Cavomycins A-C, Linear Oligomer Depsipeptides from an Annelid-Associated Streptomyces cavourensis.

Three unique linear oligomeric depsipeptides, designated as cavomycins A-C (1-3), were identified from Streptomyces cavourensis, a gut bacterium associated with the annelid Paraleonnates uschakovi. The structures of these depsipeptides were determined through a combination of spectroscopic methods and chemical derivatization techniques, including methanolysis, the modified Mosher's method, advanced Marfey's methods, and phenylglycine methyl ester derivatization. The unique dipeptidyl residue arrangements in compounds 1-3 indicate that they are not degradation products of valinomycin. Compound 2 and its methylation derivative 2a exhibited antiproliferative activity against PANC-1 pancreatic cancer cells with IC50 values of 1.2 and 1.7 µM, respectively.

Skyllamycins D and E, Non-Ribosomal Cyclic Depsipeptides from Lichen-Sourced Streptomyces anulatus.

The skyllamycins are a class of heavily modified, non-ribosomal peptides, first isolated from Streptomyces sp. KY11784. A Streptomyces strain with potent antibiotic activity against Bacillus subtilis was isolated from a sample of the New Zealand lichen Pseudocyphellaria dissimilis. Whole genome sequencing and biosynthetic gene cluster genetic analysis coupled with GNPS LCMS/MS molecular networking revealed that this strain had the capacity to produce skyllamycins, including previously undescribed congeners, and that these were likely the source of the observed biological activity. Guided by the results of the molecular networking, we isolated the previously reported skyllamycins A-C (1-3), along with two new congeners, skyllamycins D (4) and E (5). The structures of these compounds were elucidated using comprehensive 1D and 2D NMR analyses, along with HRESIMS fragmentation experiments. Antibacterial assays revealed that skyllamycin D possessed improved activity against B. subtilis E168 compared to previously reported congeners.

Pithohirolide, an antimicrobial tetradepsipeptide from a fungus Pithomyces chartarum.

Pithohirolide (1), a new depsipeptide, was isolated from an ascomycetous fungus Pithomyces chartarum TAMA 581. The planar structure of 1 was elucidated on the basis of NMR and MS analyses and the absolute configuration was determined by the advanced Marfey's analysis, chiral-phase HPLC analysis, and synthesis of degradation product. Compound 1 possesses a cyclic structure comprising (S)-2-hydroxy-3-phenylpropanoic acid, (S)-3-hydroxy-3-phenylpropanoic acid, (S)-2-hydroxyisovaleric acid, and N-methyl-L-alanine, connected via three ester and one amide linkages. Compound 1 exhibited antimicrobial activity against Staphylococcus aureus and Saccharomyces cerevisiae at MIC 3.1 µg ml-1.

Nannocystin Ax, a natural elongation factor 1α inhibitor from Nannocystis sp., suppresses epithelial-mesenchymal transition, adhesion and migration in lung cancer cells.

Epithelial-mesenchymal transition (EMT), the epithelial cells transdifferentiation into the mesenchymal cells, has been involved in cancer metastasis. Nannocystin ax (NAN) is a cyclodepsipeptide initially isolated from Myxobacterial genus, Nannocystis sp. with anticancer activities. This study was designed to explore the effect of NAN on TGF-ß1-induced EMT in lung cancer cells. The morphological alteration was observed with a microscope. Western blotting and immunofluorescence assays were used to detect the protein expression and the localization. The adhesion and migration were evaluated by adhesion assay and wound healing assay. The mRNA expression of TGF-ß receptor type I (TßRI) was determined by real-time PCR. NAN significantly restrained TGF-ß1-induced EMT morphological changes, the protein expression of E-cadherin, N-cadherin, and Vimentin, etc. TGF-ß1 activated phosphorylation and nuclear translocation of Smad2/3 were inhibited by NAN. Furthermore, NAN suppressed adhesion and migration triggered by TGF-ß1. In addition, NAN significantly down-regulated TßRI on the transcriptional level directly. In summary, these results showed that NAN restrained TGF-ß1-induced epithelial-mesenchymal transition, migration, and adhesion in human lung cancer cells. The underlying mechanism involved the inhibition of Smad2/3 and the TßRI signaling pathway. This study reveals the new anticancer effect and mechanism of NAN.

