are trying to find promising antiviral to cure the patients suffering from infection and can decrease the death. Even, different nations are using repurposing drugs to cure the symptoms and these repurposing drugs are hydroxychloroquine, remdesivir, and lopinavir, and recently, India has recently given the approval for the 2-deoxy-D-glucose for emergency purpose to cure the patients suffering from the COVID-19. Plitidepsin is a popular molecule and can be used in treatment of myeloma. Plitidepsin was explored by scientists experimentally against the COVID-19 and was given to the patient. It is found to be more a promising repurposing drug against the COVID-19 than the remdesivir. Therefore, there is a need to understand the interaction of plitidepsin with the main protease of SARS-CoV-2. Molecular docking of the plitidepsin against Mpro of SARS-CoV-2 was performed and the binding energy was found to be - 137.992 kcal/mol. Furthermore, authors have performed the molecular dynamics simulations of the main protease of SARS-CoV-2 in presence of plitidepsin at 300 and 325 K. It was found that the plitidepsin binds effectively with the main protease of SARS-CoV-2 at 300 K.

Micrococcin P1 and P2 from Epibiotic Bacteria Associated with Isolates of Moorea producens from Kenya.

Epibiotic bacteria associated with the filamentous marine cyanobacterium Moorea producens were explored as a novel source of antibiotics and to establish whether they can produce cyclodepsipeptides on their own. Here, we report the isolation of micrococcin P1 (1) (C48H49N13O9S6; obs. m/z 1144.21930/572.60381) and micrococcin P2 (2) (C48H47N13O9S6; obs. m/z 1142.20446/571.60370) from a strain of Bacillus marisflavi isolated from M. producens' filaments. Interestingly, most bacteria isolated from M. producens' filaments were found to be human pathogens. Stalked diatoms on the filaments suggested a possible terrestrial origin of some epibionts. CuSO4·5H2O assisted differential genomic DNA isolation and phylogenetic analysis showed that a Kenyan strain of M. producens differed from L. majuscula strain CCAP 1446/4 and L. majuscula clones. Organic extracts of the epibiotic bacteria Pseudoalteromonas carrageenovora and Ochrobactrum anthropi did not produce cyclodepsipeptides. Further characterization of 24 Firmicutes strains from M. producens identified extracts of B. marisflavi as most active. Our results showed that the genetic basis for synthesizing micrococcin P1 (1), discovered in Bacillus cereus ATCC 14579, is species/strain-dependent and this reinforces the need for molecular identification of M. producens species worldwide and their epibionts. These findings indicate that M. producens-associated bacteria are an overlooked source of antimicrobial compounds.

Impact of a Novel PagR-like Transcriptional Regulator on Cereulide Toxin Synthesis in Emetic Bacillus cereus.

The emetic type of foodborne disease caused by Bacillus cereus is produced by the small peptide toxin cereulide. The genetic locus encoding the Ces nonribosomal peptide synthetase (CesNRPS) multienzyme machinery is located on a 270 kb megaplasmid, designated pCER270, which shares its backbone with the Bacillus anthracis toxin plasmid pXO1. Although the ces genes are plasmid-borne, the chromosomally encoded pleiotropic transcriptional factors CodY and AbrB are key players in the control of ces transcription. Since these proteins only repress cereulide synthesis during earlier growth phases, other factors must be involved in the strict control of ces expression and its embedment in the bacterial life cycle. In silico genome analysis revealed that pCER270 carries a putative ArsR/SmtB family transcription factor showing high homology to PagR from B. anthracis. As PagR plays a crucial role in the regulation of the protective antigen gene pagA, which forms part of anthrax toxin, we used a gene-inactivation approach, combined with electrophoretic mobility shift assays and a bacterial two-hybrid system for dissecting the role of the PagR homologue PagRBc in the regulation of cereulide synthesis. Our results highlight that the plasmid-encoded transcriptional regulator PagRBc plays an important role in the complex and multilayered process of cereulide synthesis.

