Identification of an Optimized Clinical Development Candidate from Cilagicin, an Antibiotic That Evades Resistance by Dual Polyprenyl Phosphate Binding.

Cilagicin is a dual polyprenyl phosphate binding lipodepsipeptide antibiotic with strong activity against clinically relevant Gram-positive pathogens while evading antibiotic resistance. Cilagicin showed high serum binding that reduced its in vivo efficacy. Cilagicin-BP, which contains a biphenyl moiety in place of the N-terminal myristic acid found on cilagicin, showed reduced serum binding and increased in vivo efficacy but decreased potency against some pathogens. Here, we manipulated the acyl tail and the peptide core of cilagicin to identify an optimized collection of structural features that maintain potent antibiotic activity against a wide range of pathogens in the presence of serum. This led to the identification of the optimized antibiotic dodecacilagicin, which contains an N-terminal dodecanoic acid. Dodecacilagicin exhibits low MICs against clinically relevant pathogens in the presence of serum, retains polyprenyl phosphate binding, and evades resistance development even after long-term antibiotic exposure, making dodecacilagicin an appealing candidate for further therapeutic development.

Synthesis and Late-Stage Modification of (-)-Doliculide Derivatives Using Matteson's Homologation Approach.

(-)-Doliculide, a marine cyclodepsipeptide derived from the Japanese sea hare, Dolabella auricularia, exhibits potent cytotoxic properties, sparking interest in the field of synthetic chemistry. It is comprised of a peptide segment and a polyketide moiety, rendering it amenable to Matteson's homologation methodology. This technique facilitates the diversification of the distinctive polyketide side chain, thereby permitting the introduction of functional groups in late stages for modifications of the derived compounds and studies on structure-activity relationships.

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.

Synthesis, Structure-Activity Relationship Study, Bioactivity, and Nephrotoxicity Evaluation of the Proposed Structure of the Cyclic Lipodepsipeptide Brevicidine B.

The brevicidines represent a novel class of nonribosomal antimicrobial peptides that possess remarkable potency and selectivity toward highly problematic and resistant Gram-negative pathogenic bacteria. A recently discovered member of the brevicidine family, coined brevicidine B (2), comprises a single amino acid substitution (from d-Tyr2 to d-Phe2) in the amino acid sequence of the linear moiety of brevicidine (1) and was reported to exhibit broader antimicrobial activity against both Gram-negative (MIC = 2-4 µgmL-1) and Gram-positive (MIC = 2-8 µgmL-1) pathogens. Encouraged by this, we herein report the first total synthesis of the proposed structure of brevicidine B (2), building on our previously reported synthetic strategy to access brevicidine (1). In agreement with the original isolation paper, pleasingly, synthetic 2 demonstrated antimicrobial activity toward Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae (MIC = 4-8 µgmL-1). Interestingly, however, synthetic 2 was inactive toward all of the tested Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus strains. Substitution of d-Phe2 with its enantiomer, and other hydrophobic residues, yields analogues that were either inactive or only exhibited activity toward Gram-negative strains. The striking difference in the biological activity of our synthetic 2 compared to the reported natural compound warrants the re-evaluation of the original natural product for purity or possible differences in relative configuration. Finally, the evaluation of synthetic 1 and 2 in a human kidney organoid model of nephrotoxicity revealed substantial toxicity of both compounds, although 1 was less toxic than 2 and polymyxin B. These results indicate that modification to position 2 may afford a strategy to mitigate the nephrotoxicity of brevicidine.

Biosynthesis of Dolastatin 10 in Marine Cyanobacteria, a Prototype for Multiple Approved Cancer Drugs.

Dolastatin 10, a potent tubulin-targeting marine anticancer natural product, provided the basis for the development of six FDA-approved antibody-drug conjugates. Through the screening of cyanobacterial Caldora penicillata environmental DNA libraries and metagenome sequencing, we identified its biosynthetic gene cluster. Functional prediction of 10 enzymes encoded in the 39 kb cluster supports the dolastatin 10 biosynthesis. The nonheme diiron monooxygenase DolJ was biochemically characterized to mediate the terminal thiazole formation in dolastatin 10.