Structure Revision of Isocereulide A, an Isoform of the Food Poisoning Emetic Bacillus cereus Toxin Cereulide.

The emetic Bacillus cereus toxin cereulide presents an enormous safety hazard in the food industry, inducing emesis and nausea after the consumption of contaminated foods. Additional to cereulide itself, seven structurally related isoforms, namely the isocereulides A-G, have already been elucidated in their chemical structure and could further be identified in B. cereus contaminated food samples. The newly performed isolation of isocereulide A allowed, for the first time, 1D- and 2D-NMR spectroscopy of a biosynthetically produced isocereulide, revealing results that contradict previous assumptions of an l-O-Leu moiety within its chemical structure. By furthermore applying posthydrolytical dipeptide analysis, amino acid and α-hydroxy acid analysis by means of UPLC-ESI-TOF-MS, as well as MSn sequencing, the structure of previously reported isocereulide A could be corrected. Instead of the l-O-Leu as assumed to date, one l-O-Ile unit could be verified in the cyclic dodecadepsipeptide, revising the structure of isocereulide A to [(d-O-Leu-d-Ala-l-O-Val-l-Val)2(d-O-Leu-d-Ala-l-O-Ile-l-Val)].

New Hopes for Drugs against COVID-19 Come from the Sea.

The latest chapter of the historic battle of humans against pathogenic microbes is the severe acute respiratory syndrome (SARS)-like coronavirus-2 (SARS-CoV-2), responsible for COVID-19, a respiratory disease declared a global pandemic by the WHO on March 11, 2020 [...].

Gatorbulin-1, a distinct cyclodepsipeptide chemotype, targets a seventh tubulin pharmacological site.

Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.

Genomics-driven discovery of a new cyclodepsipeptide from the guanophilic fungus Amphichorda guana.

Two potential non-ribosomal peptide synthetases (NRPSs) were identified in the genome of a guanophilic fungus Amphichorda guana by bioinformatics analysis and gene knockout experiments. Liquid chromatography coupled with mass spectrometry (LC-MS) guided isolation led to the discovery of a new cyclodepsipeptide isaridin H (1) and seven known analogs, desmethylisaridin E (2), isaridin E (3), isariin A (4), iso-isariin B (5), iso-isariin D (6), isariin E (7), and nodupetide (8). The absolute configuration of isaridin H (1) was achieved by Marfey's method. Isaridin H (1) showed significant antifungal activity against Botrytis cinerea and Alternaria solani.

Detection and Isolation of Emetic Bacillus cereus Toxin Cereulide by Reversed Phase Chromatography.

The emetic toxin cereulide is a 1.2 kDa dodecadepsipeptide produced by the food pathogen Bacillus cereus. As cereulide poses a serious health risk to humans, sensitive and specific detection, as well as toxin purification and quantification, methods are of utmost importance. Recently, a stable isotope dilution assay tandem mass spectrometry (SIDA-MS/MS)-based method has been described, and an method for the quantitation of cereulide in foods was established by the International Organization for Standardization (ISO). However, although this SIDA-MS/MS method is highly accurate, the sophisticated high-end MS equipment required for such measurements limits the method's suitability for microbiological and molecular research. Thus, we aimed to develop a method for cereulide toxin detection and isolation using equipment commonly available in microbiological and biochemical research laboratories. Reproducible detection and relative quantification of cereulide was achieved, employing reversed phase chromatography (RPC). Chromatographic signals were cross validated by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS/MS). The specificity of the RPC method was tested using a test panel of strains that included non-emetic representatives of the B. cereus group, emetic B. cereus strains, and cereulide-deficient isogenic mutants. In summary, the new method represents a robust, economical, and easily accessible research tool that complements existing diagnostics for the detection and quantification of cereulide.

