Goondansamycins A-H: Benzenoid Ansamycins from an Australian Volcanic Crater Soil-Derived Actinomadura sp. S4S-00069B08.

A chemical investigation of Australian soil-derived bacteria Actinomadura sp. S4S-00069B08 yielded eight new benzenoid ansamycins, goondansamycins A-H. Goondansamycins feature rare 1,4-benzoxazin-3-one or o-diamino-p-benzoquinone moieties and can exist as both aglycones or 9-O-α-glycosides of either d-rhodinose or d-amicetose. Structures were solved on the basis of detailed spectroscopy, including X-ray analysis.

Anti-mycobacterial natural products and mechanisms of action.

Covering: up to June, 2020Tuberculosis (TB) continues to be a major disease with high mortality and morbidity globally. Drug resistance and long duration of treatment make antituberculosis drug discovery more challenging. In this review, we summarize recent advances on anti-TB natural products (NPs) and their potential molecular targets in cell wall synthesis, protein production, energy generation, nucleic acid synthesis and other emerging areas. We highlight compounds with activity against drug-resistant TB, and reveal several novel targets including Mtb biotin synthase, ATP synthase, 1,4-dihydroxy-2-naphthoate prenyltransferase and biofilms. These anti-TB NPs and their targets could facilitate target-based screening and accelerate TB drug discovery.

Genome-guided investigation of anti-inflammatory sesterterpenoids with 5-15 trans-fused ring system from phytopathogenic fungi.

Fungal terpenoids catalyzed by bifunctional terpene synthases (BFTSs) possess interesting bioactive and chemical properties. In this study, an integrated approach of genome mining, heterologous expression, and in vitro enzymatic activity assay was used, and these identified a unique BFTS sub-clade critical to the formation of a 5-15 trans-fused bicyclic sesterterpene preterpestacin I (1). The 5-15 bicyclic BFTS gene clusters were highly conserved but showed relatively wide phylogenetic distribution across several species of the diverged fungal classes Dothideomycetes and Sordariomycetes. Further genomic organization analysis of these homologous biosynthetic gene clusters from this clade revealed a glycosyltransferase from the graminaceous pathogen Bipolaris sorokiniana isolate BS11134, which was absent in other 5-15 bicyclic BFTS gene clusters. Targeted isolation guided by BFTS gene deletion led to the identification of two new sesterterpenoids (4, and 6) from BS11134. Compounds 2 and 4 showed moderate effects on LPS-induced nitrous oxide production in the murine macrophage-like cell line RAW264.7 with in vitro inhibition rates of 36.6 ± 2.4% and 24.9 ± 2.1% at 10 µM, respectively. The plausible biosynthetic pathway of these identified compounds was proposed as well. This work revealed that phytopathogenic fungi can serve as important sources of active terpenoids via systematic analysis of the genomic organization of BFTS biosynthetic gene clusters, their phylogenetic distribution in fungi, and cyclization properties of their metabolic products. KEY POINTS: • Genome mining of the first BFTS BGC harboring a glycosyltransferase. • Gene-deletion guided isolation revealed three novel 5-15 bicyclic sesterterpenoids. • Biosynthetic pathway of isolated sesterterpenoids was proposed.

Peculiarities of meroterpenoids and their bioproduction.

Meroterpenoids are a class of terpenoid-containing hybrid natural products with impressive structural architectures and remarkable pharmacological activities. Remarkable advances in enzymology and synthetic biology have greatly contributed to the elucidation of the molecular basis for their biosynthesis. Here, we review structurally unique meroterpenoids catalyzed by novel enzymes and unusual enzymatic reactions over the period of last 5 years. We also discuss recent progress on the biomimetic synthesis of chrome meroterpenoids and synthetic biology-driven biomanufacturing of tropolone sesquiterpenoids, merochlorins, and plant-derived meroterpenoid cannabinoids. In particular, we focus on the novel enzymes involved in the biosynthesis of polyketide-terpenoids, nonribosomal peptide-terpenoids, terpenoid alkaloids, and meroterpenoid with unique structures. The biological activities of these meroterpenoids are also discussed. The information reviewed here might provide useful clues and lay the foundation for developing new meroterpenoid-derived drugs. KEY POINTS: • Meroterpenoids possess intriguing structural features and relevant biological activities. • Novel enzymes are involved in the biosynthesis of meroterpenoids with unique structures. • Biomimetic synthesis and synthetic biology enable the construction and manufacturing of complex meroterpenoids.

