Serratiomycins D1-D3, Antibacterial Cyclic Peptides from a Serratia sp. and Structure Revision of Serratiomycin.

Serratiomycin (1) is an antibacterial cyclic depsipeptide, first discovered from a Eubacterium culture in 1998. This compound was initially reported to contain l-Leu, l-Ser, l-allo-Thr, d-Phe, d-Ile, and hydroxydecanoic acid. In the present study, 1 and three new derivatives, serratiomycin D1-D3 (2-4), were isolated from a Serratia sp. strain isolated from the exoskeleton of a long-horned beetle. The planar structures of 1-4 were elucidated by using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparison of the NMR chemical shifts and the physicochemical data of 1 to those of previously reported serratiomycin indeed identified 1 as serratiomycin. The absolute configurations of the amino units in compounds 1-4 were determined by the advanced Marfey's method, 2,3,4,6-tetra-O-acetyl-ß-d-glucopyranosyl isothiocyanate derivatization, and liquid chromatography-mass spectrometric (LC-MS) analysis. Additionally, methanolysis and the modified Mosher's method were used to determine the absolute configuration of (3R)-hydroxydecanoic acid in 1. Consequently, the revised structure of 1 was found to possess d-Leu, l-Ser, l-Thr, d-Phe, l-allo-Ile, and d-hydroxydecanoic acid. In comparison with the previously published structure of serratiomycin, l-Leu, l-allo-Thr, and d-Ile in serratiomycin were revised to d-Leu, l-Thr, and l-allo-Ile. The new members of the serratiomycin family, compounds 2 and 3, showed considerably higher antibacterial activities against Staphylococcus aureus and Salmonella enterica than compound 1.

Discovery of Terminal Oxazole-Bearing Natural Products by a Targeted Metabologenomic Approach.

A targeted metabologenomic method was developed to selectively discover terminal oxazole-bearing natural products from bacteria. For this, genes encoding oxazole cyclase, a key enzyme in terminal oxazole biosynthesis, were chosen as the genomic signature to screen bacterial strains that may produce oxazole-bearing compounds. Sixteen strains were identified from the screening of a bacterial DNA library (1,000 strains) using oxazole cyclase gene-targeting polymerase chain reaction (PCR) primers. The PCR amplicon sequences were subjected to phylogenetic analysis and classified into nine clades. 1H-13C coupled-HSQC NMR spectra obtained from the culture extracts of the hit strains enabled the unequivocal detection of the target compounds, including five new oxazole compounds, based on the unique 1JCH values and chemical shifts of oxazole: lenzioxazole (1) possessing an unprecedented cyclopentane, permafroxazole (2) bearing a tetraene conjugated with carboxylic acid, tenebriazine (3) incorporating two modified amino acids, and methyl-oxazolomycins A and B (4 and 5). Tenebriazine displayed inhibitory activity against pathogenic fungi, whereas methyl-oxazolomycins A and B (4 and 5) selectively showed anti-proliferative activity against estrogen receptor-positive breast cancer cells. This metabologenomic method enables the logical and efficient discovery of new microbial natural products with a target structural motif without the need for isotopic labeling.

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.

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.

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.

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.

Genomic and Spectroscopic Signature-Based Discovery of Natural Macrolactams.

The logical and effective discovery of macrolactams, structurally unique natural molecules with diverse biological activities, has been limited by a lack of targeted search methods. Herein, a targeted discovery method for natural macrolactams was devised by coupling genomic signature-based PCR screening of a bacterial DNA library with spectroscopic signature-based early identification of macrolactams. DNA library screening facilitated the efficient selection of 43 potential macrolactam-producing strains (3.6% of 1,188 strains screened). The PCR amplicons of the amine-deprotecting enzyme-coding genes were analyzed to predict the macrolactam type (α-methyl, α-alkyl, or ß-methyl) produced by the hit strains. 1H-15N HSQC-TOCSY NMR analysis of 15N-labeled culture extracts enabled macrolactam detection and structural type assignment without any purification steps. This method identified a high-titer Micromonospora strain producing salinilactam (1), a previously reported α-methyl macrolactam, and two Streptomyces strains producing new α-alkyl and ß-methyl macrolactams. Subsequent purification and spectroscopic analysis led to the structural revision of 1 and the discovery of muanlactam (2), an α-alkyl macrolactam with diene amide and tetraene chromophores, and concolactam (3), a ß-methyl macrolactam with a [16,6,6]-tricyclic skeleton. Detailed genomic analysis of the strains producing 1-3 identified putative biosynthetic gene clusters and pathways. Compound 2 displayed significant cytotoxicity against various cancer cell lines (IC50 = 1.58 µM against HCT116), whereas 3 showed inhibitory activity against Staphylococcus aureus sortase A. This genomic and spectroscopic signature-based method provides an efficient search strategy for new natural macrolactams and will be generally applicable for the discovery of nitrogen-bearing natural products.

