Study Of New Antifungal Target And Its Inhibitors Based On Fungal Epigenetics

The morbidity and mortality of invasive fungal infections are increasing.Tumorassociated fungal infection is a growing concern.The serious fungal resistance and the lack of new antifungal drugs are two major problems restricting the treatment of fungal infections.The bottleneck of overcoming fungal resistance is lack of effective drug targets.Therefore,discovering new antifungal targets and novel antifungal drugs is extremely important.It is reported that epigenetic changes are closely related to fungal resistance.In the previous studies of our group,antifungal activity of epigenetic protein inhibitors was tested.The results showed that several inhibitors of histone acetyltransferase(HD AC)and bromine domain could directly and synergistically inhibit fungi growth,which provided the basis to discover and validate new anti-fungal targets.Thus,targeting fungal epigenetic proteins to develop antifungal drugs to treat resistant fungal infections may be a new and promising research area.Therefore,this thesis focused on fungal epigenetics and resistance and completed three studies:1.BET-HDAC dual inhibitors for combinational treatment of breast cancer and concurrent Candidiasis.Inspired by the important roles of BET and HDAC in tumorigenesis and fungal resistance,a series of novel BET-HDAC dual inhibitors were designed and synthesized using the pharmacophore fusion strategy.Among them,compounds 13c and 17b showed excellent antitumor and synergistic antifungal activities.Compounds 13c and 17b also exhibited excellent and balanced inhibitory activity against both BET family protein BRD4 and HDAC1.We evaluated the BET isoform selectivity profile of compounds 13c and 17b.Compound 13c was found to be a pan-BET inhibitor,whereas compound 17b was a selective BRD4 inhibitor.Mechanism studies revealed that compounds 13c and 17b could induce the downregulation of c-Myc,increase the level of acetyl-H3,and induce MDA-MB-231 cell apoptosis.Moreover,compounds 13c and 17b blocked the morphological conversion of Candida albicans in combination with fluconazole(FLC).As compared with BRD4 or HDAC1 inhibitors,dual inhibitors 13c and 17b displayed improved in vivo antitumor efficacy in MDA-MB-231 breast cancer xenograft models.Notably,they synergized with FLC to effectively reduce the kidney fungal burden in a murine model of disseminated candidiasis.Thus,the BET-HDAC dual inhibitors represents a novel therapeutic strategy for combinational treatment of breast cancer and concurrent candidiasis.2.Discovery of fungal Hdal inhibitors and target validation of Hda1.Hdal is an important Candida albicans HD AC subtype(type II)protein,which is closely related to the resistance of azole drugs.At present,inhibitors and crystal complex of Hdal protein have not been reported.We investigated literatures and compared the sequence of Candida albicans Hdal and human HDACs.The results show that Candida albicans Hdal has high homology with human HDAC4 and HDAC6,and the binding pockets of human HDAC4,HDAC6 and Candida albicans Hdal are highly conserved.Herein,Hdal-N protein was expressed and purified.Using HDA1 enzymatic activity assay,Nexturastat A、Belinostat and AR-42 were identified to be Hdal inhibitors,which had excellent activity against drug-resistant fungi.For the first time,the three-dimensional structure of Nexturastat A,Belinostat and AR-42 in complex with Candida albicans Hdal were successfully solved,and the mode of action was elucidated.Moreover,Hdal mutant proteins were designed and expressed.The key role of amino acid residues in the catalytic binding pocket of Hdal protein,such as His151,His152,Phe161,His 193,Tyr222 and Ser109,were verified by enzyme inhibition activity assay.These results preliminarily prove that Hdal is a novel potential antifungal target.3.Design,synthesis and target validation of selective fungal Hdal inhibitorsThe above discovered Hdal inhibitors are derived from human HD AC inhibitors,which had poor fungal selectivity,potential host toxicity and side effects.Therefore,improving affinity and selectivity to fungal Hdal is a major challenge in structural optimization of Hdal inhibitors.Structure-based rational drug design was carried out based on the crystal structures of Hdal-inhibitor complex.As a result,selective Hdal inhibitors LA1 and LA5 were successfully designed and synthesized.Furthermore,crystal structures of LA1 and LA5 in complex with Hdal-N terminal proteins were obtained,and the binding mode,key pharmacophore of Hdal inhibitors and key amino acid residues in the catalytic binding pocket were elucidated.In addition,the selectivity against human HDACs and the sequences of Candida albicans Hdal and human HDAC6/7/8 were compared and analyzed.Tyr222 and Ser109 are found to be key amino acid residues that determine the specific binding of inhibitors with Hda1.These results provide valuable information to further discovery of novel fungal selective Hdal inhibitors.In conclusion,novel antifungal lead compounds with intellectual propriety rights were obtained,and the results provided a theoretical basis for the discovery and verification of new antifungal drug targets.