Antimicrobial Benzyltetrahydroisoquinoline-Derived Alkaloids from the Leaves of Doryphora aromatica.

Four new alkaloids, (R)-nomimantharine trifluoroacetate (2), 12-demethylphaeantharine trifluoroacetate (3), nominanthranal trifluoroacetate (4), and the enolic form of 1-hydroxy-6,7-dimethoxy-2-methylisoquinoline trifluoroacetate (5), together with the known dimeric alkaloid phaeantharine trifluoroacetate (1), have been isolated from the extract of the leaves of the rainforest tree Doryphora aromatica (Monimiaceae). The structures of these compounds were elucidated by HRMS and 1D and 2D NMR data. (R)-Nomimantharine trifluoroacetate (2) contains an ether linkage connecting a benzylisoquinoline unit with a tetrahydroisoquinoline, a novel class of dimeric alkaloid. The absolute configuration of (R)-nomimantharine trifluoroacetate (2) was established via electronic circular dichroism data. The compounds isolated were subjected to in vitro antimicrobial assays against a panel of pathogenic microorganisms, including Mycobacterium smegmatis, M. tuberculosis, Escherichia coli, Staphylococcus aureus (SA), and five clinical isolates of oxacillin/methicillin-resistant S. aureus (MRSA). Phaeantharine trifluoroacetate (1) and (R)-nomimantharine trifluoroacetate (2) showed moderate inhibitory activities against Mycobacteria and MRSA strains.

Chemical constituents from Macleaya cordata (Willd) R. Br. and their phenotypic functions against a Parkinson's disease patient-derived cell line.

Cytological profiling (CP) assay against a human olfactory neuroshpere-derived (hONS) cell line using a library of traditional Chinese medicinal plant extracts gave indications that the ethanolic extract of Macleaya cordata (Willd) R. Br. elicited strong perturbations to various cellular components. Further chemical investigation of this extract resulted in the isolation of two new benzo[c]phenanthridine alkaloids, (6R)-10-methoxybocconoline (1) and 6-(1-hydroxyethyl)-10-methoxy-5,6-dihydrochelerythrine (2). Their planar structures were elucidated by extensive 1D and 2D NMR studies, together with MS data. The absolute configuration for position C-6 of 1 and relative configurations for position C-6 and C-1' of 2 were assigned by density functional theory (DFT) calculations of ECD and NMR data, respectively. Also isolated were fourteen known metabolites, including ten alkaloids (3-12) and four coumaroyl-containing compounds (13-16). Cytological profiling of the isolates against Parkinson's Disease (PD) patient-derived olfactory cells revealed bocconoline (3) and 6-(1-hydroxyethyl)-5,6-dihydrochelerythrine (4) significantly perturbated the features of cellular organelles including early endosomes, mitochondria and autophagosomes. Given that hONS cells from PD patients model some functional aspects of the disease, the results suggested that these phenotypic profiles may have a role in the mechanisms underlying PD and signified the efficacy of CP in finding potential chemical tools to study the biological pathways in PD.

Genome-Inspired Chemical Exploration of Marine Fungus Aspergillus fumigatus MF071.

