Origin and characterization of cyclodepsipeptides: Comprehensive structural approaches with focus on mass spectrometry analysis of alkali-cationized molecular species.

Cyclodepsipeptides (CDPs) represent a huge family of chemically and structurally diverse molecules with a wide ability for molecular interactions. CDPs are cyclic peptide-related natural products made up of both proteinogenic and nonproteinogenic amino acids linked by amide and ester bonds. The combined use of different analytical methods is required to accurately determine their integral structures including stereochemistry, thus allowing deeper insights into their often-intriguing bioactivities and their possible usefulness. Our goal is to present the various methods developed to accurately characterize CDPs. Presently, Marfey's method and NMR (nuclear magnetic resonance) are still considered the best for characterizing CDP configuration. Nevertheless, electrospray-high resolution tandem mass spectrometry (ESI-HRMS/MS) is of great value for efficiently resolving CDP's composition and sequences. For instance, recent data shows that the fragmentation of cationized CDPs (e.g., [M + Li]+ and [M + Na]+) leads to selective cleavage of ester bonds and specific cationized product ions (b series) useful to get unprecedented sequence information. Thus, after a brief presentation of their structure, biological functions, and biosynthesis, we also provide a historic overview of these various analytical approaches as well as their advantages and limitations with a special emphasis on the emergence of methods based on HRMS/MS through recent fundamental works and applications.

Optimization of fermentation medium and conditions for enhancing valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Valinomycin is a cyclodepsipeptide antibiotic with a broad spectrum of biological activities, such as antiviral, antitumor, and antifungal activities. However, the low yield of valinomycin often limits its applications in medicine, agriculture, and industry. In our previous report, Streptomyces sp. ZJUT-IFE-354 was identified as a high-yielding strain of valinomycin. In this study, Plackett-Burman design (PBD) and response surface methodology (RSM) were used to optimize components of medium. The optimal medium contained 31 g/L glucose, 22 g/L soybean meal, and 1.6 g/L K2HPO4·3H2O, which could generate 262.47 ± 4.28 mg/L of valinomycin. Then, the culture conditions were optimized by a one-factor-at-a-time (OFAT) approach. The optimal conditions for the strain included a seed age of 24 h, an inoculum size of 8% (v/v), an incubation temperature of 28 °C, an initial pH of 7.2, an elicitor of 0.1% Bacillus cereus feeding at 24 h cultivation, and the feeding of 0.6% L-valine at 36 h cultivation. The final valinomycin production increased to 457.23 ± 9.52 mg/L, which was the highest yield ever reported. It highlights that RSM and OFAT may be efficient methods to enhance valinomycin production by Streptomyces sp. ZJUT-IFE-354.

Conformational changes in tubulin upon binding cryptophycin-52 reveal its mechanism of action.

Cryptophycin-52 (Cp-52) is potentially the most potent anticancer drug known, with IC50 values in the low picomolar range, but its binding site on tubulin and mechanism of action are unknown. Here, we have determined the binding site of Cp-52, and its parent compound, cryptophycin-1, on HeLa tubulin, to a resolution of 3.3 Å and 3.4 Å, respectively, by cryo-EM and characterized this binding further by molecular dynamics simulations. The binding site was determined to be located at the tubulin interdimer interface and partially overlap that of maytansine, another cytotoxic tubulin inhibitor. Binding induces curvature both within and between tubulin dimers that is incompatible with the microtubule lattice. Conformational changes occur in both α-tubulin and ß-tubulin, particularly in helices H8 and H10, with distinct differences between α and ß monomers and between Cp-52-bound and cryptophycin-1-bound tubulin. From these results, we have determined: (i) the mechanism of action of inhibition of both microtubule polymerization and depolymerization, (ii) how the affinity of Cp-52 for tubulin may be enhanced, and (iii) where linkers for targeted delivery can be optimally attached to this molecule.

Largazole Inhibits Ocular Angiogenesis by Modulating the Expression of VEGFR2 and p21.

