Retinestatin, a Polyol Polyketide from a Termite Nest-Derived Streptomyces sp.

A new polyol polyketide, named retinestatin (1), was obtained and characterized from the culture of a Streptomyces strain, which was isolated from a subterranean nest of the termite Reticulitermes speratus kyushuensis Morimoto. The planar structure of 1 was elucidated on the basis of the cumulative analysis of ultraviolet, infrared, mass spectrometry, and nuclear magnetic resonance spectroscopic data. The absolute configuration of 1 at 12 chiral centers was successfully assigned by employing a J-based configuration analysis in combination with ROESY correlations, a quantum mechanics-based computational approach to calculate NMR chemical shifts, and a 3 min flash esterification by Mosher's reagents followed by NMR analysis. Biological evaluation of retinestatin (1) using an in vitro model of Parkinson's disease revealed that 1 protected SH-SY5Y dopaminergic cells from MPP+-induced cytotoxicity, indicating its neuroprotective effects.

Exploring the Diverse Landscape of Biaryl-Containing Peptides Generated by Cytochrome P450 Macrocyclases.

Cytochrome P450 enzymes (P450s) catalyze diverse oxidative cross-coupling reactions between aromatic substrates in the natural product biosynthesis. Specifically, P450s install distinct biaryl macrocyclic linkages in three families of ribosomally synthesized and post-translationally modified peptides (RiPPs). However, the chemical diversity of biaryl-containing macrocyclic RiPPs remains largely unexplored. Here, we demonstrate that P450s have the capability to generate diverse biaryl linkages on RiPPs, collectively named "cyptides". Homology-based genome mining for P450 macrocyclases revealed 19 novel groups of homologous biosynthetic gene clusters (BGCs) with distinct aromatic residue patterns in the precursor peptides. Using the P450-modified precursor peptides heterologously coexpressed with corresponding P450s in Escherichia coli, we determined the NMR structures of three novel biaryl-containing peptides─the enzymatic products, roseovertin (1), rubrin (2), and lapparbin (3)─and confirmed the formation of three unprecedented or rare biaryl linkages: Trp C-7'-to-His N-τ in 1, Trp C-7'-to-Tyr C-6 in 2, and Tyr C-6-to-Trp N-1' in 3. Biochemical characterization indicated that certain P450s in these pathways have a relaxed substrate specificity. Overall, our studies suggest that P450 macrocyclases have evolved to create diverse biaryl linkages in RiPPs, promoting the exploration of a broader chemical space for biaryl-containing peptides encoded in bacterial genomes.

Tandocyclinones A and B, Ether Bridged C-Glycosyl Benz[a]anthracenes from an Intertidal Zone Streptomyces sp.

Two new proton-deficient metabolites, tandocyclinones A and B (1 and 2), were discovered via the chemical profiling of the Streptomyces sp. strain TDH03, which was isolated from a marine sediment sample collected from the intertidal mudflat in Tando Port, the Republic of Korea. The structures of 1 and 2 were elucidated as new ether-bridged C-glycosyl benz[a]anthracenes by using a combination of spectroscopic analyses of ultraviolet (UV) and mass spectrometry (MS) data, along with nuclear magnetic resonance (NMR) spectra, which were acquired in tetrahydrofuran (THF)-d8 selected after an extensive search for a solvent, resulting in mostly observable exchangeable protons in the 1H NMR spectrum. Their configurations were successfully assigned by applying a J-based configuration analysis, rotating-frame Overhauser enhancement spectroscopy (ROESY) NMR correlations, chemical derivatization methods based on NMR (a modified version of Mosher's method) and circular dichroism (CD) (Snatzke's method using Mo2(OAc)4-induced CD), as well as quantum-mechanics-based computational methods, to calculate the electronic circular dichroism (ECD). Tandocyclinones A and B (1 and 2) were found to have weak antifungal activity against Trichophyton mentagrophytes IFM40996 with an MIC value of 128 µg/mL (244 and 265 µM for 1 and 2, respectively). A further biological evaluation revealed that tandocyclinone A (1) displayed inhibitory activity against Mycobacterium avium (MIC50 = 40.8 µM) and antiproliferative activity against SNU638 and HCT116 cancer cells, with IC50 values of 31.9 µM and 49.4 µM, respectively.

Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures.

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N-N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H-15N heteronuclear multiple bond correlation (HMBC) along with 1H-15N heteronuclear single quantum coherence (HSQC) and 1H-15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade-structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.

Discovery and Biosynthesis of Cihunamides, Macrocyclic Antibacterial RiPPs with a Unique C-N Linkage Formed by CYP450 Catalysis.

Cihunamides A-D (1-4), novel antibacterial RiPPs, were isolated from volcanic-island-derived Streptomyces sp. The structures of 1-4 were elucidated by 1 H, 13 C, and 15 N NMR, MS, and chemical derivatization; they contain a tetrapeptide core composed of WNIW, cyclized by a unique C-N linkage between two Trp units. Genome mining of the producer strain revealed two biosynthetic genes encoding a cytochrome P450 enzyme and a precursor peptide. Heterologous co-expression of the core genes demonstrated the biosynthesis of cihunamides through P450-mediated oxidative Trp-Trp cross-linking. Further bioinformatic analysis uncovered 252 homologous gene clusters, including that of tryptorubins, which possess a distinct Trp-Trp linkage. Cihunamides do not display the non-canonical atropisomerism shown in tryptorubins, which are the founding members of the "atropitide" family. Therefore, we propose to use a new RiPP family name, "bitryptides", for cihunamides, tryptorubins, and their congeners, wherein the Trp-Trp linkages define the structural class rather than non-canonical atropisomerism.

Chromatographic Determination of the Absolute Configuration in Sanjoinine A That Increases Nitric Oxide Production.

A chiral derivatization strategy with phenylglycine methyl ester (PGME) was employed to develop a straightforward method to determine the absolute configurations of N,N-dimethyl amino acids. The PGME derivatives were analyzed using liquid chromatography-mass spectrometry to identify the absolute configurations of various N,N-dimethyl amino acids based on their elution time and order. The established method was applied to assign the absolute configuration of the N,N-dimethyl phenylalanine in sanjoinine A (4), a cyclopeptide alkaloid isolated from Zizyphi Spinosi Semen widely used as herbal medicine for insomnia. Sanjoinine A displayed production of nitric oxide (NO) in LPS-activated RAW 264.7 cells.

Total Synthesis of Melicoptelines C-E: Antiviral Cyclopeptides Containing a Hexahydropyrrolo[2,3-b]indole Moiety.

Melicoptelines, natural cyclopeptides containing a 3a-hydroxy hexahydropyrrolo[2,3-b]indole (HPI) moiety, exhibit anti-influenza activity. Herein, we report the first total synthesis of melicoptelines C-E (1-3, respectively). The core 3a-hydroxy HPIs were synthesized in a diastereoselective manner from l-tryptophan using dimethyldioxirane-mediated oxidation. Subsequently, sequential peptide couplings and cyclization completed the synthesis of melicoptelines C-E and unnatural melicopteline 4. The synthesized melicoptelines were evaluated for their anti-influenza activity, and melicopteline E showed the most potent inhibition of cytopathic effects.

Epoxinnamide: An Epoxy Cinnamoyl-Containing Nonribosomal Peptide from an Intertidal Mudflat-Derived Streptomyces sp.

Cinnamoyl-containing nonribosomal peptides (CCNPs) form a unique family of actinobacterial secondary metabolites and display various biological activities. A new CCNP named epoxinnamide (1) was discovered from intertidal mudflat-derived Streptomyces sp. OID44. The structure of 1 was determined by the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) data along with a mass spectrum. The absolute configuration of 1 was assigned by the combination of advanced Marfey's method, 3JHH and rotating-frame overhauser effect spectroscopy (ROESY) analysis, DP4 calculation, and genomic analysis. The putative biosynthetic pathway of epoxinnamide (1) was identified through the whole-genome sequencing of Streptomyces sp. OID44. In particular, the thioesterase domain in the nonribosomal peptide synthetase (NRPS) biosynthetic gene cluster was proposed as a bifunctional enzyme, which catalyzes both epimerization and macrocyclization. Epoxinnamide (1) induced quinone reductase (QR) activity in murine Hepa-1c1c7 cells by 1.6-fold at 5 µM. It also exhibited effective antiangiogenesis activity in human umbilical vein endothelial cells (IC50 = 13.4 µM).