The marine-derived fungus Aspergillus fumigatus MF071, isolated from sediment collected from the Bohai Sea, China, yielded two new compounds 19S,20-epoxy-18-oxotryprostatin A (1) and 20-hydroxy-18-oxotryprostatin A (2), in addition to 28 known compounds (3-30). The chemical structures were established on the basis of 1D, 2D NMR and HRESIMS spectroscopic data. This is the first report on NMR data of monomethylsulochrin-4-sulphate (4) and pseurotin H (10) as naturally occurring compounds. Compounds 15, 16, 20, 23, and 30 displayed weak antibacterial activity (minimum inhibitory concentration: 100 µg/mL). Compounds 18 and 19 exhibited strong activity against S. aureus (minimum inhibitory concentration: 6.25 and 3.13 µg/mL, respectively) and E. coli (minimum inhibitory concentration: 6.25 and 3.13 µg/mL, respectively). A genomic data analysis revealed the putative biosynthetic gene clusters ftm for fumitremorgins, pso for pseurotins, fga for fumigaclavines, and hel for helvolinic acid. These putative biosynthetic gene clusters fundamentally underpinned the enzymatic and mechanistic function study for the biosynthesis of these compounds. The current study reported two new compounds and biosynthetic gene clusters of fumitremorgins, pseurotins, fumigaclavines and helvolinic acid from Aspergillus fumigatus MF071.

Discovery of a Natural Product That Binds to the Mycobacterium tuberculosis Protein Rv1466 Using Native Mass Spectrometry.

Elucidation of the mechanism of action of compounds with cellular bioactivity is important for progressing compounds into future drug development. In recent years, phenotype-based drug discovery has become the dominant approach to drug discovery over target-based drug discovery, which relies on the knowledge of a specific drug target of a disease. Still, when targeting an infectious disease via a high throughput phenotypic assay it is highly advantageous to identifying the compound's cellular activity. A fraction derived from the plant Polyalthia sp. showed activity against Mycobacterium tuberculosis at 62.5 µge/µL. A known compound, altholactone, was identified from this fraction that showed activity towards M. tuberculosis at an minimum inhibitory concentration (MIC) of 64 µM. Retrospective analysis of a target-based screen against a TB proteome panel using native mass spectrometry established that the active fraction was bound to the mycobacterial protein Rv1466 with an estimated pseudo-Kd of 42.0 ± 6.1 µM. Our findings established Rv1466 as the potential molecular target of altholactone, which is responsible for the observed in vivo toxicity towards M. tuberculosis.

Genome-based mining of new antimicrobial meroterpenoids from the phytopathogenic fungus Bipolaris sorokiniana strain 11134.

Polyketide-terpenoid hybrid compounds are one of the largest families of meroterpenoids, with great potential for drug development for resistant pathogens. Genome sequence analysis of secondary metabolite gene clusters of a phytopathogenic fungus, Bipolaris sorokiniana 11134, revealed a type I polyketide gene cluster, consisting of highly reducing polyketide synthase, non-reducing polyketide synthase, and adjacent prenyltransferase. MS- and UV-guided isolations led to the isolation of ten meroterpenoids, including two new compounds: 19-dehydroxyl-3-epi-arthripenoid A (1) and 12-keto-cochlioquinone A (2). The structures of 1-10 were elucidated by the analysis of NMR and high-resolution electrospray ionization mass spectroscopy data. Compounds 5-8 and 10 showed moderate activity against common Staphylococcus aureus and methicillin-resistant S. aureus, with minimum inhibitory concentration (MIC) values of 12.5-100 µg/mL. Compound 5 also exhibited activity against four clinical resistant S. aureus strains and synergistic antifungal activity against Candida albicans with MIC values of 12.5-25 µg/mL. The biosynthetic gene cluster of the isolated compounds and their putative biosynthetic pathway are also proposed. KEY POINTS: • Ten meroterpenoids were identified from B. sorokiniana, including two new compounds. • Cochlioquinone B (5) showed activity against MRSA and synergistic activity against C. albicans. • The biosynthetic gene cluster and biosynthetic pathway of meroterpenoids are proposed. • Genome mining provided a new direction to uncover the diversity of meroterpenoids.

Brocaeloid D, a novel compound isolated from a wheat pathogenic fungus, Microdochium majus 99049.