Discovery of a nitroaromatic nannocystin with potent in vivo anticancer activity against colorectal cancer by targeting AKT1.

The development of targeted chemotherapeutic agents against colorectal cancer (CRC), one of the most common cancers with a high mortality rate, is in a constant need. Nannocystins are a family of myxobacterial secondary metabolites featuring a 21-membered depsipeptide ring. The in vitro anti-CRC activity of natural and synthetic nannocystins was well documented, but little is known about their in vivo efficacy and if positive, the underlying mechanism of action. In this study we synthesized a nitroaromatic nannocystin through improved preparation of a key fragment, and characterized its in vitro activity and in vivo efficacy against CRC. We first described the total synthesis of compounds 2-4 featuring Heck macrocyclization to forge their 21-membered macrocycle. In a panel of 7 cancer cell lines from different tissues, compound 4 inhibited the cell viability with IC values of 1-6 nM. In particular, compound 4 (1, 2, 4 nM) inhibited the proliferation of CRC cell lines (HCT8, HCT116 and LoVo) in both concentration and time dependent manners. Furthermore, compound 4 concentration-dependently inhibited the colony formation and migration of CRC cell lines. Moreover, compound 4 induced cell cycle arrest at sub-G1 phase, apoptosis and cellular senescence in CRC cell lines. In three patient-derived CRC organoids, compound 4 inhibited the PDO with IC values of 3.68, 28.93 and 11.81 nM, respectively. In a patient-derived xenograft mouse model, injection of compound 4 (4, 8 mg/kg, i.p.) every other day for 12 times dose-dependently inhibited the tumor growth without significant change in body weight. We conducted RNA-sequencing, molecular docking and cellular thermal shift assay to elucidate the anti-CRC mechanisms of compound 4, and revealed that it exerted its anti-CRC effect at least in part by targeting AKT1.

Mebamamide C, a deoxy analogue of mebamamides in Bryopsis marine green algae and Elysia sacoglossan mollusks.

Kahalalides, originally isolated from the sacoglossan mollusk Elysia rufescens, have been found in various Elysia and Bryopsis species, with over 20 variants identified to date. These compounds are biosynthesized by Candidatus Endobryopsis kahalalidefaciens within Bryopsis species. In this study, we report the isolation and structural determination of a new cyclic depsipeptide, mebamamide C (1), from Bryopsis sp. The planar structure was determined by spectroscopic data analyses, and the absolute configurations were determined using Marfey's method and modified Mosher's method. Additionally, our study explores the chemical relationship between Bryopsis algae and Elysia mollusks. The individual chemical profiles of these marine organisms highlight a fascinating aspect of marine chemical ecology. The distinct, species-specific chemical profiles observed in Elysia species imply the possibility of a symbiotic relationship with the kahalalide-producing bacteria.

In Vitro Module Editing Of NRPS Enables Production Of Highly Potent Gq -Signaling Inhibitor FR900359 Derived From Unculturable Plant Symbiont.

Heterotrimeric G proteins are key mediators in the signaling of G protein-coupled receptors (GPCR) that are involved in a plethora of important physiological processes and thus major targets of pharmaceutical drugs. The cyclic depsipeptides YM-254890 and FR900359 are strong and selective inhibitors of the Gq subfamily of G proteins. FR900359 was first reported to be produced by unculturable plant symbiont, however, a culturable FR900359 producer was discovered recently by the standard strategy, screening of the producing strain from the environment. As another strategy, we introduce herein the different way to supply natural compounds of unculturable microorganism origin. We therefore embarked on constructing an artificial biosynthetic gene cluster (BGC) for FR900359 with YM-254890 BGC as a template using "in vitro module editing" technology, first developed for the modification of type-I PKS BGCs, to edit YM-254890 BGC. The resulting artificial BGCs coding FR900359 were heterologously expressed in the Pseudomonas putida KT2440 host strain.

A GSH-resistant FK228 analogue containing a stable disulfide bond.

