Bioactive Components Isolated From Cyathus And Qinling-mountain-derived Streptomyces And Their Preliminary Biosynthesis Study

Owing to diversity of structures,significance of bioactivities,and excellent reproducibility of secondary metabolites,the natural products of microorganisms have been an important source of drugs or lead compounds.In this paper,we aim to discover the active compounds from microbes on the basis of the laboratory research platform.Mainly,the chemical constituents and their bioactivities of Cyathus hookeri and Streptomyces cacaoi subsp.asoensis were studied through multiple chromatographies,including silica gel,reversedphase C18,Sephadex LH-20,HSCCC and HPLC.Derivatives of one pair of tautomers isolated from Cyathus were obtained in order to enrich the chemical diversity.Ten new natural products and fourteen derivatives,together with 24 known compounds were obtained.The structures were elucidated by extensive spectral data?1D/2D NMR,MS,IR,and UV?.The absolute configurations of new compounds were established by CD,ECD,X-ray diffraction and chemical reaction.Various bioactivities of most of compounds were evaluated and their mechanisms of action were also discussed.Besides,the metabolites potential of S.cacaoi subsp.asoensis H2S5 was analyzed based on the genome sequence,and the genetic transformation system of strain H2S5 was explored preliminary.Key results are as follows.1.Based on the chemical epigenetic manipulation strategy,six new cyathane diterpenes,cyahookerin A-F?3-1³-6?,along with 17 known compounds were isolated from the liquid broth of the medicinal fungus Cyathus hookeri Berk CGMCC 5.1116.Compounds 3-1 and 3-2 represent the first unusual cyathane acetals featuring a dioxolane ring.The absolute configurations of new compounds were determined by single-crystal X-ray crystallographic analysis.Compound 3-3 displayed the most potent nerve growth factor?NGF?-induced neurite outgrowth-promoting activity in PC-12 cells with rate of 32.02%at 10?M,which was about twice of the NGF control.Compound 3-15 showed the highest inhibition in H₂O₂-metiated neuronal damage of PC-12 cells with an IC₅₀ value of 7.81?M.Compound 3-9 performed significant nitric oxide?NO?production inhibition in lipopolysaccharide-induced?LPS?-induced BV-2 microglial cells with an IC₅₀ value of 6.9?M.In addition,the structure-activity relationship?SAR?tendency implied the presence of a free aldehyde group or a low polar acetal unit at C-12,a hydrogen at C-13,a hydroxyl group at C-14,and the?,?-unsaturated aldehyde moiety between C-12 and C-13 in the molecules are beneficial to the improvement of anti-neuroinflammatory activity.The activity mechanisms of these compounds were explored preliminarily by molecular docking simulation with iNOS.These bioassay results indicate cyathane diterpenes could be used as leading compounds for the treatment of neurodegenerative diseases,especially 3-3.2.Based on the diversity-oriented synthesis strategy,14 new derivatives of a tautomer 3-16 were obtained.Compound 3-16 showed the higher activities than 12 derivatives in neurite outgrowth activity and anti-neuroinflammatory activity.Detailedly,3-16 showed the promotion rate of 23.65%about NGF-induced PC-12 neurite outgrowth-promoting activity at10?M,which was about 1.5 times of the NGF control,and positively correlated with the concentration.Derivatives 4-3a,4-3b,4-4a,4-4b,4-6a and 4-6b displayed potent neuroprotection against H₂O₂-metiated neuronal damage of PC-12 cells with IC₅₀ values of13.96 to 33.56?M,which were higher than 3-16.3.According to HPLC analysis and bioassays,we identified the moss soil-derived actinomycete S.cacaoi subsp.asoensis H2S5 as the object strain from 80 actinomycetes of Qinling Mountain.11 ansamycins,including four new compounds,trienomycins J-M?5-1⁵-3,and 5-11?,were isolated from liquid culture of strain H2S5.Compound 5-11 was the second trienomycin containing glucose moiety,and the absolute configuration of new compounds were determined using chemical reaction.Compounds 5-1⁵-10 had significant antiproliferative activities against PC-3 and HepG2 cells,particularly,5-4 and 5-7 performed higher activity than the positive control doxorubicin,with IC₅₀ values of less than 0.7?M.Given that compound 5-1 had the greatest activity against HepG2 cells(IC₅₀=0.1?M),the induction of apoptosis of HepG2 cells by 5-1 was investigated by flow cytometry and evaluation of nuclear morphology using Hoechst staining,and the apoptosis induced by 5-1was related to the block of cells in S phase.Except for 5-11,other ten compounds exhibited significant anti-neuroinflammation activity,and eight of them much stronger than the positive control quercentin,especially 5-1,5-4,and 5-7,with values of 0.09,0.02,and 0.03?M,respectively.SAR analysis implied the longer length of the carboxylic acid moiety attached to the alanine residue at C-11 and the exposure of the 13-OH group leads to an increase of the anti-neuroinflammation activity.Theoretically,molecular docking simulation with iNOS revealed that the phenol moiety of these trienomycins were stacked against the aromatic rings of Trp463.4.The genome sequence of S.cacaoi subsp.asoensis H2S5 was completed,and the chromosome of strain H2S5 contains 10.04 Mb with 71.0%GC content.31 secondary metabolites biosynthetic gene clusters were predicted,involving type I,II,III PKS,NRPS,and PKS-NRPS,which showed great metabolic potentials.Comparative analysis of the tym gene cluster and the reported myc gene cluster of mycotrienin,the tym was identified as the biosynthetic gene cluster for trienomycins from S.cacaoi subsp.asoensis H2S5.5.Qualitative calibration of the multiple components of the crude extract of S.cacaoi subsp.asoensis H2S5 was performed on LC-MS.The theoretical yield of trienomycin A from the WT strain H2S5 was calculated as 5.7 mg/mL,through plotting the standard curve of trienomycin A.Additionally,the antibiotic sensitivity of strain H2S5 was evaluated,and the spore cultural medium was screened.Attempts on establishment of genetic transformation system for strain H2S5 had been carried out from some aspects.Meanwhile,five genetic modification vectors had been constructed,which related to the biosynthetic genes of regulators,PKS,and precursors.Taken together,this study exploited the new candidates for the treatment of neurodegenerative disease,as well as prostate cancer and liver cancer,from one fungus and one special habitat Streptomyces,and laid foundations for the the further biosynthesis of trienomycins from S.cacaoi subsp.asoensis H2S5.