Microbes serve as the most important resource for drug discovery. During our screening for bioactive compounds from our natural products library, a pathogenic fungus, Microdochium majus strain 99049, from wheat was selected for further investigation. A new alkaloid named brocaeloid D (1), together with six previously characterized compounds (2-7) were identified. Compound 1 belongs to 4-oxoquinoline with C-2 reversed prenylation and a succinimide substructure. All the structures of these newly isolated compounds were determined by different means in spectroscopic experiments. The absolute configurations of 1 was further deduced from comparison of its CD spectrum with that of known compound 2. The bioactivities of these identified compounds were evaluated against several pathogenic microorganisms and cancer cell lines. Compounds 1-5 showed activity against HUH-7 human hepatoma cells with IC50 values of 80 µg/mL. Compound 6 showed mild activity against HeLa cells (IC50 = 51.9 µg/mL), weak anti-MTB activity (MIC = 80  µg/mL), and moderate anti-MRSA activity (MIC = 25 µg/mL), and compound 7 showed weak anti-MRSA activity (MIC = 100 µg/mL).

Genome- and MS-based mining of antibacterial chlorinated chromones and xanthones from the phytopathogenic fungus Bipolaris sorokiniana strain 11134.

Halogen substituents are important for biological activity in many compounds. Genome-based mining of halogenase along with its biosynthetic gene cluster provided an efficient approach for the discovery of naturally occurring organohalogen compounds. Analysis of the genome sequence of a phytopathogenic fungus Bipolaris sorokiniana 11134 revealed a polyketide gene cluster adjacent to a flavin-dependent halogenase capable of encoding halogenated polyketides, which are rarely reported in phytopathogenic fungi. Furthermore, MS- and UV-guided isolation and purification led to the identification of five chlorine-containing natural products together with seven other chromones and xanthones. Two of the chlorinated compounds and four chromones are new compounds. Their structures were elucidated by NMR spectroscopic analysis and HRESIMS data. The biosynthetic gene clusters of isolated compounds and their putative biosynthetic pathway are also proposed. One new chlorinated compound showed activity against Staphylococcus aureus, methicillin-resistant S. aureus, and three clinical-resistant S. aureus strains with a shared minimum inhibitory concentration (MIC) of 12.5 µg/mL. Genome-based mining of halogenases combined with high-resolution MS- and UV-guided identification provides an efficient approach to discover new halogenated natural products from microorganisms.

A systems approach using OSMAC, Log P and NMR fingerprinting: An approach to novelty.

The growing number of sequenced microbial genomes has revealed a remarkably large number of secondary metabolite biosynthetic clusters for which the compounds are still unknown. The aim of the present work was to apply a strategy to detect newly induced natural products by cultivating microorganisms in different fermentation conditions. The metabolomic analysis of 4160 fractions generated from 13 actinomycetes under 32 different culture conditions was carried out by 1H NMR spectroscopy and multivariate analysis. The principal component analysis (PCA) of the 1H NMR spectra showed a clear discrimination between those samples within PC1 and PC2. The fractions with induced metabolites that are only produced under specific growth conditions was identified by PCA analysis. This method allows an efficient differentiation within a large dataset with only one fractionation step. This work demonstrates the potential of NMR spectroscopy in combination with metabolomic data analysis for the screening of large sets of fractions.

Fungal biotransformation of tanshinone results in [4+2] cycloaddition with sorbicillinol: evidence for enzyme catalysis and increased antibacterial activity.

The biotransformation of tanshinone IIA to a new antibacterial agent tanshisorbicin (1) by the fungus Hypocrea sp. (AS 3.17108) is described. The structure of tanshisorbicin is a hybrid of tanshinone IIA (2) and sorbicillinol (3). The latter is a metabolite produced by Hypocrea sp. The structure of tanshisorbicin was determined using mass spectrometry, NMR spectroscopy, and ECD calculations. The anti-MRSA activity of 1 was found to be significantly higher than that of the parent substrate Tan IIA. Preliminary experiments indicate that tanshisorbicin is formed via a [4+2] cycloaddition reaction that is likely catalyzed by microbial enzyme.

Anti-MRSA and anti-TB metabolites from marine-derived Verrucosispora sp. MS100047.