Identification of Cyclopropane Formation in the Biosyntheses of Hormaomycins and Belactosins: Sequential Nitration and Cyclopropanation by Metalloenzymes.

Hormaomycins and belactosins are peptide natural products that contain unusual cyclopropane moieties. Bioinformatics analysis of the corresponding biosynthetic gene clusters showed that two conserved genes, hrmI/belK and hrmJ/belL, were potential candidates for catalyzing cyclopropanation. Using in vivo and in vitro assays, the functions of HrmI/BelK and HrmJ/BelL were established. HrmI and BelK, which are heme oxygenase-like dinuclear iron enzymes, catalyze oxidation of the ϵ-amino group of l-lysine to afford l-6-nitronorleucine. Subsequently, HrmJ and BelL, which are iron- and α-ketoglutarate-dependent oxygenases, effectively convert l-6-nitronorleucine into 3-(trans-2-nitrocyclopropyl)-alanine through C4-C6 bond installation. These observations disclose a novel pathway of cyclopropane ring construction and exemplify the new chemistry involving metalloenzymes in natural product biosynthesis.

Rearrangement of Thiodepsipeptides by S → N Acyl Shift Delivers Homodetic Autoinducing Peptides.

Group behavior in many bacteria relies on chemically induced communication called quorum sensing (QS), which plays important roles in the regulation of colonization, biofilm formation, and virulence. In Gram-positive bacteria, QS is often mediated by cyclic ribosomally synthesized and posttranslationally modified peptides (RiPPs). In staphylococci, for example, most of these so-called autoinducing peptides (AIPs) contain a conserved thiolactone functionality, which has also been predicted to constitute a structural feature of AIPs from other genera. Here, we show that pentameric AIPs from Lactiplantibacillus plantarum, Clostridium perfringens, and Listeria monocytogenes that were previously presumed to be thiolactone-containing structures readily rearrange to become homodetic cyclopeptides. This finding has implications for the developing understanding of cross-species and potential cross-genus communication of bacteria and may help guide the discovery of peptide ligands to perturb their function.

Stereodivergent Nitrocyclopropane Formation during Biosynthesis of Belactosins and Hormaomycins.

Belactosins and hormaomycins are peptide natural products containing 3-(2-aminocyclopropyl)alanine and 3-(2-nitrocyclopropyl)alanine residues, respectively, with opposite stereoconfigurations of the cyclopropane ring. Herein we demonstrate that the heme oxygenase-like enzymes BelK and HrmI catalyze the N-oxygenation of l-lysine to generate 6-nitronorleucine. The nonheme iron enzymes BelL and HrmJ then cyclize the nitroalkane moiety to the nitrocyclopropane ring with the desired stereochemistry found in the corresponding natural products. We also show that both cyclopropanases remove the 4-proS-H of 6-nitronorleucine during the cyclization, establishing the inversion and retention of the configuration at C4 during the BelL and HrmJ reactions, respectively. This study reveals the unique strategy for stereocontrolled cyclopropane synthesis in nature.

The chromodepsins - chemistry, biology and biosynthesis of a selective Gq inhibitor natural product family.

Covering: up to April 2021The bacterial cyclic depsipeptides FR900359 (FR) and YM-254890 (YM) were shown to selectively inhibit Gαq proteins with high potency and selectivity and have recently emerged as valuable pharmacological tools due to their effective mechanism of action. Here, we summarize important aspects of this small and specialized natural product family, for which we propose the name chromodepsins, starting from their discovery, producing organisms and structural variety. We then review biosynthesis, structure-activity relationships and ecological and evolutionary aspects of the chromodepsins. Lastly, we discuss their mechanism of action, potential medicinal applications and future opportunities and challenges for further use and development of these complex inhibitor molecules from nature.

Chemical structure of hydrolysates of cereulide and their time course profile.