Hamuramicin C, a Cytotoxic Bicyclic Macrolide Isolated from a Wasp Gut Bacterium.

A new bicyclic macrolide, hamuramicin C (1), was isolated from Streptomyces sp. MBP16, a gut bacterial strain of the wasp Vespa crabro flavofasciata. Its 22-membered macrocyclic lactone structure was determined by NMR and mass spectrometry. The relative configurations of hamuramicin C (1) were assigned by J-based configuration analysis utilizing 1H rotating frame Overhauser effect spectroscopy and heteronuclear long-range coupling NMR spectroscopy. Genomic and bioinformatic analyses of the bacterial strain enabled identification of the type-I polyketide synthase pathway, which employs a trans-acyltransferase system. The absolute configurations of 1 were proposed based on the analysis of the sequences of ketoreductases in the modular gene cluster. Moreover, hamuramicin C (1) demonstrated significant inhibitory activity against diverse human cancer cell lines (HCT116, A549, SNU-638, SK-HEP-1, and MDA-MB-231).

Nyuzenamide C, an Antiangiogenic Epoxy Cinnamic Acid-Containing Bicyclic Peptide from a Riverine Streptomyces sp.

A new nonribosomal peptide, nyuzenamide C (1), was discovered from riverine sediment-derived Streptomyces sp. DM14. Comprehensive analysis of the spectroscopic data of nyuzenamide C (1) revealed that 1 has a bicyclic backbone composed of six common amino acid residues (Asn, Leu, Pro, Gly, Val, and Thr) and four nonproteinogenic amino acid units, including hydroxyglycine, ß-hydroxyphenylalanine, p-hydroxyphenylglycine, and 3,ß-dihydroxytyrosine, along with 1,2-epoxypropyl cinnamic acid. The absolute configuration of 1 was proposed by J-based configuration analysis, the advanced Marfey's method, quantum mechanics-based DP4 calculations, and bioinformatic analysis of its nonribosomal peptide synthetase biosynthetic gene cluster. Nyuzenamide C (1) displayed antiangiogenic activity in human umbilical vein endothelial cells and induced quinone reductase in murine Hepa-1c1c7 cells.

Ligiamycins A and B, Decalin-Amino-Maleimides from the Co-Culture of Streptomyces sp. and Achromobacter sp. Isolated from the Marine Wharf Roach, Ligia exotica.

Streptomyces sp. GET02.ST and Achromobacter sp. GET02.AC were isolated together from the gut of the wharf roach, Ligia exotica, inhabiting the intertidal zone of the west coast of Korea. The co-cultivation of these two strains significantly induced the production of two new metabolites, ligiamycins A (1) and B (2), which were barely detected in the single culture of Streptomyces sp. GET02.ST. The planar structures of ligiamycins A (1) and B (2) were elucidated as new decalins coupled with amino-maleimides by the analysis of various spectroscopic data, including nuclear magnetic resonance (NMR), ultraviolet (UV), and mass (MS) data. The assignment of two nitrogen atoms in amino-maleimide in 1 was accomplished based on 1H-15N heteroatom single quantum coherence spectroscopy (HSQC) NMR experiments. The relative configurations of the ligiamycins were determined using rotating frame Overhauser effect spectroscopy (ROESY) NMR data, and their absolute configurations were deduced by comparing their experimental and calculated optical rotations. Ligiamycin A (1) displayed antibacterial effects against Staphylococcus aureus and Salmonella enterica, while ligiamycin B (2) exhibited mild cell cytotoxicity against human colorectal cancer cells.

Dumulmycin, an Antitubercular Bicyclic Macrolide from a Riverine Sediment-Derived Streptomyces sp.