Inhibition of Streptococcus mutans adhesion and biofilm formation with small-molecule inhibitors of sortase A from Juniperus chinensis.

Background: Streptococcus mutans, an important Gram-positive pathogen in dental caries, uses sortase A (SrtA) to anchor surface proteins to the bacterial cell wall, thereby promoting biofilm formation and attachment to the tooth surface. Design: Based on activity-guided separation, inhibitors of S. mutans SrtA were isolated from Juniperus chinensis and identified through combined spectroscopic analysis. Further effects of isolated SrtA inhibitor on S. mutans were evaluated on bacterial aggregation, adherence and biofilm formation. Results: Six compounds (1-6) were isolated from the dried heartwood of J. chinensis. A novel compound designated 3',3"-dihydroxy-(-)-matairesinol (1) was identified, which exhibited potent inhibitory activity toward S. mutans SrtA (IC50 = 16.1 µM) without affecting microbial viability (minimum inhibitory concentration > 300 µM). The results of subsequent bioassays using compound 1 indicated that this compound inhibits S. mutans aggregation, adhesion and biofilm formation on solid surfaces by inhibiting SrtA activity. The onset and magnitude of inhibition of adherence and biofilm formation in S. mutans treated with compound 1 at 4× the SrtA IC50 are comparable to the behaviors of the untreated srtA-deletion mutant. Conclusion: Our findings suggest that small-molecule inhibitors of S. mutans SrtA may be useful for the prevention of dental plaque and treatment of dental microbial diseases.

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.

Inhibitory Effects of Nitrogenous Metabolites from a Marine-Derived Streptomyces bacillaris on Isocitrate Lyase of Candida albicans.

Two nitrogenous metabolites, bacillimide (1) and bacillapyrrole (2), were isolated from the culture broth of the marine-derived actinomycete Streptomyces bacillaris. Based on the results of combined spectroscopic and chemical analyses, the structure of bacillimide (1) was determined to be a new cyclopenta[c]pyrrole-1,3-dione bearing a methylsulfide group, while the previously reported bacillapyrrole (2) was fully characterized for the first time as a pyrrole-carboxamide bearing an alkyl sulfoxide side chain. Bacillimide (1) and bacillapyrrole (2) exerted moderate (IC50 = 44.24 µM) and weak (IC50 = 190.45 µM) inhibitory effects on Candida albicans isocitrate lyase, respectively. Based on the growth phenotype using icl-deletion mutants and icl expression analyses, we determined that bacillimide (1) inhibits the transcriptional level of icl in C. albicans under C2-carbon-utilizing conditions.

Gwanakosides A and B, 6-Deoxy-α-l-talopyranose-Bearing Aromatic Metabolites from a Streptomyces sp. and Coculture with Pandoraea sp.