Thioesterase-mediated side chain transesterification generates potent Gq signaling inhibitor FR900359.

The potent and selective Gq protein inhibitor depsipeptide FR900359 (FR), originally discovered as the product of an uncultivable plant endosymbiont, is synthesized by a complex biosynthetic system comprising two nonribosomal peptide synthetase (NRPS) assembly lines. Here we characterize a cultivable bacterial FR producer, enabling detailed investigations into biosynthesis and attachment of the functionally important FR side chain. We reconstitute side chain assembly by the monomodular NRPS FrsA and the non-heme monooxygenase FrsH, and characterize intermolecular side chain transesterification to the final macrocyclic intermediate FR-Core, mediated by the FrsA thioesterase domain. We harness FrsA substrate promiscuity to generate FR analogs with altered side chains and demonstrate indispensability of the FR side chain for efficient Gq inhibition by comparative bioactivity, toxicity and docking studies. Finally, evolution of FR and side chain biosynthesis is discussed based on bioinformatics analyses. Side chain transesterification boosts potency and target affinity of selective Gq inhibitor natural products.

Isolation and structure determination of new chymotrypsin inhibitory peptides streptopeptolins B and C.

New chymotrypsin inhibitory peptides named streptopeptolins B and C were isolated from Streptomyces olivochromogenes. Structures of streptopeptolins B and C were determined to be cyclic depsipeptides possessing 3-amino-6-hydroxy-2-piperidone unit by interpretation of NMR spectra and ESI-MS. Streptopeptolins B and C showed inhibitory activities to chymotrypsin with IC50 of 8.0 and 12.0 µg/mL, respectively.

A pipecolic acid-rich branched cyclic depsipeptide ulleungamide C from a Streptomyces species induces G0/G1 cell cycle arrest in promyelocytic leukemia cells.

In this study, screening by LC-MS and cytotoxicity-guided isolation led to the identification of ulleungamide C (1), a previously unknown pipecolic acid-rich branched cyclic depsipeptide, from a soil actinobacterium Streptomyces sp. KCB13F003. The structure of 1 was determined by interpretation of spectroscopic and spectrometric data from 1D and 2D NMR and HRESIMS experiments. Antiproliferative assays using mammalian cancerous cells revealed that 1 inhibits the proliferation of HL-60 human promyelocytic leukemia cells. Cell cycle analysis showed an increased accumulation of cells in the G0/G1 phase after treatment with 1. Results of immunoblotting assays revealed that 1 reduced the expression levels of cyclin-dependent kinase 4 (CDK4), CDK6, retinoblastoma protein (Rb), and phosphorylated Rb, whereas it induced cyclin-dependent kinase inhibitor 1B (p27/Kip1) expression.

Effects of Modification of Light Parameters on the Production of Cryptophycin, Cyanotoxin with Potent Anticancer Activity, in Nostoc sp.

Cryptophycin-1 is a cyanotoxin produced by filamentous cyanobacteria. It has been evaluated as an anticancer agent with great potential. However, its synthesis provides insufficient yield for industrial use. An alternative solution for metabolite efficient production is to stress cyanobacteria by modifying the environmental conditions of the culture (Nostoc sp. ATCC 53789). Here, we examined the effects of light photoperiod, wavelength, and intensity. In light photoperiod, photoperiods 24:0 and 16:8 (light:dark) were tested while in wavelength, orange-red light was compared with blue. Medium, high, and very high light intensity experiments were performed to test the effect of light stress. For a 10-day period, growth was measured, metabolite concentration was calculated through HPLC, and the related curves were drawn. The differentiation of light wavelength had a major effect on the culture, as orange-red filter contributed to noticeable increase in both growth and doubled the cyanotoxin concentration in comparison to blue light. Remarkably, constant light provides higher cryptophycin yield, but slightly lower growth rate. Lastly, the microorganism prefers medium light intensities for both growth and metabolite expression. The combination of these optimal conditions would contribute to the further exploitation of cryptophycin.