Dumulmycin (1) was isolated from Streptomyces sp. DM28, a bacterial strain from a riverine sediment sample. The structure of 1 was elucidated as a bicyclic macrolide possessing 19-membered and 5-membered rings by spectroscopic analysis. The stereochemistry of 1 was determined by J-based configuration analysis, ROESY NMR data, DP4 calculations, and the modified Mosher's method. Genetic analysis identified a trans-acyltransferase polyketide biosynthetic gene cluster for 1. Dumulmycin exhibited in vitro antitubercular activity (MIC50 = 27.1 µM).

Lenzimycins A and B, Metabolites With Antibacterial Properties From Brevibacillus sp. Associated With the Dung Beetle Onthophagus lenzii.

Symbiotic microorganisms associated with insects can produce a wide array of metabolic products, which provide an opportunity for the discovery of useful natural products. Selective isolation of bacterial strains associated with the dung beetle, Onthophagus lenzii, identified two strains, of which the antibiotic-producing Brevibacillus sp. PTH23 inhibited the growth of Bacillus sp. CCARM 9248, which is most closely related to the well-known entomopathogen, Bacillus thuringiensis. A comprehensive chemical investigation based on antibiotic activity discovered two new antibiotics, named lenzimycins A and B (1-2), which inhibited growth of Bacillus sp. CCARM 9248. The 1H and 13C NMR, MS, MS/MS, and IR analyses elucidated the structures of 1 and 2, which comprised a novel combination of fatty acid (12-methyltetradecanoic acid), glycerol, sulfate, and N-methyl ethanolamine. Furthermore, the acid hydrolysis of 1 revealed the absolute configuration of 12-methyltetradecanoic acid as 12S by comparing its optical rotation value with authentic (R)- and (S)-12-methyltetradecanoic acid. In addition to inhibition of Bacillus sp. CCARM 9248, lenzimycins A and B were found to inhibit the growth of some human pathogenic bacteria, including Enterococcus faecium and certain strains of Enterococcus faecalis. Furthermore, the present study elucidated that lenzimycins A and B activated a reporter system designed to detect the bacterial cell envelope stress, thereby indicating an activity against the integrity of the bacterial cell wall.

Formicolides A and B, Antioxidative and Antiangiogenic 20-Membered Macrolides from a Wood Ant Gut Bacterium.

Two new macrolides, formicolides A (1) and B (2), were isolated from Streptomyces sp. BA01, a gut bacterial strain of the wood ant (Formica yessensis). Their 20-membered macrocyclic lactone structures were established using NMR and mass spectrometric data. The relative configurations of the formicolides were determined by J-based configuration analysis utilizing ROESY, HETLOC, and HECADE NMR spectroscopic data. Genomic and bioinformatics analysis of the bacterial strain enabled us to identify the type-I polyketide synthase pathway employing a trans-acyltransferase system. The absolute configurations of 1 and 2 are proposed based on detailed analysis of the sequences of the ketoreductases in the modular gene cluster and statistical comparative analysis of the experimental NMR chemical shifts and quantum mechanical calculations. Formicolides A and B (1 and 2) induced quinone reductase activity in murine Hepa-1c1c7 cells and antiangiogenic activity by suppression of tube formation in human umbilical vein endothelial cells.

Rhizolutin, a Novel 7/10/6-Tricyclic Dilactone, Dissociates Misfolded Protein Aggregates and Reduces Apoptosis/Inflammation Associated with Alzheimer's Disease.

Rhizolutin (1) was discovered as a natural product of ginseng-rhizospheric Streptomyces sp. WON17. Its structure features an unprecedented 7/10/6-tricyclic dilactone carbon skeleton composed of dimethylcyclodecatriene flanked by a 7-membered and a 6-membered lactone ring based on spectroscopic analysis. During an unbiased screening of natural product libraries, this novel compound was found to dissociate amyloid-ß (Aß) plaques and tau tangles, which are key pathological hallmarks of Alzheimer's disease (AD). Rhizolutin treatment of APP/PS1 double transgenic mice with AD significantly dissociated hippocampal plaques. In vitro, rhizolutin substantially decreased Aß-induced apoptosis and inflammation in neuronal and glial cells. Our findings introduce a unique chemical entity that targets Aß and tau concurrently by mimicking misfolded protein clearance mechanisms of immunotherapy, which is prominently investigated in clinical trials.

