Structural Study On The Working Mechanism Of Inhibitors On Candida Albicans Deacetylase HDA1

Invasive fungal infections have become a major threat to public health.Candida albicans infection is more severe clinically and has higher mortality rate.At present,azoles are commonly used in clinical treatment of Candida albicans.The emergence of drug resistance makes the therapeutic effect of existing treatment continue to decrease.Reducing the drug resistance of Candida albicans and discovering new treatment strategies are important to control Candida albicans infection.Studies have shown that HDA1 protein is an essential drug target.HDA1 is related to the virulence characteristics of Candida albicans such as hyphal morphological transition and white-opaque transition.Deletion of hda1 gene can reduce the medicinal resistance of Candida albicans to azole drugs.This thesis studies the inhibitory mechanism of HDA1 protein by the methods of structural biology,which provides strong support for further design and screening of highly selective inhibitors based on structural analysis.In order to reduce the side effects of the inhibitor on the human counterpart proteins to improve the selectivity of inhibitor on HDA1,the ability of the inhibitors to bind to the HDAC proteins was tested by thermal shift assay.The most selective inhibitors were screened to provide direction for further inhibitor structure design.In this thesis,the plasmid of p ET21a-sumo-hdac6 cd2,p ET21a-sumo-hdac7 a and p ET21a-sumo-hdac8 were constructed by molecular biology technology,and the eukaryotic proteins were expressed by prokaryotic system.Using mature protein purification technology,the protein is separated by column chromatography,and finally high-purity HDA1-N protein and HDAC8 protein were obtained.HDAC6 CD2 and HDAC7 A proteins are prone to form multimers but not monomers.First,the binding experiments of HDA1-N protein and 11 different inhibitors were carried out.Thermal shift experiments showed that R1,D1,A0,C8,A2,A3 and E2 inhibitors could all bind to HDA1-N,and R1 might have the highest binding affinity.Similarly,in the binding experiments of HDAC8 and the 11 inhibitors,it was found that R1,A0,C8,C3,A2,A3,E2 might bind HDAC8 with high affinity.Through differential analysis,the binding ability of A0,R1,C3 to HDA1-N was much higher than that of HDAC8,while A2 and A3 showed higher selectivity to HDAC8.In the crystallization section,the method of soaking can obtain the co-crystals of HDA1-N with the inhibitors.The high-resolution structure of HDA1-N Apo and the complex structure of HDA1-N with five inhibitors A0,C8,R1,D1,and E2 were obtained from the data collected at the Shanghai Synchrotron Radiation Facility.By analyzing the structures of HDA1-N,HDAC6 CD2,HDAC7 A and HDAC8,it was found that the binding pocket volumn of HDA1-N was larger than that of HDAC6 CD2 and HDAC7 A,and the surface of the pocket was hydrophobic.There are 4 more amino acids in HDA1 in the Loop1 sequence outside the pocket,which provides a structural basis for designing selective inhibitors.Through the analysis of the five complex structures,the inhibitor interacts with His151,His152,Phe161,His193,Tyr222 and Ser109 in the binding pocket of HDA1-N,which provides an important reference for further design of mutations to verify the inhibition mechanism.Through the analysis of the structure of E2 inhibitor and Loop1 of HDA1-N,E2 may form a hydrogen bond interaction with Glu24 of Loop1,indicating that the interaction between Loop1 of HDA1-N and the inhibitor is the key to improve selectivity.