Trichodestruxins A-D: Cytotoxic Cyclodepsipeptides from the Endophytic Fungus Trichoderma harzianum.

Four new cyclodepsipeptides, trichodestruxins A-D (1-4), together with destruxin E2 chlorohydrin (5) and destruxin A2 (6), were isolated from the plant endophytic fungus Trichoderma harzianum by a bioassay-guided fractionation method. Their planar structures were elucidated on the basis of 1D and 2D NMR and MS/MS spectroscopic analyses. The stereochemical configuration was established by application of the advanced Marfey's method, J-based configuration analysis, Mosher's method, and chemical derivatizations. Trichodestruxin B contains hydroxy acid fragments of the THPA unit, while trichodestruxins A and C contain the ß-methylproline moiety. All cyclodepsipeptides displayed cytotoxicity against HT-29, A549, and/or P388 cell lines with IC50 values of 0.7-19.1 µM.

Application of untargeted tandem mass spectrometry with molecular networking for detection of enniatins and beauvericins from complex samples.

An untargeted LC-MS/MS-based molecular networking method was established for the automatic determination of variants of enniatin and beauvericin from both fungal cultures and naturally contaminated samples. Using this method, a large number of samples can be efficiently analyzed for the presence of enniatin- and beauvericin-related compounds. As proof of concept, 26 cultures, derived from 13 fungal strains in the genera of Fusarium, Beauveria, and Diaporthe, as well as 46 food samples were analyzed. Four enniatin- and three beauvericin-producing fungi were newly discovered. Among them, the production of beauvericin by Fusarium sp. 190-20-2 was further confirmed by the presence of a beauvericin biosynthesis gene cluster in its genomic sequence. Additionally, 17 enniatin congeners, including one new isomer of enniatin A, and three previously unreported bassianolide analogues were detected from an enniatin-producing fungus, Fusarium sp. 17-048, and a beauvericin-producing fungus, Beauveria sp. 186-069, respectively. The structures of the detected compounds were tentatively determined by a series of product ions of their sodium adducts. The new isomer of enniatin A was further confirmed by NMR spectra. A preliminary survey of food samples showed that enniatins were prevalent in the tested wheat flour and noodle samples, whereas beauvericin was only discovered in cornflour powder samples.

Virtual Screening of Marine Natural Compounds by Means of Chemoinformatics and CDFT-Based Computational Peptidology.

This work presents the results of a computational study of the chemical reactivity and bioactivity properties of the members of the theopapuamides A-D family of marine peptides by making use of our proposed methodology named Computational Peptidology (CP) that has been successfully considered in previous studies of this kind of molecular system. CP allows for the determination of the global and local descriptors that come from Conceptual Density Functional Theory (CDFT) that can give an idea about the chemical reactivity properties of the marine natural products under study, which are expected to be related to their bioactivity. At the same time, the validity of the procedure based on the adoption of the KID (Koopmans In DFT) technique, as well as the MN12SX/Def2TZVP/H2O model chemistry is successfully verified. Together with several chemoinformatic tools that can be used to improve the process of virtual screening, some additional properties of these marine peptides are identified related to their ability to behave as useful drugs. With the further objective of analyzing their bioactivity, some useful parameters for future QSAR studies, their predicted biological targets, and the ADMET (Absorption, Distribution, Metabolism, Excretion and Toxicity) parameters related to the theopapuamides A-D pharmacokinetics are also reported.

Bioactivities of Lyngbyabellins from Cyanobacteria of Moorea and Okeania Genera.

