Discovery And Antitumor Activity Evaluation Of HSP90 C-terminal Inhibitor

The client protein of HSP90 is involved in the occurrence and development of a variety of cancers,so HSP90 is a favorable target for the development of anti-cancer drugs.Compared with N-terminal inhibitors,HSP90 C-terminal inhibitors can regulate the biological functions of HSP90 without causing heat shock response.However,there are still scientific problems in the research of HSP90 C-terminal inhibitors,such as relatively weak anti-tumor activity,lack of clinical research drugs for tumor treatment,and unclear specific sites of action.Based on the current problems of HSP90 C-terminal inhibitors,this paper established a virtual screening method to quickly obtain small molecules that have strong binding ability to HSP90 C-terminal.In addition,virtual screening of small molecule libraries of clinical drugs on the market has been carried out to accelerate the clinical research of HSP90 C-terminal inhibitors.In this study,based on the structure of human HSP90,the CASTp online server and blind docking method were used to determine the active site of the C-terminus of HSP90.And a virtual screening method was established.The virtual screening of 280 monomers of licorice was performed.And the ranking was performed according to the docking binding energy,and getting 13 kinds of small molecules.Use 13 selected small molecules to confirm the virtual screening method.The verification of the virtual screening method is divided into two steps.The first step is verified by molecular dynamics simulation,and it is found that MOL2(3’-hydroxy-4’-O-methylglabridin)and MOL12(shinpterocarpin)small molecule monomers can combine stably with the C-terminal of HSP90.The complex with a stable conformation preliminarily proves the reliability of the established method.The second step was confirmed by network pharmacology.It was found that 8 of the 13 small molecules obtained by the screening could directly target HSP90 and exert its anti-cancer activity,further verifying the reliability of the established method.In this study,through the virtual screening strategy established above,1818 drugs in the FDA clinical drug small molecule database were virtual screened,and the anti-tumor cell proliferation test was performed on the top 10 compounds with binding energy.And found that miltefosine(alkylphosphocholine)and octenidine have good anti-tumor proliferation activities.Later,they were identified as C-terminal inhibitor of HSP90 through physical experiments.According to the MTT experiment,both miltefosine and octenidine show strong and broadspectrum anti-cancer activity.Apoptosis experiments found that miltefosine and octenidine can induce apoptosis of MCF-7 cells.Western Blot experiments showed that both miltefosine and octenidine can significantly down-regulate the expression levels of HSP90 client proteins(including p-AKT or AKT,CDK6 and ERK)in MCF-7 cells.And neither of the two induced overexpression of heat shock proteins(including HSP70 and HSP90).The above results indicate that mitefosine and octenidine are two C-terminal inhibitors of HSP90.In addition,Miltefosine can significantly down-regulate the expression level of HSF-1(Heat Shock Factor-1),thereby reducing the expression level of HSP70.In summary,this study has obtained two HSP90 C-terminal inhibitors(miltefosine and octenidine)with strong anti-tumor activity,which can accelerate the clinical research of HSP90C-terminal inhibitors,and lay the foundation for the further development of new anti-tumor drugs.