FK228 is a potent natural pan HDAC inhibitor approved by the FDA for the treatment of cutaneous T-cell lymphoma as well as peripheral T-cell lymphoma. It is generally believed that the mechanism of FK228 acting on HDACs is by reducing its disulfide bond after entering the cell, and the dithiol group may chelate with Zn2+ and form a weak reversible covalent bond with cysteine in the catalytic pocket of HDACs, therefore inhibiting the activity of HDACs. However, due to the weak stability of the disulfide bond in FK228, it has been difficult to obtain direct evidence for the above conjecture. Thus, improving the stability of the FK228 disulfide bond will help to explore the exact mechanism of FK228. In this study, based on the stability and target-induced covalent properties of the Cysteine-Penicillamine (Cys-Pen) disulfide bond reported previously, the Pen was introduced into the modification of FK228. Specifically, the d-Cys in FK228 was replaced by d-Pen, the total synthetic pathway was optimized, and the novel synthetic FK228 analogue (FK-P) stability was verified. FK-P can also be used as a new drug molecule in the future to participate in the research of related biological mechanisms or the treatment of diseases.

Beauvericin Immunotoxicity Prevention by Gentiana lutea L. Flower In Vitro.

Beauvericin (BEA) is an emerging mycotoxin produced by some species of Fusarium genera that widely contaminates food and feed. Gentiana lutea is a protected medicinal plant known for its antioxidant and anti-inflammatory properties, which are attributed to its rich content of bioactive compounds. In order to evaluate the beneficial effects of G. lutea flower against BEA cytotoxicity, the aim of this study is to evaluate changes in protein expression after Jurkat cell exposure through a proteomics approach. To carry out the experiment, cells were exposed to intestinally digested G. lutea flower alone or in combination with the BEA standard (100 nM) over 7 days. Differentially expressed proteins were statistically evaluated (p < 0.05), revealing a total of 172 proteins with respect to the control in cells exposed to the BEA standard, 145 proteins for G. lutea alone, and 139 proteins when exposing the cells to the combined exposure. Bioinformatic analysis revealed processes implicated in mitochondria, ATP-related activity, and RNA binding. After careful analysis of differentially expressed proteins, it was evident that G. lutea attenuated, in most cases, the negative effects of BEA. Furthermore, it decreased the presence of major oncoproteins involved in the modulation of immune function.

Macrocyclization strategies for the total synthesis of cyclic depsipeptides.

Cyclic depsipeptides are an important class of peptide natural products that are defined by the presence of ester and amide bonds within the macrocycle. The structural diversity of depsipeptides has required the development of a broad range of synthetic strategies to access these biologically active compounds. Solid phase peptide synthesis (SPPS) strategies have been an invaluable tool in their synthesis. The key aspect of their synthesis is the macrocyclization strategy. Three main strategies are used, solution phase macrolactamization of acyclic ester containing peptide, on-resin macrolactamization of a sidechain-anchored peptide, and the solution phase macrolactonization of a linear peptide. Additionally, biocatalysts have been used to produce these compounds in a regio- and chemo-selective manner. Each compound offers unique challenges, requiring careful synthetic design to avoid undesirable side reactivity or unwanted epimerization during the esterification and macrocyclizing steps. This focused review analyzes these three strategies for cyclic depsipeptide natural product total synthesis with selected examples from the literature between 2001-2023.

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.

Factors influencing on-resin depsipeptide bond formation: case studies on daptomycin- and brevicidine-derived sequences.

Depsipeptides are an important class of bioactive natural products, where a growing number of genome-mined structures that display anti-microbial activity are macrocyclic depsipeptides. Chemically, peptide ester (depsipeptide) bond formation often displays low yields, and thereby hampers efforts to access these structures for structure-activity studies. Herein, we present a systematic study of the variables that influence depsipeptide bond formation on-resin, using simplified sequences derived from antibiotic peptides, daptomycin and brevicidine, prepared via Fmoc-based solid-phase synthesis. Our study highlights reaction solvent as the key determinant, where switching the solvent from DMF to DCM in almost all cases increased the amount of depsipeptide product. Limiting the number of amino-acids N-terminal to the reactive alcohol was also noted to significantly improve the acylation efficiency. The impact of different N-terminal and side-chain protecting groups, as well as stereochemistry, was also investigated. Additives to the reaction, such as inclusion of surfactants for esterification of long hydrophobic sequences, did not improve conversion. 6-ClHOBt, often added to improve acylation efficiency, notably decreased the amount of depsipeptide observed. Lastly, no significant difference between polystyrene and Tentagel® (PEG-decorated) resins were found for these sequences.

