Discovery of P450-Modified Sesquiterpenoids Levinoids A-D through Global Genome Mining.

Cytochrome P450-modified bacterial terpenoids remain in a vast chemical space to be explored. In the present study, we conducted global genome mining of 223,829 bacterial genomes and identified 2892 bacterial terpenoid biosynthetic gene clusters (BGCs) with cytochrome P450 genes. Among these, we selected 562 with multiple P450 enzymes, which were further clustered as 355 gene cluster families by sequence similarity analysis. We then chose lev, a BGC from Streptomyces levis MCCC1A01616, for heterologous expression and discovered four new α-amorphene-type sesquiterpenoids, levinoids A-D (1-4). The structures and absolute configurations of these four new compounds were determined by employing extensive NMR analysis, NMR chemical shift calculations with DP4+, and ECD calculations. Furthermore, levinoid C (3) exhibited a moderate level of neuroprotective activity (EC50 = 21 µM) in the glutamate-induced excitotoxicity cell model. Our findings highlight the untapped chemical diversity of P450-modified bacterial terpenoids, opening new avenues for further exploration and discovery.

Mechanistic Insight into the Inhibitory Activity of Elasnin-Based Coating against Early Marine Biofilms.

Marine biofilms are multispecies microbial communities on surfaces that are crucial to the marine environment. They cause marine corrosion, biofouling, and transmission of marine pathogens and thus pose a great threat to public health and the maritime industry. To control marine biofilms, effective and environmentally friendly antibiofilm compounds are highly needed. Elasnin is a potent antibiofilm compound that exhibits high efficiency in inhibiting marine biofilms and biofouling, but its mode of action remains unclear. In the present study, multiomic analysis combined with quorum-sensing assays and in silico study revealed that elasnin acted as a signaling molecule in the microbial community. Elasnin promoted the growth of dominant species in the biofilm but deprived their ability of sensing and responding to environmental changes by disturbing their regulations of the two-component system, i.e., the ATP-binding cassette transport system and the bacterial secretion system. Consequently, biofilm maturation and subsequent biofouler settlement were inhibited. Elasnin also exhibited higher antibiofilm efficiency than dichlorooctylisothiazolinone and had low toxicity potential on the embryos and adults of marine medaka fish. Overall, this study provided molecular and ecological insights into elasnin's mode of action, highlighting its application potential in controlling marine biofilms and the feasibility and advantages of using signal molecules to develop eco-friendly technologies.

Bathiapeptides: Polythiazole-Containing Peptides from a Marine Biofilm-Derived Bacillus sp.

Bacteria in marine biofilms are a rich reservoir of natural products. To facilitate novel secondary metabolite discovery, we investigated the metabolic profile of a marine biofilm-derived Bacillus sp. B19-2 by combining bioinformatics and LC-UV-MS analyses. After dereplication and purification of putatively unknown compounds, a new family of compounds 1-8 was uncovered and named bathiapeptides. Structural elucidation using NMR, HRESIMS, ozonolysis, advanced Marfey's analysis, and X-ray diffraction revealed that bathiapeptides are polypeptides that contain a rare polythiazole moiety. These compounds exhibited strong cytotoxicity against Hep G2, HeLa, MCF-7, and MGC-803 cell lines, and the lowest IC50 value was 0.5 µM. An iterative biosynthesis logic in bathiapeptides' biosynthesis was proposed based on the identified chemical structures and putative gene cluster analysis.

Optimization of an ultrasonic-assisted aqueous two-phase extraction method for four flavonoids from Lysionotus pauciflorus.