Ocular angiogenic diseases, characterized by abnormal blood vessel formation in the eye, are the leading cause of blindness. Although Anti-VEGF therapy is the first-line treatment in the market, a substantial number of patients are refractory to it or may develop resistance over time. As uncontrolled proliferation of vascular endothelial cells is one of the characteristic features of pathological neovascularization, we aimed to investigate the role of the class I histone deacetylase (HDAC) inhibitor Largazole, a cyclodepsipeptide from a marine cyanobacterium, in ocular angiogenesis. Our study showed that Largazole strongly inhibits retinal vascular endothelial cell viability, proliferation, and the ability to form tube-like structures. Largazole strongly inhibits the vessel outgrowth from choroidal explants in choroid sprouting assay while it does not affect the quiescent choroidal vasculature. Largazole also inhibits vessel outgrowth from metatarsal bones in metatarsal sprouting assay without affecting pericytes coverage. We further demonstrated a cooperative effect between Largazole and an approved anti-VEGF drug, Alflibercept. Mechanistically, Largazole strongly inhibits the expression of VEGFR2 and leads to an increased expression of cell cycle inhibitor, p21. Taken together, our study provides compelling evidence on the anti-angiogenic role of Largazole that exerts its function through mediating different signaling pathways.

Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin.

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective ß-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin.

Rocking Aspergillus: morphology-controlled cultivation of Aspergillus niger in a wave-mixed bioreactor for the production of secondary metabolites.

BACKGROUND: Filamentous fungi including Aspergillus niger are cell factories for the production of organic acids, proteins and bioactive compounds. Traditionally, stirred-tank reactors (STRs) are used to cultivate them under highly reproducible conditions ensuring optimum oxygen uptake and high growth rates. However, agitation via mechanical stirring causes high shear forces, thus affecting fungal physiology and macromorphologies. Two-dimensional rocking-motion wave-mixed bioreactor cultivations could offer a viable alternative to fungal cultivations in STRs, as comparable gas mass transfer is generally achievable while deploying lower friction and shear forces. The aim of this study was thus to investigate for the first time the consequences of wave-mixed cultivations on the growth, macromorphology and product formation of A. niger. RESULTS: We investigated the impact of hydrodynamic conditions on A. niger cultivated at a 5 L scale in a disposable two-dimensional rocking motion bioreactor (CELL-tainer®) and a BioFlo STR (New Brunswick®), respectively. Two different A. niger strains were analysed, which produce heterologously the commercial drug enniatin B. Both strains expressed the esyn1 gene that encodes a non-ribosomal peptide synthetase ESYN under control of the inducible Tet-on system, but differed in their dependence on feeding with the precursors D-2-hydroxyvaleric acid and L-valine. Cultivations of A. niger in the CELL-tainer resulted in the formation of large pellets, which were heterogeneous in size (diameter 300-800 µm) and not observed during STR cultivations. When talcum microparticles were added, it was possible to obtain a reduced pellet size and to control pellet heterogeneity (diameter 50-150 µm). No foam formation was observed under wave-mixed cultivation conditions, which made the addition of antifoam agents needless. Overall, enniatin B titres of about 1.5-2.3 g L-1 were achieved in the CELL-tainer® system, which is about 30-50% of the titres achieved under STR conditions. CONCLUSIONS: This is the first report studying the potential use of single-use wave-mixed reactor systems for the cultivation of A. niger. Although final enniatin yields are not competitive yet with titres achieved under STR conditions, wave-mixed cultivations open up new avenues for the cultivation of shear-sensitive mutant strains as well as high cell-density cultivations.

Fusaripeptide A: new antifungal and anti-malarial cyclodepsipeptide from the endophytic fungus Fusarium sp.