Cyanobacteria are reported as rich sources of secondary metabolites that provide biological activities such as enzyme inhibition and cytotoxicity. Ten depsipeptide derivatives (lyngbyabellins) were isolated from a Malaysian Moorea bouillonii and a Red Sea Okeania sp.: lyngbyabellins G (1), O (2), P (3), H (4), A (7), 27-deoxylyngbyabellin A (5), and homohydroxydolabellin (6). This study indicated that lyngbyabellins displayed cytotoxicity, antimalarial, and antifouling activities. The isolated compounds were tested for cytotoxic effect against human breast cancer cells (MCF7), for antifouling activity against Amphibalanus amphitrite barnacle larvae, and for antiplasmodial effect towards Plasmodium falciparum. Lyngbyabellins A and G displayed potent antiplasmodial effect against Plasmodium, whereas homohydroxydolabellin showed moderate effect. For antifouling activity, the side chain decreases the activity slightly, but the essential feature is the acyclic structure. As previously reported, the acyclic lyngbyabellins are less cytotoxic than the corresponding cyclic ones, and the side chain increases cytotoxicity. This study revealed that lyngbyabellins, despite being cytotoxic agents as previously reported, also exhibit antimalarial and antifouling activities. The unique chemical structures and functionalities of lyngbyabellin play an essential role in their biological activities.

Discovery and Biosynthesis of Bolagladins: Unusual Lipodepsipeptides from Burkholderia gladioli Clinical Isolates*.

Two Burkholderia gladioli strains isolated from the lungs of cystic fibrosis patients were found to produce unusual lipodepsipeptides containing a unique citrate-derived fatty acid and a rare dehydro-ß-alanine residue. The gene cluster responsible for their biosynthesis was identified by bioinformatics and insertional mutagenesis. In-frame deletions and enzyme activity assays were used to investigate the functions of several proteins encoded by the biosynthetic gene cluster, which was found in the genomes of about 45 % of B. gladioli isolates, suggesting that its metabolic products play an important role in the growth and/or survival of the species. The Chrome Azurol S assay indicated that these metabolites bind ferric iron, which suppresses their production when added to the growth medium. Moreover, a gene encoding a TonB-dependent ferric-siderophore receptor is adjacent to the biosynthetic genes, suggesting that these metabolites may function as siderophores in B. gladioli.

Boholamide A, an APD-Class, Hypoxia-Selective Cyclodepsipeptide.

Calcium homeostasis is implicated in some cancers, leading to the possibility that selective control of calcium might lead to new cancer drugs. On the basis of this idea, we designed an assay using a glioblastoma cell line and screened a collection of 1000 unique bacterial extracts. Isolation of the active compound from a hit extract led to the identification of boholamide A (1), a 4-amido-2,4-pentadieneoate (APD)-class peptide. Boholamide A (1) applied in the nanomolar range induces an immediate influx of Ca2+ in glioblastoma and neuronal cells. APD-class natural products are hypoxia-selective cytotoxins that primarily target mitochondria. Like other APD-containing compounds, 1 is hypoxia selective. Since APD natural products have received significant interest as potential chemotherapeutic agents, 1 provides a novel APD scaffold for the development of new anticancer compounds.

Swinhopeptolides A and B: Cyclic Depsipeptides from the Sponge Theonella swinhoei That Inhibit Ras/Raf Interaction.

Two new cyclic depsipeptides named swinhopeptolides A (1) and B (2) have been isolated from the marine sponge Theonella swinhoei cf. verrucosa, collected from Papua New Guinea. They each contain 11 diverse amino acid residues and 13-carbon polyketide moieties attached at the N-terminus. Compounds 1 and 2 each exist as two conformers in DMSO-d6 due to cis/trans isomerism of the proline residue, and their structures were successfully assigned by extensive NMR analyses complemented by chemical degradation and derivatization studies. Swinhopeptolide B (2) contains a previously undescribed 2,6,8-trimethyldeca-(2E,4E,6E)-trienoic acid moiety N-linked to a terminal serine residue. Swinhopeptolides A (1) and B (2) showed significant inhibition of the Ras/Raf signaling pathway with IC50 values of 5.8 and 8.5 µM, respectively.