Structure-based Virtual Screening And Activity Verification Of Candida Albicans F₁F_o-ATP Synthase β Subunit Inhibitors

Invasive fungal infections（IFIs）are an underestimated global health problem,killing an estimated 1.5 million people worldwide each year,more than tuberculosis or malaria.The most important pathogen causing IFIs is Candida,which can cause30-40%mortality.The increase in clinically resistant strains and the limited availability of drugs urgently require the discovery of novel antifungal agents.At present,the research and development of antifungal drugs is still mainly to obtain compounds that inhibit the growth of fungi by screening synthetic compounds or natural product libraries.Our previous study confirmed that theβsubunit of Candida albicans F₁F_o-ATP synthase is crucial to the pathogenesis,but there is no antifungal drug that acts on theβsubunit.To this end,we combined the advantages of HTS and SBVS to carried out activity verification to obtain an effective and broad-spectrum inhibitor targeting this subunit.ObjectiveIn this study,the homologous structure of Candida albicans F₁F_o-ATP synthaseβsubunit have been constructed.And we performed structure-based virtual screening,activity verification,molecular docking and structure optimization of Candida albicans F₁F_o-ATP synthaseβsubunit inhibitors.And to discovery a broad-spectrum inhibitor targeting theβsubunit.MethodsPart 1:（1）Homology modeling using S.cerevisiaeβsubunits as a template to perform homology modeling to obtain the 3D structure of C.albicansβsubunits,and protein active sites through the Site Finder module in MOE to determine the binding pockets of compounds of theβsubunits were performed.（2）Virtual screening preprocess the target protein and Chem Bridge compound library,and conduct experimental molecular docking to determine the optimal mode,and rigid molecular docking,compounds in Chem Bridge compound library were evaluated by SBVS based on force field scoring function GBVI/WSA d G.Part 2:（1）Compounds activity validation:detect the antifungal activity in vitro of compounds by microdilution checkerboard techniques and time-kill curves.（2）The influence of compounds to main virulence factors were measured by hyphae formation,biofilm formation and adhesion experiment.（3）The influence of compounds to ATP level were measured by ATP concentration.（4）Detect the toxicity of the compounds by CCK-8 and LDH methods.Part 3:（1）Application of MOE software to analyze binding mode of the compounds with target protein.（2）Design inhibitor derivatives by replacing inactive essential groups by electronic isometric,and perform molecular docking and scoring.（3）Inhibitor derivative activity validation:detect the antifungal activity in vitro of compounds by microdilution checkerboard techniques.Detect the toxicity of the derivative by CCK-8 and LDH methods.ResultPart 1:（1）Take theβsubunits of S.cerevisiae as a template,obtain the 3D structure of theβsubunits of C.albicans through homology modeling;and analyze the binding pocket of theβsubunits surface through the Site Finder module in MOE.（2）The compounds selected through rigid and flexible molecular docking were evaluated and GBVI/WSA d G scoring functions were analyzed by molecular fingerprint cluster analysis（20 hits）.Part 2:（1）Antifungal activity in vitro found that the MIC₅₀ of S2-16 and S2-13 was0.25、0.5μg/m L.The time-kill curves showed that S2-13 and S2-16 had strong fungicidal effects and had a dose-dependent effect.（2）S2-13 could reduce the ability of hyphae formation,biofilm formation and adhesion of Candida albicans.And its inhibitory ability was better than S2-16.（3）S2-13 could inhibit the activity of the cell’s ATP.（4）The IC₅₀of S2-13,S2-16 to liver,kidney and macrophages is 16.1,25.64,34.16μg/m L with high toxicity.Part 3:（1）It was showed binding mode of the compound with target protein well.（2）Inhibitor derivatives were designed by electronic isometric.（3）The MIC₅₀ of inhibitor derivative was 0.25μg/m L.The IC₅₀of inhibitor derivative to liver,kidney and macrophages is 12.15,20.89,29.1μg/ml.ConclusionIn this study,a broad-spectrum inhibitor targeting theβsubunit was discovered through virtual screening of Candida albicans F₁F_o-ATP synthaseβsubunit,combined with activity verification and structure optimization.It provides a practical basis for further screening of highly selectiveβsubunit inhibitors and provides a direction for the development of new antifungal drugs.