An effective approach to extracting four flavonoids (lysioside C, nevadensin-7-sambubioside, ikarisoside B, and nevadensin) from Lysionotus pauciflorus using ultrasonic-assisted aqueous two-phase extraction (UAATPE) technology was studied. Ethanol/K2HPO4 system was selected and the influence of several important parameters, including composition of the aqueous two-phase system (ATPS), extraction temperature and time, particle size, and solvent to material ratio, were investigated by single factor experimentss. Then three key parameters (ethanol concentration, solvent to material ratio and extraction temperature) were further optimized by response surface methodology (RSM). The optimal process was that 1 g 120 mesh herb powders were extracted with 45 g ATPS (made of 30% ethanol/18% K2HPO4) in 43 °C for 30 min, and yields of four flavonoids could reach 2.56, 2.06, 3.62, and 6.28 mg/g, respectively. Compared with ultrasonic-assisted extraction (UAE) in three conventional solvents (water, ethanol and 60% ethanol), the UAATPE displayed comparatively higher extraction capability. Therefore, UAATPE in ethanol/K2HPO4 system could be an alternative technology for integrated extraction of the flavonoids from L. pauciflorus.

Ultrasound-assisted aqueous two-phase extraction of synephrine, naringin, and neohesperidin from Citrus aurantium L. fruitlets.

The ultrasound-assisted aqueous two-phase extraction (UA-ATPE) was first employed to develop an effective technique for simultaneous extraction and preliminary purification of synephrine, naringin, and neohesperidin from Citrus aurantium L. fruitlets. Five types of ethanol/salts of aqueous two-phase system (ATPS) were investigated and then the extraction conditions were further optimized using single-factor experiments and response surface methodology (RSM) via Box-Behnken Design (BBD). The optimum process parameters were concluded as follows: 20.60% (w/w) K2CO3, 27% (w/w) ethanol, solvent-to-material ratio of 45.17:1 (g:g), 120-mesh particle size of fruitlets powder, extraction temperature of 50 °C, extraction time of 30 min, and ultrasonic power of 80 W. Under these conditions, the extraction yields of synephrine, naringin, and neohesperidin were up to 11.17 mg/g, 7.39 mg/g, and 89.27 mg/g, respectively. The yield of neohesperidin extracted by the optimal UA-ATPE was over eight times higher than that extracted by the ultrasound-assisted extraction (UAE) using conventional solvents, and the total yield of target compounds was over twice higher while the impurity content in the extract was much lower. Therefore, UA-ATPE appeared to be a highly effective and promising approach for the extraction of synephrine, naringin, and neohesperidin from C. aurantium fruitlets.

Marine natural products as antifouling molecules - a mini-review (2014-2020).

In the present review, 182 antifouling (AF) natural products from marine microorganisms, algae and marine invertebrates reported from August 2014 to May 2020 are presented. Amongst these compounds, over half were isolated from marine-derived microorganisms, including 70 compounds from fungi and 31 compounds from bacteria. The structure-relationship of some of these compounds is also briefly discussed. Based on the work reported, a general workflow was drafted to refine the procedures for the commercialization of any novel AF compounds. Finally, butenolide, which is considered a potential environmentally friendly antifoulant, is used as a case study to show the procedures involved in AF compound work from the aspect of discovery, structure optimization, toxicity, stability, AF mechanism and coating incorporation, which highlight the current challenges and future perspectives in AF compound research.

Bioactivity-guided identification of flavonoids with cholinesterase and ß-amyloid peptide aggregation inhibitory effects from the seeds of Millettia pachycarpa.

Millettia pachycarpa Benth, a widely used anthelminthic drug in folk, is rich in flavonoids with various bioactivities. This study aimed to identify active flavonoids with anti-Alzheimer's disease (AD) effect from its seeds by a bioassay-guided isolation. A novel rotenoid with unusual oxidative ring-opening skeleton (10) and nine known flavonoids (1-9) were obtained, and their structures were elucidated by NMR and HR-ESIMS analysis. Among all isolates, 7 and 8 showed selective butyrylcholinesterase (BChE) inhibitory activities (IC50 = 2.34 and 11.49 µM, respectively), while 3 was classified as a dual-action inhibitor against acetylcholinesterase (AChE) and BChE (IC50 AChE = 17.14 µM, IC50 BChE = 5.68 µM). Further kinetic study revealed that 3, 7, and 8 were mixed-type BChE inhibitors, but 3 was a competitive AChE inhibitor. Their strong binding affinities to BChE were confirmed by fluorescence quenching analysis. Additionally, 3 and 8 exhibited potent inhibitory effects against ß-amyloid peptide aggregation. These results revealed M. pachycarpa could be a valuable source for anti-AD leads development, and compounds 3, 7 and 8 were worthy of further study as multifunctional or specific agents for AD treatment.