Valhidepsin Lipopeptides from Chromobacterium vaccinii: Structures, Biosynthesis, and Coregulation with FR900359 Production.

Microbial secondary metabolites continue to provide a valuable source of both chemical matter and inspiration for drug discovery in a broad range of therapeutic areas. Beyond this, the corresponding microorganisms represent a sustainable modality for biotechnological production of structurally complex molecules at the quantities required for drug development or even commercial manufacturing. Chromobacterium vaccinii, which has recently been reported as a producer of the pharmacologically highly important Gq inhibitor FR900359 (FR), represents such an example. The characterization of an orphan biosynthetic gene cluster (BGC) located directly downstream of the frs BCG led to the discovery of eight new lipopeptides, valhidepsins A-H (1-8), produced by C. vaccinii. Their chemical structures were elucidated through analysis of 1D and 2D NMR data and high-resolution MS/MS fragmentation methods. The valhidepsins did not display significant antibiotic nor cytotoxic activities but showed surfactant properties. The cluster-compound correlation was demonstrated by generation of a knockout mutant, which abolished production of valhidepsins. This knockout mutant yielded a significantly increased isolated yield of FR.

A Survey of Didemnin Depsipeptide Production in Tistrella.

As one of the first families of marine natural products to undergo clinical trials, the didemnin depsipeptides have played a significant role in inspiring the discovery of marine drugs. Originally developed as anticancer therapeutics, the recent re-evaluation of these compounds including synthetically derived dehydrodidemnin B or Aplidine, has led to their advancement towards antiviral applications. While conventionally associated with production in colonial tunicates of the family Didemnidae, recent studies have identified their biosynthetic gene clusters from the marine-derived bacteria Tistrella mobilis. While these studies confirm the production of didemnin X/Y, the low titer and general lack of understanding of their biosynthesis in Tistrella currently prevents the development of effective microbial or synthetic biological approaches for their production. To this end, we conducted a survey of known species of Tistrella and report on their ability to produce the didemnin depsipeptides. These data were used to develop conditions to produce didemnin B at titers over 15 mg/L.

Marine Organisms as a Prolific Source of Bioactive Depsipeptides.

Depsipeptides, an important group of polypeptides containing residues of hydroxy acids and amino acids linked together by amide and ester bonds, have potential applications in agriculture and medicine. A growing body of evidence demonstrates that marine organisms are prolific sources of depsipeptides, such as marine cyanobacteria, sponges, mollusks, microorganisms and algae. However, these substances have not yet been comprehensively summarized. In order to enrich our knowledge about marine depsipeptides, their biological sources and structural features, as well as bioactivities, are highlighted in this review after an extensive literature search and data analysis.

Depsipeptides Targeting Tumor Cells: Milestones from In Vitro to Clinical Trials.

Cancer is currently considered one of the most threatening diseases worldwide. Diet could be one of the factors that can be enhanced to comprehensively address a cancer patient's condition. Unfortunately, most molecules capable of targeting cancer cells are found in uncommon food sources. Among them, depsipeptides have emerged as one of the most reliable choices for cancer treatment. These cyclic amino acid oligomers, with one or more subunits replaced by a hydroxylated carboxylic acid resulting in one lactone bond in a core ring, have broadly proven their cancer-targeting efficacy, some even reaching clinical trials and being commercialized as "anticancer" drugs. This review aimed to describe these depsipeptides, their reported amino acid sequences, determined structure, and the specific mechanism by which they target tumor cells including apoptosis, oncosis, and elastase inhibition, among others. Furthermore, we have delved into state-of-the-art in vivo and clinical trials, current methods for purification and synthesis, and the recognized disadvantages of these molecules. The information collated in this review can help researchers decide whether these molecules should be incorporated into functional foods in the near future.

High-yield production of FK228 and new derivatives in a Burkholderia chassis.