From the culture of the endophytic fungus Fusarium sp. isolated from the roots of Mentha longifolia L. (Labiatae) growing in Saudi Arabia, a new cyclodepsipeptide, namely fusaripeptide A (1), along with three known compounds adenosine (2), 2[(2-hydroxypropionyl)amino]benzamide (3), and cyclopentanol (4), have been isolated. Their structures were determined, using extensive 1D and 2D NMR and HRESI and GC mass spectral data. That is the first report for the isolation of compound 4 from natural source. In addition, compounds 2 and 3 are reported here for the first time from Fusarium sp. The absolute configuration of the amino acid residues of 1 was assigned by chiral GCMS and Marfey's analysis after acid hydrolysis. Fusaripeptide A differs from the reported ones from Fusarium sp. in the length of fatty acidic alkyl chain. Compound 1 was evaluated for its antifungal, anti-malarial, and cytotoxic activities. It exhibited potent antifungal activity toward C. albicans, C. glabrata, C. krusei, and A. fumigates with IC50 values of 0.11, 0.24, 0.19, and 0.14 µM, respectively. Furthermore, it had significant anti-malarial activity toward P. falciparum (D6 clone) with IC50 value of 0.34 µM. However, it showed cytotoxic activity toward the tested cell lines.

Potent oxazoline analog of apratoxin C: Synthesis, biological evaluation, and conformational analysis.

In this research, the synthesis, biological evaluation, and conformational analysis of an apratoxin C oxazoline analog (3) have been demonstrated. The preparation of synthetic key intermediate 9 was achieved using an improved strategy that involves commercially available 3-methylglutaric anhydride (12), an enzymatic enantioselective alcoholysis, and a diastereoselective reduction. The Pro-Dtrina (3,7-dihydroxy-2,5,8-trimethylnonanoic acid) moiety 8 was successfully synthesized in a similar manner as our previously reported synthesis of apratoxin C (1). The cyclization precursor 5 was formed after the coupling of Pro-Dtrina 8 with a known tetrapeptide 7 to afford a linear peptide 6, the formation of an oxazoline, and the removal of the protecting groups. Finally, the macrolactamization of 5 with O-(7-aza-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIEA) furnished an apratoxin C oxazoline analog (3), which exhibited a potent cytotoxicity against HeLa cells (IC50 value of 22 nM) that was comparable with the cytotoxicity of apratoxin C (1) (IC50 value of 4.2 nM). Conformational analyses of 1 and 3 through NMR experiments showed that oxazoline analog 3 formed a tertiary structure that was similar to the apratoxin C (1) structure in CD3 CN, which provided a probable explanation for their comparable cytotoxicities. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 404-414, 2016.

Charge-solvated versus protonated salt forms of cyclodepsipeptide toxins in electrospray: Dissociation of alkali-cationized forms enables straightforward sequencing of cereulide.

Bacillus cereus is responsible for foodborne outbreaks worldwide. Among the produced toxins, cereulide induces nausea and vomiting after 30 min to 6 h following the consumption of contaminated foods. Cereulide, a cyclodepsipeptide, is an ionophore selective to K+ in solution. In electrospray, the selectivity is reduced as [M + Li]+; [M + Na]+ and [M + NH4]+ can also be detected without adding corresponding salts. Two forms are possible for alkali-cationized ions: charge-solvated (CS) that exclusively dissociates by releasing a bare alkali ion and protonated salt (PS), yielding alkali product ions by covalent bond cleavages (CBC) promoted by mobile proton. Based on a modified peptide cleavage nomenclature, the PS product ion series (b, a, [b + H2O] and [b + CnH2nO] [n = 4, 5]) are produced by Na+/Li+/K+-cationized cereulide species that specifically open at ester linkages followed by proton mobilization promoting competitive ester CBC as evidenced under resonant collision activation. What is more, unlike the sodiated or lithiated cereulide, which regenerates little or no alkali cation, the potassiated forms lead to an abundant K+ regeneration. This occurs by splitting of (i) the potassiated CS forms with an appearance threshold close to that of the PS first fragment ion generation and (ii) eight to four potassiated residue product ions from the PS forms. Since from Na+/Li+-cationized cereulide, (i) the negligible Na+/Li+ regeneration results in a higher sensibility than that of potassiated forms that abundantly releasing K+, and (ii) a better sequence recovering, the use of Na+ (or Li+) should be more pertinent to sequence isocereulides and other cyclodepsipeptides.

Recent advances on cyclodepsipeptides: biologically active compounds for drug research.