Hydrolysates of an emetic toxin cereulide were found in the broth of Bacillus cereus. The ester cleaved depsipeptides of cereulide were synthesized using liquid phase fragment condensation method starting from commercially available amino acids. The chemical structure of hydrolysates was verified tetradepsipeptide l-O-Val-l-Val-D-O-Leu-d-Ala and dodecadepsipeptide (D-O-Leu-d-Ala-l-O-Val-l-Val)3 using LC-TOFMS. Quantitative analysis of cereulide in the broth revealed production of cereulide in the stationary phase and decomposition in the death phase. The increase in tetradepsipeptide continued after the stationary phase until decomposition occurred.

Ahp-Cyclodepsipeptides as tunable inhibitors of human neutrophil elastase and kallikrein 7: Total synthesis of tutuilamide A, serine protease selectivity profile and comparison with lyngbyastatin 7.

We describe the total synthesis of tutuilamide A, a potent porcine pancreatic elastase (PPE) inhibitor and a representative member of the 3-amino-6-hydroxy-2-piperidone (Ahp) cyclodepsipeptide family, isolated from marine cyanobacteria. The Ahp unit serves as a pharmacophore and the adjacent 2-amino-2-butenoic acid (Abu) is a main driver of the selectivity among serine proteases. We adapted our previous convergent strategy to generate the macrocycle, common with lyngbyastatin 7 and related elastase inhibitors, and then appended the tutuilamide A-specific side chain bearing a vinyl chloride. Tutuilamide A and lyngbyastatin 7 were evaluated side by side for the inhibition of the disease-relevant human neutrophil elastase (HNE). Tutuilamide A and lyngbyastatin 7 were approximately equipotent against HNE, while tutuilamide A was previously shown to be more active against PPE compared with lyngbyastatin 7, further demonstrating that the side chain provides opportunities to not only modulate potency but also selectivity among proteases of the same function from different organisms. Profiling of tutuilamide A against mainly human serine proteases revealed high selectivity for HNE (IC50 0.73 nM) and pleiotropic activity against kallikrein 7 (KLK7, IC50 5.0 nM), without affecting other kallikreins, similarly to lyngbyastatin 7 (IC50 0.85 nM for HNE and 3.1 nM for KLK7). A comprehensive molecular docking study for elastases and KLK7 afforded deeper insight into the intricate differences between inhibitor interactions with HNE and PPE, accounting for the differential activities for both compounds. The synthesis and molecular studies serve as a proof-of-concept that the macrocyclic scaffold can be diversified to fine-tune the activity of serine protease inhibitors.

Microviridin 1777: A Toxic Chymotrypsin Inhibitor Discovered by a Metabologenomic Approach.

The toxicity of the cyanobacterium Microcystis aeruginosa EAWAG 127a was evaluated against the sensitive grazer Thamnocephalus platyurus, and the extract possessed strong activity. To investigate the compounds responsible for cytotoxicity, a series of peptides from this cyanobacterium were studied using a combined genomic and molecular networking approach. The results led to the isolation, structure elucidation, and biological evaluation of microviridin 1777, which represents the most potent chymotrypsin inhibitor characterized from this family of peptides to date. Furthermore, the biosynthetic gene clusters of microviridin, anabaenopeptin, aeruginosin, and piricyclamide were located in the producing organism, and six additional natural products were identified by tandem mass spectrometry analyses. These results highlight the potential of modern techniques for the identification of natural products, demonstrate the ecological role of protease inhibitors produced by cyanobacteria, and raise ramifications concerning the presence of novel, yet uncharacterized, toxin families in cyanobacteria beyond microcystin.

Genome Mining, Microbial Interactions, and Molecular Networking Reveals New Dibromoalterochromides from Strains of Pseudoalteromonas of Coiba National Park-Panama.