Highly selective carbamate-based butyrylcholinesterase inhibitors derived from a naturally occurring pyranoisoflavone.

This current study described the design and synthesis of a series of derivatives based on a natural pyranoisaflavone, which was obtained from the seeds of Millettia pachycarpa and displayed attractive BChE inhibition and high selectivity in our previous study. The inhibitory potential of all derivatives against two cholinesterases was evaluated. Only a few compounds demonstrated AChE inhibitory activity at the tested concentrations, while 26 compounds showed significant inhibition on BChE (the IC50 values varied from 9.34 µM to 0.093 µM), most of them presented promising selectivity to ward BChE. Prediction of ADME properties for 7 most active compounds was performed. Among them, 9g (IC50 = 222 nM) and 9h (IC50 = 93 nM) were found to be the most potent BChE inhibitors with excellent selectivity over AChE (SI ratio = 1339 and 836, respectively). The kinetic analysis demonstrated both of them acted as mixed-type BChE inhibitors, while the molecular docking results indicated that they interacted with both residues in the catalytic active site. A cytotoxicity test on PC12 cells showed that both 9g and 9h had a therapeutic safety range similar to tacrine. Overall, the results indicate that 9h could be a good candidate of BChE inhibitors.

Two new stilbenoids from Bletilla striata.

Two new stilbenoids bletilol D (1) and bletilol E (2), together with five known compounds were isolated from Bletilla striata. Three of them (3, 4, and 7) were obtained from this genus for the first time. Their structures were elucidated by spectroscopic methods and comparing with data reported in literatures. The cytotoxic activities of compounds 1-7 against MCF-7 (human breast cancer) and A549 (human lung carcinoma) cell lines were evaluated by MTT assay. Compound 2 showed weak cytotoxicity against MCF-7 and A549 cell lines with IC50 values of 36.32 ± 1.17 and 36.48 ± 1.12 µM, respectively, and 5 exhibited weak cytotoxicity against MCF-7 cell line with IC50 value of 57.09 ± 2.03 µM.

Abi Family Protein, DidK, Is Involved in the Maturation of Anticancer Depsipeptide, Didemnin B.

Didemnin B is a marine-derived depsipeptide with potent antiviral and anticancer activities. A prodrug activation mechanism was previously proposed for the biosynthesis of didemnin B by the nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly line, but the enzyme involved in the maturation process remained unknown. Herein, we demonstrated that DidA, a dimodular NRPS predicted with unrelated activity to didemnin B structure assembly, was indispensable to produce didemnin B, confirming the prodrug mechanism in didemnin B biosynthesis. We further identified an Abi family transmembrane protease, DidK, that functioned as an esterase in the maturation step of didemnin B by in vivo gene knockout and heterologous expression. DidK is structurally distinct from other known hydrolytic enzymes involved in the maturation of bacterial nonribosomal peptides and is the first Abi family protein known to participate in NRPS/PKS-derived natural product production. Further bioinformatic analysis revealed more than 20 DidK homologues encoded in bacterial NRPS/PKS BGCs, suggesting that the involvement of Abi family proteins in natural product biosynthesis might not be rare. These results not only clarify the priming and maturation steps of didemnin B biosynthesis but also expand the function scope of Abi family proteins.