Single-strain cultivation of a mountain soil-derived Streptomyces sp. GA02 and its coculture with Pandoraea sp. GA02N produced two aromatic products, gwanakosides A and B (1 and 2, respectively). Their spectroscopic analysis revealed that 1 is a new dichlorinated naphthalene glycoside and 2 is a pentacyclic aromatic glycoside. The assignment of the two chlorine atoms in 1 was confirmed by the analysis of its band-selective CLIP-HSQMBC spectrum. The sugars in the gwanakosides were identified as 6-deoxy-α-l-talopyranose based on 1H-1H coupling constants, Rotating frame Overhauser enhancement spectroscopy (ROESY) NMR correlations, and chemical derivatization followed by spectroscopic and chromatographic analyses. The absolute configuration of 2, whose production was enhanced approximately 100-fold in coculture, was proposed based on a quantum mechanics-based chemical shift analysis method, DP4 calculations, and the chemically determined configuration of 6-deoxy-α-l-talopyranose. Gwanakoside A displayed inhibitory activity against pathogenic bacteria, including Staphylococcus aureus (MIC = 8 µg/mL) and Mycobacterium tuberculosis (MIC50 = 15 µg/mL), and antiproliferative activity against several human cancer cell lines (IC50 = 5.6-19.4 µM).

Ochraceopetalin, a Mixed-Biogenetic Salt of Polyketide and Amino Acid Origins from a Marine-Derived Aspergillus ochraceopetaliformis Fungus.

Ochraceopetalin (1), a mixed-biogenetic salt compound and its component 2 were isolated from the culture broths of a marine-derived fungus, Aspergillus ochraceopetaliformis. Based on combined spectroscopic and chemical analyses, the structure of 1 was determined to be a sulfonated diphenylether-aminol-amino acid ester guanidinium salt of an unprecedented structural class, while 2 was determined to be the corresponding sulfonated diphenylether. Ochraceopetaguanidine (3), the other guanidine-bearing aminol amino acid ester component, was also prepared and structurally elucidated. Compound 1 exhibited significant cytotoxicity against K562 and A549 cells.

Psammocindoles A-C: Isolation, Synthesis, and Bioactivity of Indole-γ-lactams from the Sponge Psammocinia vermis.

Psammocindoles A-C (1-3), a new class of indole alkaloids, were isolated from a Psammocinia vermis sponge. By combined spectroscopic analyses, the structures of these compounds were determined to be the indole-γ-lactams derived from three amino acid residues. In addition, an enantiomer psammocindole D (4), and the N-lactam isomers isopsammocindoles A-D (5-8) were also synthesized. These natural products and synthetic analogues were found to significantly stimulate adiponectin secretion in human bone marrow mesenchymal stem cells.

Inhibitory Effects of Epipolythiodioxopiperazine Fungal Metabolites on Isocitrate Lyase in the Glyoxylate Cycle of Candida albicans.

Four epipolythiodioxopiperazine fungal metabolites (1-4) isolated from the sponge-derived Aspergillus quadrilineatus FJJ093 were evaluated for their capacity to inhibit isocitrate lyase (ICL) in the glyoxylate cycle of Candida albicans. The structures of these compounds were elucidated using spectroscopic techniques and comparisons with previously reported data. We found secoemestrin C (1) (an epitetrathiodioxopiperazine derivative) to be a potent ICL inhibitor, with an inhibitory concentration of 4.77 ± 0.08 µM. Phenotypic analyses of ICL-deletion mutants via growth assays with acetate as the sole carbon source demonstrated that secoemestrin C (1) inhibited C. albicans ICL. Semi-quantitative reverse-transcription polymerase chain reaction analyses indicated that secoemestrin C (1) inhibits ICL mRNA expression in C. albicans under C2-assimilating conditions.

Antibacterial Activity and Mode of Action of Lactoquinomycin A from Streptomyces bacillaris.

This study aims to isolate and identify the structure of antibacterial compounds having potent activity on methicillin-resistant Staphylococcus aureus (MRSA) from marine actinomycetes, and also to identify their mode of action. Lactoquinomycin A (LQM-A) (compound 1) and its derivatives (2-4) were isolated from marine-derived Streptomyces bacillaris strain MBTC38, and their structures were determined using extensive spectroscopic methods. These compounds showed potent antibacterial activities against Gram-positive bacteria, with MIC values of 0.06-4 µg/mL. However, the tested compounds exhibited weak inhibitory activity against Gram-negative bacteria, although they were effective against Salmonella enterica (MIC = 0.03-1 µg/mL). LQM-A exhibited the most significant inhibitory activity against methicillin-resistant Staphylococcus aureus (MRSA) (MIC = 0.25-0.5 µg/mL), with a low incidence of resistance. An in vivo dual-reporter assay designed to distinguish between compounds that inhibit translation and those that induce DNA damage was employed to assess the mode of action of LQM-A. LQM-A-induced DNA damage and did not inhibit protein synthesis. The gel mobility shift assay showed that LQM-A switched plasmid DNA from the supercoiled to relaxed form in a time- and concentration-dependent manner. These data suggest that LQM-A intercalated into double-stranded DNA and damaged DNA repair.