Structural Revision of Lydiamycin A by Reinvestigation of the Stereochemistry.

Lydiamycin A (1) is a previously reported piperazic acid-bearing cyclic peptide from Streptomyces. The absolute configuration of lydiamycin A has not been fully determined despite the fact that multiple total syntheses have been reported. The absolute configuration of 1 was reinvestigated by the advanced Marfey's method, chemical derivatization, and quantum-mechanics-based computational analysis, eventually resulting in a structural revision and establishment of the complete configuration. Lydiamycin A displayed weak antituberculosis activity in vitro.

Donghaecyclinones A-C: New Cytotoxic Rearranged Angucyclinones from a Volcanic Island-Derived Marine Streptomyces sp.

Chemical profiling of the Streptomyces sp. strain SUD119, which was isolated from a marine sediment sample collected from a volcanic island in Korea, led to the discovery of three new metabolites: donghaecyclinones A-C (1-3). The structures of 1-3 were found to be rearranged, multicyclic, angucyclinone-class compounds according to nuclear magnetic resonance (NMR) and mass spectrometry (MS) analyses. The configurations of their stereogenic centers were successfully assigned using a combination of quantum mechanics-based computational methods for calculating the NMR shielding tensor (DP4 and CP3) as well as electronic circular dichroism (ECD) along with a modified version of Mosher's method. Donghaecyclinones A-C (1-3) displayed cytotoxicity against diverse human cancer cell lines (IC50: 6.7-9.6 µM for 3).

Donghaesulfins A and B, Dimeric Benz[ a]anthracene Thioethers from Volcanic Island Derived Streptomyces sp.

The chemical analysis of a Streptomyces strain, from a Korean volcanic island, discovered new benz[ a]anthracene dimers linked by a thioether bond. The structures of donghaesulfins A and B (1 and 2) were elucidated by spectroscopic analysis including energy-dispersive X-ray. Their configurations were determined by ROESY NMR data, DP4 calculations, the modified Mosher's method, and ECD calculations. Donghaesulfins A (1) induced quinone reductase, whereas donghaesulfin B (2) displayed antiangiogenesis activity.

Camporidines A and B: Antimetastatic and Anti-inflammatory Polyketide Alkaloids from a Gut Bacterium of Camponotus kiusiuensis.

Chemical studies of gut bacteria of the carpenter ant Camponotus kiusiuensis led to the discovery of two new alkaloids, camporidines A and B (1 and 2), from Streptomyces sp. STA1. The structures of 1 and 2 were established as new polyketide alkaloids bearing a piperidine-cyclopentene-epoxide 6/5/3 tricyclic system based on NMR spectroscopic and mass spectrometric analysis. The relative configurations of the camporidines were determined by their 1H-1H NOESY/ROESY and 1D NOE NMR correlations. The experimental ECD spectra of 1 and 2 were compared with their calculated ECD spectra to assign their absolute configurations. Camporidine A (1) displayed antimetastatic activity by suppression of cell invasion against the metastatic breast cancer cell line MDA-MB-231 and showed an anti-inflammatory effect by suppressing nitric oxide production induced by lipopolysaccharide. In addition, the putative biosynthetic gene cluster of the camporidines was identified, and the biosynthetic pathway of the camporidines was proposed based on bioinformatic analysis of the full genome of Streptomyces sp. STA1. Camporidines A and B (1 and 2) could be biosynthesized by a modular type I PKS containing an acyl transferase domain that accepts an unusual extender unit, which becomes the (C1'-C6') hexyl side chain. The post-PKS modification enzymes were predicted to perform an amination and an oxidation along with spontaneous Schiff base formation and generate the unique piperidine-cyclopentene-epoxide 6/5/3 tricyclic framework.