Pterosin sesquiterpenoids from Pteris laeta Wall. ex Ettingsh. protect cells from glutamate excitotoxicity by modulating mitochondrial signals.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Pteris (Pteridaceae) has been used as a traditional herb for a long time. In particular, Pteris laeta Wall. ex Ettingsh. has been widely used in traditional Chinese medicine to treat nervous system diseases and some pterosin sesquiterpenes from Pteris show neuroprotective activity, but their underlying molecular mechanisms remain elusive. Therefore, to investigate the neuroprotective activity and working mechanism of pterosin sesquiterpenes from P. laeta Wall. ex Ettingsh. will provide a better understanding and guidance in using P. laeta Wall. ex Ettingsh. as a traditional Chinese medicine. AIM OF THE STUDY: We aim to develop effective treatments for neurodegenerative diseases from pterosin sesquiterpenes by evaluating their neuroprotective activity and investigating their working mechanisms. MATERIALS AND METHODS: Primary screening on the glutamate-induced excitotoxicity cell model was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Fluorescent-activated cell sorting (FACS) was used to analyze the activation level of glutamate receptors and mitochondria membrane potential after treatment. Transcriptomics and proteomics analysis was performed to identify possible targets of pterosin B. The key pathways were enriched by the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis through the Database for Annotation, Visualization, and Integrated Discovery (DAVID). The core targets were visualized by a protein-protein interaction network using STRING. The mRNA and protein expressions were evaluated using real-time quantitative polymerase chain reaction (Q-PCR) and western blot, respectively. Immunocytochemistry was performed to monitor mitochondrial and apoptotic proteins. Cellular reactive oxygen species (ROS) were measured by ROS assay, and Ca2+ was stained with Fluo-4 AM to quantify intracellular Ca2+ levels. RESULTS: We found pterosin B from Pteris laeta Wall. ex Ettingsh. showed significant neuroprotective activity against glutamate excitotoxicity, enhancing cell viability from 43.8% to 105% (p-value: <0.0001). We demonstrated that pterosin B worked on the downstream signaling pathways of glutamate excitotoxicity rather than directly blocking the activation of glutamate receptors. Pterosin B restored mitochondria membrane potentials, alleviated intracellular calcium overload from 107.4% to 95.47% (p-value: 0.0006), eliminated cellular ROS by 36.55% (p-value: 0.0143), and partially secured cells from LPS-induced inflammation by increasing cell survival from 46.75% to 58.5% (p-value: 0.0114). Notably, pterosin B enhanced the expression of nuclear factor-erythroid factor 2-related factor 2 (NRF2) and heme oxygenase-1 (HO-1) by 2.86-fold (p-value: 0.0006) and 4.24-fold (p-value: 0.0012), and down-regulated Kelch-like ECH-associated protein 1 (KEAP1) expression by 2.5-fold (p-value: 0.0107), indicating that it possibly promotes mitochondrial biogenesis and mitophagy to maintain mitochondria quality control and homeostasis, and ultimately inhibits apoptotic cell death. CONCLUSIONS: Our work revealed that pterosin B protected cells from glutamate excitotoxicity by targeting the downstream mitochondrial signals, making it a valuable candidate for developing potential therapeutic agents in treating neurodegenerative diseases.

Biological evaluation, molecular modeling and dynamics simulation of phenanthrenes isolated from Bletilla striata as butyrylcholinesterase inhibitors.

As part of our continuous studies on natural cholinesterase inhibitors from plant kingdom, the 95% ethanol extract from tubers of Bletilla striata showed promising butyrylcholinesterase (BChE) inhibition (IC50 = 8.6 µg/mL). The extracts with different polarities (petroleum ether, ethyl acetate, n-butanol, and water) were prepared and evaluated for their inhibition of cholinesterases. The most active ethyl acetate extract was subjected to a bioassay-guided isolation and afforded twenty-two bibenzyls and phenanthrenes (1-22). All isolates were further evaluated for their BChE inhibition activity, and five phenanthrenes presented promising capacity (IC50 < 10 µM). Further kinetic studies indicated their modes of inhibition. Compounds 6, 8, and 14 were found to be mixed-type inhibitors, while compounds 10 and 12 could be classified as non-competitive inhibitors. The potential interaction mechanism of them with BChE was demonstrated by molecular docking and molecular dynamics simulation, showing that they could interact with catalytic active site and peripheral anionic site of BChE. These natural phenanthrenes provide new scaffold for the further design and optimization, with the aim to discover new selective BChE inhibitors for the treatment of AD.

Griseocazines: Neuroprotective Multiprenylated Cyclodipeptides Identified through Targeted Genome Mining.

