Studies On The Bioactive Secondary Metabolites From Fungi With Antibacterial Activity Against Drug-resistance Bacteria

Bacterial resistance crisis is a growing threat to human health.It directly leads to the failure of medical treatment,the increase of medical expenses,and the increase of mortality rate.What's more,further development of antibiotic-resistance may cause the human to face the threat of infectious diseases effectively regressing to the pre-antibiotic era.To respond to this emerging crisis,global organizations have urged the scientific community to identify new approaches to combat antibiotic resistance.The chemical and biological diversity of the fungal metabolites are inexhaustible sources of new antibacterial drugs.In recent years,with the development of life science,the mechanisms related to bacterial resistance have been clarified,the in vitro large-scale drug screening model have been established and technology innovation such as computer simulation and virtual docking,there has been a worldwide upsurge of finding antibacterial leading compounds from microbial metabolites.Five chapters were included in the dissertation.In the first chapter,the background and the significance of this study were introduced.Fifty strains in the in-house fungal library which were isolated from medicinal plant(Anoectochilus formosanus;Hyperforin perforatum and Lycoris radiata)and the Yangtze river tidal flats,were screened against antibiotic-resistance bacteria.Three talented fungal strains Aspergillus sp.Z12,Aspergillus sp.TJ23,and Emericella sp.TJ29,which could produce abundant chemical metabolites with potential antibacterial activities,were chosen as the aimed strains to investigate their bioactive metabolites in this study.In the second chapter,bioassay-guided isolation of cultures of Aspergillus sp.Z12 yielded eight fungal naphtho-y-pyrones 1-8.The antimicrobial effects of naphtho-y-pyrones 1-8 against drug-resistant bacteria were firtsly investigated.Fonsecinones A and C,and aurasperones A and E were identified with potential antibacterial activities.Among which,fonsecinone A exhibited the most potent antibacterial activity,with minimum inhibitory concentrations(MICs)of 4.26,17.04,and 4.26 ?g/mL against ESBL-producing E.coli,P.aeruginosa,and E.faecalis,respectively.We also performed structure-based virtual screening and confirmatory bioassays to identify the possible antibacterial targets of these bioactive naphtho-y-pyrones.Molecular docking-based target identification of naphtho-y-pyrones 1-8 revealed bacterial enoyl-acyl carrier protein reductase(FabI)as an antibacterial target,which was further validated by FabI affinity and inhibition assays.In addition,this work provides an example of the successful application of in silico methods in target identification and validation for natural products and lies the foundation of our subsequent activity and target finding of the bioactive natural products.In the chapter three,the secondary metabolites of Aspergillus sp.TJ23 were systematically investigated.Antibacterial activities against drug-resistance pathogens and anti-tumor activities of all these isolates were evaluated.Fourteen compounds which included five new compounds were isolated in the rice solid culture of Aspergillus sp.TJ23.The five new compouds included spiroaspertrione A,which has a novel spiro[bicyclo[3.2.2]nonane-2,1'-cyclohexane]carbocyclic skeleton;two meroterpenoids(andiconin B and asperspiropene A);one steride;and one alkaloid.The structures and absolute configurations of these new compounds were elucidated by spectroscopic analyses,single-crystal X-ray diffraction,and ECD calculations.In the bioactivity assays,spiroaspertrione A,with an intriguing bridged spirocyclic structure,showed anti-MRSA activity and resulted in potent activity in suppression of oxacillin resistance in MRSA by reducing the oxacillin MIC to 1 ?g/mL.With the help of virtual molecular docking,MST and Western Bolt,the synergism of the anti-MRSA effect is associated with the inhibition of PBP2a expression.Moreover,in the screening experiment of potential inhibitor of mutant IDH1(IDH1R132H/R132C),asperspiropene A was idetified as a potent candicate.To the best of our knowledge,asperspiropene A is the first fungal metabolite reported to exhibit IDH1-inhibitory activity.Inhibitors of mutant IDH1 are being intensely pursued,and these findings may provide a promising lead compound for promising drug candidates in personalized cancer treatment.In the chapter four,the secondary metabolites of Emericella sp.TJ29 were systematically investigated.Fourteen compounds including five new compounds were isolated from the rice solid cultures of Emericella sp.TJ29.The five new compouds included three new meroterpenoids(emervaridones A-C)and two polyketides(varioxiranediols A and B).The structures and absolute configurations of these new compounds were elucidated by spectroscopic analyses,single-crystal X-ray diffraction,Mo2(OAc)4-induced electronic circular dichroism(ECD)data,and ECD calculations.To date,only one case bearing the emervaridone-type carbocyclic skeleton has been reported.The structures of emervaridones A-C expand new members of this type of natural product,and emervaridone A represented the first example of an a-directional H-7' in this structural category.Emervaridone A and varioxiranediol A exhibited potent inhibitory effects against ESBL-E.coli and P.aeruginosa,as well as with low toxicities to mammalian cells,these two compounds may provide novel chemical scaffolds for the discovery of antibacterial agents for drug-resistant microbial pathogens.In the last chapter,the antibacterial drug discovery in the resistance era was reviewed.We described five aspects in detail:Current situation of bacteria antibiotic resistance;Mechanisms and origins of antibiotics;Mechanism of bacterial resistance formation and diffusion;The strategies of modern antimicrobial discovery and the future development of antimicrobial drugs.We got conclusions that,natural products have provided unparalleled sources of small molecules that have continue to play a prominent role in the discovery of new antimicrobials.In the bacterial resistance era,the antibacterial drug discovery need innovation.That is combination of new computational and biological technology in the screening of natural products.The strategy of developing or searching antibiotics also need innovation,which should move the focus of the field from broad-spectrum agents to narrow,disease-specific antibiotics,from bioactive chemical matter for single use to combination studies,and from aim at one definit molecular target to effect on multiple cellular targets.