Cyclodepsipeptides are a large family of peptide-related natural products consisting of hydroxy and amino acids linked by amide and ester bonds. A number of cyclodepsipeptides have been isolated and characterized from fungi and bacteria. Most of them showed antitumor, antifungal, antiviral, antimalarial, and antitrypanosomal properties. Herein, this review summarizes the recent literatures (2010-2022) on the progress of cyclodepsipeptides from fungi and bacteria except for those of marine origin, in order to enrich our knowledge about their structural features and biological sources.

Heterotrimeric G Protein α-Subunits - Structures, Peptide-Derived Inhibitors, and Mechanisms.

G protein-coupled receptors are the largest protein family in the human body and represent the most important class of drug targets. They receive extracellular signals and transduce them into the cytosol. The guanine nucleotide-binding Gα proteins represent the main relays by which GPCRs induce intracellular effects. More than 800 different GPCRs interact with 16 Gα proteins belonging to 4 families, Gαi, Gαs, Gαq, and Gα12/13. The direct inhibition of Gα protein subunits rather than the modulation of GPCR subtypes has been proposed as a novel strategy for the treatment of complex diseases, including inflammation and cancer. This mini-review presents an introduction to G protein structure and function and describes achievements in the development of peptidic and peptide-derived Gα protein inhibitors. They have become indispensable pharmacological tools, and some of them exhibit significant potential as future drugs.

Natural Thiazoline-Based Cyclodepsipeptides from Marine Cyanobacteria: Chemistry, Bioefficiency and Clinical Aspects.

BACKGROUND: Peptides and peptide-based therapeutics are biomolecules that demarcate a significant chemical space to bridge small molecules with biological therapeutics, such as antibodies, recombinant proteins, and protein domains. INTRODUCTION: Cyclooligopeptides and depsipeptides, particularly cyanobacteria-derived thiazoline-based polypeptides (CTBCs), exhibit a wide array of pharmacological activities due to their unique structural features and interesting bioactions, which furnish them as promising leads for drug discovery. METHODS: In the present study, we comprehensively review the natural sources, distinguishing chemistries, and pertinent bioprofiles of CTBCs. We analyze their structural peculiarities counting the mode of actions for biological portrayals which render CTBCs as indispensable sources for emergence of prospective peptide-based therapeutics. In this milieu, metal organic frameworks and their biomedical applications are also briefly discussed. To boot, the challenges, approaches, and clinical status of peptide-based therapeutics are conferred. RESULTS: Based on these analyses, CTBCs can be appraised as ideal drug targets that have always remained a challenge for traditional small molecules, like those involved in protein- protein interactions or to be developed as potential cancer-targeting nanomaterials. Cyclization-induced reduced conformational freedom of these cyclooligopeptides contribute to improved metabolic stability and binding affinity to their molecular targets. Clinical success of several cyclic peptides provokes the large library-screening and synthesis of natural product-like cyclic peptides to address the unmet medical needs. CONCLUSION: CTBCs can be considered as the most promising lead compounds for drug discovery. Adopting the amalgamation of advanced biological and biopharmaceutical strategies might endure these cyclopeptides to be prospective biomolecules for futuristic therapeutic applications in the coming times.

Production of Diverse Beauveriolide Analogs in Closely Related Fungi: a Rare Case of Fungal Chemodiversity.