FK228 (romidepsin) is the only natural histone deacetylases (HDACs) inhibitor approved by FDA to treat cutaneous and peripheral T-cell lymphoma. However, the limited supply and severe cardiotoxicity of FK228 underscore the importance to develop an effective synthetic biology platform for the manufacturing and fine-tuning of this drug lead. In this work, we constructed a Burkholderia chassis for the high-yield production of FK228-family (unnatural) natural products. By virtue of the optimized Burkholderia-specific recombineering system, the biosynthetic gene cluster (BGC) encoding the FK228-like skeleton thailandepsins (tdp) in Burkholderia thailandensis E264 was replaced with an attB integration site to afford the basal chassis KOGC1. The tdp BGC directly captured from E264 was hybridized with the FK228-encoding BGC (dep) using the versatile Red/ET technology. The hybrid BGC (tdp-dep) was integrated into the attB site of KOGC1, resulting in the heterologous expression of FK228. Remarkably, the titer reached 581 mg/L, which is 30-fold higher than that of native producer Chromobacterium violaceum No. 968. This success encouraged us to further engineer the NRPS modules 4 or 6 of hybrid tdp-dep BGC by domain units swapping strategy, and eight new FK228 derivatives (1-8) varying in the composition of amino acids were generated. Especially, the titers of 2 and 3 in KOGC1 were up to 985 mg/L and 453 mg/L, respectively. 2 and 3 displayed stronger cytotoxic activity than FK228. All in all, this work established a robust platform to produce FK228 and its new derivatives in sufficient quantities for anticancer drug development.

Biosynthesis of Lyngbyastatins 1 and 3, Cytotoxic Depsipeptides from an Okeania sp. Marine Cyanobacterium.

Lyngbyastatins (Lbns) 1 (1) and 3 (2) belong to a group of cyclic depsipeptides that inhibit cancer cell proliferation. These compounds have been isolated from different marine cyanobacterial collections, while further development of these compounds relies on their lengthy total synthesis. Biosynthetic studies of these compounds can provide viable strategies to access these compounds and develop new analogs. In this study, we report the identification and characterization of one Lbn biosynthetic gene cluster (BGC) from the marine cyanobacterium Okeania sp. VPG18-21. We initially identified 1 and 2 in the organic extract by mass spectrometry and performed the targeted isolation of these compounds, which feature a (2S,3R)-3-amino-2-methylpentanoic acid (MAP) and a (2S,3R)-3-amino-2-methylhexanoic acid (Amha) moiety, respectively. Parallel metagenomic sequencing of VPG18-21 led to the identification of a putative Lbn BGC that encodes six megaenzymes (LbnA-F), including one polyketide synthase (PKS, LbnE), four nonribosomal peptide synthetases (NRPSs, LbnB-D and -F), and one PKS-NRPS hybrid (LbnA). Bioinformatic analysis of these enzymes suggested that the BGC produces 1 and 2. Furthermore, our biochemical studies of three recombinant adenylation domains uncovered their substrate specificities, supporting the identity of the BGC. Finally, we identified near-complete Lbn-like BGCs in the genomes of two other marine cyanobacteria.

Cytotoxic Cyclodepsipeptides and Cyclopentane Derivatives from a Plant-Associated Fungus Fusarium sp.

In this work, four new cyclodepsipeptides, fusarihexins C-E (1-3) and enniatin Q (4), four new cyclopentane derivatives, fusarilins A-D (5-8), together with eight known compounds (9-16), were isolated from cultures of the endophytic fungus Fusarium sp. The structures of the isolated compounds were elucidated by analysis of HRMS and NMR spectroscopic data. The absolute configurations were determined using Marfey's method, a modified Mosher's method, single-crystal X-ray diffraction analysis, and ECD analysis. The antitumor activities of the isolated compounds in vitro were evaluated. Cyclodepsipeptides displayed cytotoxicities against the Huh-7, MRMT-1, and HepG-2 cell lines. Compounds 4, 9, 10, and 12 with IC50 values of 1.0-9.1 µM exhibited the most potent cytotoxicities against the three cell lines as compared to the positive control-5-fluorouracil. Compounds 1-3 and 11 exhibited moderate cytotoxic activities (IC50 values of 10.7-20.1 µM).