Bioactive Bis(indole) Alkaloids from a Spongosorites sp. Sponge.

Six new bis(indole) alkaloids (1-6) along with eight known ones of the topsentin class were isolated from a Spongosorites sp. sponge of Korea. Based on the results of combined spectroscopic analyses, the structures of spongosoritins A-D (1-4) were determined to possess a 2-methoxy-1-imidazole-5-one core connecting the indole moieties, and these were linked by a linear urea bridge for spongocarbamides A (5) and B (6). The absolute configurations of spongosoritins were assigned by electronic circular dichroism (ECD) computation. The new compounds exhibited moderate inhibition against transpeptidase sortase A and weak inhibition against human pathogenic bacteria and A549 and K562 cancer cell lines.

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.

Antibacterial Activity of Chromomycins from a Marine-Derived Streptomyces microflavus.

A marine-derived actinomycete (Streptomyces sp. MBTI36) exhibiting antibacterial activities was investigated in the present study. The strain was identified using genetic techniques. The 16S rDNA sequence of the isolate indicated that it was most closely related to Streptomyces microflavus. Furthermore, a new chromomycin A9 (1), along with chromomycin Ap (2), chromomycin A2 (3), and chromomycin A3 (4), were isolated from the ethyl acetate extract. Their structures were determined using extensive spectroscopic methods including 1D and 2D NMR, and HRMS, as well as comparisons with previously reported data. Compounds 1-4 showed potent antibacterial activities against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). During a passage experiment, minimum inhibitory concentration (MIC) values for compounds 1-4 showed no more than a 4-fold increase from the starting MIC value, indicating that no resistance was detected over the 21 passages.

Sortase A-Inhibitory Coumarins from the Folk Medicinal Plant Poncirus trifoliata.

Thirteen coumarins (1-13), including five new compounds (1-5), were isolated from the folk medicinal plant Poncirus trifoliata. Combined spectroscopic analyses revealed that coumarins 1-4 are bis-isoprenylated coumarins with diverse oxidation patterns, while 5 is an enantiomeric di-isoprenylated coumarin. The absolute configurations of the stereogenic centers in the isoprenyl chains were assigned through MTPA and MPA methods, and those of the known compounds triphasiol (6) and ponciol (7) were also assigned using similar methods. These coumarins inhibited significantly Staphylococcus aureus-derived sortase A (SrtA), a transpeptidase responsible for anchoring surface proteins to the peptidoglycan cell wall in Gram-positive bacteria. The present results obtained indicated that the bioactivity and underlying mechanism of action of these coumarins are associated with the inhibition of SrtA-mediated S. aureus adhesion to eukaryotic cell matrix proteins including fibrinogen and fibronectin, thus potentially serving as SrtA inhibitors.

Bioactive Bianthraquinones and Meroterpenoids from a Marine-Derived Stemphylium sp. Fungus.

Three new bianthraquinones, alterporriol Z1-Z3 (1-3), along with three known compounds of the same structural class, were isolated from the culture broth of a marine-derived Stemphylium sp. fungus. Based upon the results of spectroscopic analyses and ECD measurements, the structures of new compounds were determined to be the 6-6'- (1 and 2) and 1-5'- (3) C-C connected pseudo-dimeric anthraquinones, respectively. Three new meroterpenoids, tricycloalterfurenes E-G (7-9), isolated together with the bianthraquinones from the same fungal culture broth, were structurally elucidated by combined spectroscopic methods. The relative and absolute configurations of these meroterpenoids were determined by modified Mosher's, phenylglycine methyl ester (PGME), and computational methods. The bianthraquinones significantly inhibited nitric oxide (NO) production and suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW 264.7 cells.