Fungal chemodiversity is well known in part due to the production of diverse analogous compounds by a single biosynthetic gene cluster (BGC). Usually, similar or the same metabolites are produced by closely related fungal species under a given condition, the foundation of fungal chemotaxonomy. Here, we report a rare case of the production of the cyclodepsipeptide beauveriolides (BVDs) in three insect-pathogenic fungi. We found that the more closely related fungi Beauveria bassiana and Beauveria brongniartii produced structurally distinct analogs of BVDs, whereas the less-close relatives B. brongniartii and Cordyceps militaris biosynthesized structurally similar congeners under the same growth condition. It was verified that a conserved BGC containing four genes is responsible for BVD biosynthesis in three fungi, including a polyketide synthase (PKS) for the production of 3-hydroxy fatty acids (FAs) with chain length variations. In contrast to BVD production patterns, phylogenetic analysis of the BGC enzymes or enzyme domains largely resulted in the congruence relationship with fungal speciation. Feeding assays demonstrated that an FA with a chain length of eight carbon atoms was preferentially utilized, whereas an FA with a chain longer than 10 carbon atoms could not be used as a substrate for BVD biosynthesis. Insect survival assays suggested that the contribution of BVDs to fungal virulence might be associated with the susceptibility of insect species. The results of this study enrich the knowledge of fungal secondary metabolic diversity that can question the reliability of fungal chemotaxonomy.IMPORTANCE Fungal chemotaxonomy is an approach to classify fungi based on the fungal production profile of metabolites, especially the secondary metabolites. We found an atypical example that could question the reliability of fungal chemical classifications in this study, i.e., the more closely related entomopathogenic species Beauveria bassiana and Beauveria brongniartii produced structurally different congeners of the cyclodepsipeptide beauveriolides, whereas the rather divergent species B. brongniartii and Cordyceps militaris biosynthesized similar analogs under the same growth condition. The conserved biosynthetic gene cluster (BGC) containing four genes present in each species is responsible for beauveriolide production. In contrast to the compound formation profiles, the phylogenies of biosynthetic enzymes or enzymatic domains show associations with fungal speciation. Dependent on the insect species, production of beauveriolides may contribute to fungal virulence against the susceptible insect hosts. The findings in this study augment the diversity of fungal secondary metabolisms.

Characterization of multimeric daptomycin bound to lipid nanodiscs formed by calcium-tolerant styrene-maleic acid copolymer.

Daptomycin is a lipopeptide antibiotic that is important in the treatment of infections with Gram-positive bacteria. In the presence of calcium, daptomycin binds to phosphatidylglycerol in the bacterial cytoplasmic membrane and then forms oligomers that mediate its bactericidal effect. The structure of these bactericidal oligomers has not been elucidated. We here explore the feasibility of structural studies on the oligomer by solution-state NMR. To this end, we use nanodiscs that contain DMPC and DMPG, stabilized with a styrene-maleic acid copolymer that has been modified to minimize calcium chelation. We show that these nanodiscs bind daptomycin and induce the formation of stable oligomers under physiologically relevant conditions. The findings suggest that this membrane model is suitable for structural and functional characterization of oligomeric daptomycin, and possibly of other calcium-dependent lipopeptide antibiotics. We show that these nanodiscs bind daptomycin and induce the formation of stable oligomers, under conditions that are suitable for biomolecular NMR. The findings suggest that this membrane model is suitable for structural elucidation of oligomeric daptomycin, and possibly of other calcium-dependent lipopeptide antibiotics.

Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria.

Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A, WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system. These molecules are already known as receptor antagonists, the first two for tachykinin and the last one for endothelin. WS9326A also inhibited the transcription of pfoA regulated by the VirSR two-component system in Clostridium perfringens. Receptor-binding assays using a fluorescence-labeled autoinducer (FITC-GBAP) showed that WS9326A and WS9326B act as receptor antagonists in this system. In addition, an ex vivo assay showed that WS9326B substantially attenuated the toxicity of S. aureus for human corneal epithelial cells. These results suggest that these three natural cyclodepsipeptides have therapeutic potential for targeting the cyclic peptide-mediated quorum sensing of Gram-positive pathogens.

Discovery of a new class of immunosuppressants from Trichothecium roseum co-inspired by cross-kingdom similarity in innate immunity and pharmacophore motif.

The limited selection of immunosuppressants in the clinic hampers the efficient management of immune disorders such as rejections after organ transplantations. However, the search for new immunosuppressive compounds remains random and creates inevitably financial and laborious wastes. Herein, we present an immunity-inspired discovery strategy that rationally allows an efficient identification of immunosuppressive compounds from the endophyte culture, as exemplified by the new peptide trichomide A. This compound exerts its immunosuppressive action more selectively than cyclosporin A. It was found that trichomide A decreases the expression of Bcl-2, increases the expression of Bax, and has a small or negligible effect on the expressions of p-Akt, CD25, and CD69. Our study strengthens the idea that the cross-kingdom similarity in immunity among living things could provide a shorter route towards the identification of natural products valuable for the development of new immunosuppressants.