Microbes belonging to the genus Verrucosispora possess significant chemical diversity and biological properties. They have attracted the interests of many researchers and are becoming promising resources in the marine natural product research field. A bioassay-guided isolation from the crude extract of Verrucosispora sp. strain MS100047, isolated from sediments collected from the South China Sea, has led to the identification of a new salicylic derivative, glycerol 1-hydroxy-2,5-dimethyl benzoate (1), along with three known compounds, brevianamide F (2), abyssomicin B (3), and proximicin B (4). Compound 1 showed selective activity against methicillin-resistant Staphylococcus aureus (MRSA) with a minimum inhibitory concentration (MIC) value of 12.5 µg/mL. Brevianamide F (2), which was isolated from actinomycete for the first time, showed a good anti-BCG activity with a MIC value of 12.5 µg/mL that has not been reported previously in literatures. Proximicin B (4) showed significant anti-MRSA (MIC = 3.125 µg/mL), anti-BCG (MIC = 6.25 µg/mL), and anti-tuberculosis (TB) (MIC = 25 µg/mL) activities. This is the first report on the anti-tubercular activities of proximicins. In addition, Verrucosispora sp. strain MS100047 was found to harbor 18 putative secondary metabolite gene clusters based on genomic sequence analysis. These include the biosynthetic loci encoding polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) consistent with abyssomicins and proximicins, respectively. The biosynthetic pathways of these isolated compounds have been proposed. These results indicate that MS100047 possesses a great potential as a source of active secondary metabolites.

Drinking water: a risk factor for high incidence of esophageal cancer in Anyang, China.

Anyang is known to be a high-incidence area of esophageal cancer (EC) in China. Among a long list of risk factors, the quality of drinking water was evaluated. We have selected 3806 individuals and collected 550 drinking water samples correspondent with this not-matched case-control survey. There are 531 EC patients included based on Population Cancer Registry from 92 townships, of which 3275 controls with long-lived aged over 90 years and free from EC are used as controls in the same regions. Our result suggests that the quality of drinking water is a highly associated risk factor for EC. The residential ecological environment and the quality of water resource positively link with each other. The analysis of water samples also demonstrated that the concentrations of methyl ethylamine, morpholine, N-methylbenzylamine, nitrate and chloride in water from springs and rivers are higher than those in well and tap water (P = 0.001). Micronuclei formation tests show that well water and tap water in these regions have no mutagenicity.

Discovery of tanshinone derivatives with anti-MRSA activity via targeted bio-transformation.

Two potent anti-MRSA tanshinone glycosides (1 and 2) were discovered by targeted microbial biotransformation, along with rapid identification via MS/MS networking. Serial reactions including dehydrogenation, demethylations, reduction, glycosylation and methylation have been observed after incubation of tanshinone IIA and fungus Mucor rouxianus AS 3.3447. In addition, tanshinosides B (2) showed potent activities against serial clinical isolates of oxacillin-resistant Staphylococcus aureus with MIC values of 0.78 µg/mL. This is the first study that shows a significant increase in the level and activities of tanshinone glycosides relative to the substrate tanshinone IIA.

Extraction methods of natural products from Traditional Chinese medicines.

In recent years, many research activities have focused on Natural Products (NPs) derived from Traditional Chinese medicines (TCMs), thus making a renaissance in the drug discovery process of TCMs. Maximizing the diversity of extracts from those plants is the key for the chemical biology process. Methods for the preparation and pretreatment of plant extracts are very important for further purification and discovery of active compounds present in minor quantities. In this chapter, two methods of extraction, including one of the most broadly applicable method (solvent extraction) and one newly developed technique (supercritical fluid extraction), have been described in detail.

Bioassay-guided identification of bioactive molecules from traditional Chinese medicines.

Traditional Chinese medicines (TCMs) serve as a major source of a variety of drug lead compounds. In the process of natural products development, bioassay-guided isolation is a rapid and validated method for isolation of compounds with bioactivities. This chapter describes bioassay-guided separation and purification of compounds from the crude extracts of TCMs. Two approaches including size-exclusion chromatography (SEC) and high performance liquid chromatography (HPLC) are described in detail.