The marine bacterial genus Pseudoalteromonas is known for their ability to produce antimicrobial compounds. The metabolite-producing capacity of Pseudoalteromonas has been associated with strain pigmentation; however, the genomic basis of their antimicrobial capacity remains to be explained. In this study, we sequenced the whole genome of six Pseudoalteromonas strains (three pigmented and three non-pigmented), with the purpose of identifying biosynthetic gene clusters (BGCs) associated to compounds we detected via microbial interactions along through MS-based molecular networking. The genomes were assembled and annotated using the SPAdes and RAST pipelines and mined for the identification of gene clusters involved in secondary metabolism using the antiSMASH database. Nineteen BGCs were detected for each non-pigmented strain, while more than thirty BGCs were found for two of the pigmented strains. Among these, the groups of genes of nonribosomal peptide synthetases (NRPS) that code for bromoalterochromides stand out the most. Our results show that all strains possess BGCs for the production of secondary metabolites, and a considerable number of distinct polyketide synthases (PKS) and NRPS clusters are present in pigmented strains. Furthermore, the molecular networking analyses revealed two new molecules produced during microbial interactions: the dibromoalterochromides D/D' (11-12).

Neo-Aplysiatoxin A Isolated from Okinawan Cyanobacterium Moorea Producens.

A new aplysiatoxin derivative, neo-aplysiatoxin A (1), along with seven known compounds, neo-debromoaplysiatoxin A (2), dolastatin 3 (3), lyngbic acid (4), malyngamide M (5), hermitamide A (6), (-)-loliolide (7), and (+)-epiloliolide (8), was isolated from the Okinawan cyanobacterium Moorea producens. Their structures were elucidated on the basis of spectroscopic data, including high-resolution mass spectrometry and nuclear magnetic resonance. The compounds were evaluated for cytotoxic and diatom growth inhibition activities.

[Prevalence of Cereulide-Producing Bacillus cereus in Pasteurized Milk].

To recognize the risk of Bacillus cereus in pasteurized milk, we investigated the prevalence of B. cereus and the rate of the production of cereulide from B. cereus isolates. B. cereus was found in 66 out of 101 (65.3%) domestically pasteurized milk samples in Japan. The ces gene was identified in 3 out of 90 B. cereus isolates that were isolated from three samples (one product) among the 101 samples. The ces gene positive isolate, the reference strain F4810/72 and a B. cereus isolate collected in a food poisoning incident were shown the productivity of cereulide using an LC-MS/MS analysis. The LC-MS/MS analysis was confirmed the ability of identification and quantification of cereulide produced in the milk samples. In this study, it was shown that B. cereus strains are prevalent in pasteurized milk, some of these strains produce cereulide, and confirmed usefulness of LC-MS/MS analysis to detect cereulide in milk.

Synthesis of the reported structure of homocereulide and its vacuolation assay.

Homocereulide, isolated from marine bacterium Bacillus cereus, is an analog of emetic toxin cereulide. There is no report on its structure determination and involvement in B. cereus-associated food poisoning. Homocereulide is a cyclic dodecadepsipeptide composed of l-O-Val-l-Val-d-O-Leu-d-Ala and l-O-allo-Ile-d-Val-d-O-Leu-d-Ala. Here, we synthesized homocereulide using liquid phase fragment condensation. The NMR spectrum of synthesized homocereulide confirmed the intended structure and LC-MS results were consistent with natural products. Morphological evaluation using HEp-2 cells showed higher toxicity with homocereulide (1.39 nM) than cereulide (3.95 nM). Though cereulide is the main component in broth culture, homocereulide is also likely involved in B. cereus-associated food poisoning.

The Necessity of d-Thr in the New Antibiotic Teixobactin: A Molecular Dynamics Study.

Ever since the discovery of the new antibiotic teixobactin, studies of its structure-activity relationships have never ceased. Here we focus on the chirality of the threonine (Thr) residue, which belongs to the ring motif of teixobactin and plays an important role in the binding with its target, lipid II molecule. We study the structural propensity of the open and closed ring motifs with different chiral Thr residues as well as the teixobactin-lipid II complex with the help of molecular dynamics simulations. Our results suggest that different chiralities lead to different NH orientations of Thr with respect to the ring plane. Only in the closed ring motif with d-Thr is a favored binding cavity achievable with all four NH groups facing the same side of the ring plane. This study develops a deeper understanding of the binding mechanism of teixobactin and lipid II and is expected to be beneficial to new teixobactin-based drug design.