Establishment Of NAE-UBE2F Inhibitors Screening Method And Activity Evaluation Of Inhibitors

Neddylation modification is a kind of ubiquitination like modification,which is similar to ubiquitination modification,and can target its specific substrate to modify a series of substrate proteins.The process of modification is to transfer the ubiquitin like molecule of neural precursor cell expressed down regulated protein 8(NEDD8)to specific substrate for modification through the participation of three enzymes.NAE as the only activator of NEDD8,if it is inhibited by inhibitors,the whole process of neddylation will be completely blocked,leading tumor cells to death.Mln4924 is a specific inhibitor for NAE,which is currently in the clinical trial stage.It has a good effect on a variety of tumors,but in some cancer cells,NAE mutations make the cancer cells tolerant to mln4924.In order to solve the problem of tolerance of cancer cells caused by NAE mutation,it is of great value to develop a screening method of inhibitors for the interaction of NAE-ubiquitin binding enzyme E2 F(UBE2F).This thesis mainly focused on the construction of NAE-UBE2F interaction inhibitor screening method.Firstly,the prokaryotic expression vectors of NAE,UBE2F and NEDD8 were constructed.The target genes of NAE,UBE2F and NEDD8 were cloned into pET28b,pGEX-6p-1 and pCOLADuet-1 respectively,and the recombinant protein expression was successfully separated and purified by purification technology.The activity of the three recombinant proteins was studied by HTRF method,and the HTRF screening method was evaluated by the positive compound MLN4924.Finally,the inhibitor screening method for the interaction between NAE and UBE2F was successfully constructed.The activity screening of enzyme level revealed that the IC50 of the antifungal compounds Micafungin Sodium and Caspofugin Acetate were 21.08±1.695 ?M and 16.25±1.655 ?M,respectively.The protein thermal shift assay verified that the compounds could target and bind to UBE2F,the antifungal compounds could inhibit the interaction between NAE and UBE2F at the cellular and molecular levels.At the cell level,it is found that Micafungin Sodium and Caspofungin Acetate have little effect on the proliferation of cells,but could lead to the accumulation of target proteins downstream of Neddylation,which can inhibit the ability of clonal formation and wound healing of gastric cancer cells.