Prenylation can impart pharmacological advantages to bioactive compounds. Global genome mining for prenylated cyclodipeptides identified a gczABC BGC from Streptomyces griseocarneus 132 containing a cyclodipeptide synthase and two prenyltransferase genes. Subsequent heterologous expression allowed isolation and characterization of griseocazines, which displayed potent neuroprotective activity. Further biotransformation analyses revealed that prenyltransferases GczB and GczC catalyzed the stereospecific prenylation of cWW and attached geranyl and farnesyl groups to a cyclodipeptide scaffold, respectively.

Global Genome Mining Reveals the Distribution of Diverse Thioamidated RiPP Biosynthesis Gene Clusters.

Thioamidated ribosomally synthesized and post-translationally modified peptides (RiPPs) are recently characterized natural products with wide range of potent bioactivities, such as antibiotic, antiproliferative, and cytotoxic activities. These peptides are distinguished by the presence of thioamide bonds in the peptide backbone catalyzed by the YcaO-TfuA protein pair with its genes adjacent to each other. Genome mining has facilitated an in silico approach to identify biosynthesis gene clusters (BGCs) responsible for thioamidated RiPP production. In this work, publicly available genomic data was used to detect and illustrate the diversity of putative BGCs encoding for thioamidated RiPPs. AntiSMASH and RiPPER analysis identified 613 unique TfuA-related gene cluster families (GCFs) and 797 precursor peptide families, even on phyla where the presence of these clusters have not been previously described. Several additional biosynthesis genes are colocalized with the detected BGCs, suggesting an array of possible chemical modifications. This study shows that thioamidated RiPPs occupy a widely unexplored chemical landscape.

Neolignans and Diarylheptanoids with Anti-Inflammatory Activity from the Rhizomes of Alpinia zerumbet.

The dried leaves and rhizomes of Alpinia zerumbet have been traditionally used as food and medicine. Anti-inflammatory activity-guided phytochemical investigation into the rhizomes of A. zerumbet led to the isolation of 17 compounds including 10 neolignans (1-10, 1a, 1b, 2a, 2b, 3a, 3b, 4, and 5 are new compounds) and seven diarylheptanoids (11-17) in which 1-3 were three pairs of enantiomers. 4 was only one enantiomer and 5 was a racemic mixture. Compounds 1a, 1b, 2a, and 2b incorporated an 8',9'-dinorneolignan skeleton, which was rare in the lignan family. The planar structures of these compounds were elucidated by extensive analyses of spectroscopic data. The relative and absolute configurations were determined by the time-dependent density functional theory (TDDFT)-based electronic circular dichroism (ECD) calculation method. The 95% ethanol extract and ethyl acetate extract of A. zerumbet were found to show anti-inflammatory activity against croton oil-induced ear edema in mice with inhibition rates of 20.0 and 47.6% at a dose of 80 mg/kg, respectively. Bioassays showed that compounds 1a, 1b, 2a, 2b, and 12 moderately inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-induced RAW264.7 cells with IC50 values of 3.62, 7.63, 6.51, 5.60, and 8.33 µM, respectively.

Novel nortriterpenoids with new skeletons and limonoids from the fruits of Evodia rutaecarpa and their bioactivities.

Two novel nortriterpenoids together with 7 known compounds were isolated from the fruits of Evodia rutaecarpa. The structures of the new compounds were elucidated by spectroscopic analysis, X-ray, and electronic circular dichroism (ECD) calculations. Compound 1 is the first example of triterpenoid with a 27 (17 â†’ 12)-abeo-five-ring skeleton. In turn, compound 2 possesses a unique C/D/E linear fused ring system and a methyl on C-21. Plausible biogenetic pathway for the new compounds 1 and 2 are also proposed. Compound 1 exhibited significantly antitumor activity against A549 and LoVo cells with IC50 values of 2.0 µM and 1.9 µM, respectively. Colony formation inhibition, cell cycle arrest and cell apoptosis of compound 1 were also evaluated. Compound 2, 6, 7 and 9 showed potent neuroprotective activities against serum-deprivation induced P12 cell damage.