Discovery Of DC_H31 As Potential Mutant IDH1 Inhibitor Through NADPH-based High Throughput Screening

The disrupted metabolism has long been studied to play key role in carcinogenesis.Warburg et al.observed that cancer cells generate energy by metabolizing glucose into lactic acid,even under the presence of sufficient oxygen.He has predicted the metabolic difference between non-cancer cells and cancer cells nearly 100 years ago,and in recent years,the metabolic difference has been identified that plays an important role in the carcinogenic process,as a potential therapeutic target.Isocitrate dehydrogenase(IDH)mutation,one of the main enzymes in the tricarboxylic acid cycle,has a significant effect on cell metabolism.The mutation of IDH1 gene has been found in many malignant tumors.The disruption of IDH protein and the abnormal 2-HG(2-Hydroxyglutarate)activity caused by the mutation are poteintial initiation for the different malignant tumors,which are implicated in various disordered biological processes.The mutation plays an important role in cancer initiation and progression,and has become an important therapeutic target.In our work,a new reliable and robust NADPH-based high throughput screening(HTS)assay targeting mutant IDH1 was developed and optimized to identify mutantspecific inhibitor.The feasibility of this HTS method was further verified by testing the Z factor and the inhibitory activities of the positive compound AGI-5198.A library containing 25,000 compounds with diverse structures was screened by this NADPHbased high throughput screening assay,and through selectivity screening and dosedependent screening,DC_H31 was identified as a potential lead compound.In addition,Protein Thermal Shift(PTS)and Surface Plasmon Resonance were employed to further investigating the interaction of compound DC_H31 and mutant IDH1,suggesting that DC_H31 is a potential selective and on-target inhibitor to bind mutant IDH1 directly.After that,molecular docking study was employed to reveal the binding mode and structural details of its interactions with mutant IDH1.We also synthesized a series of derivatives and evaluated the structure-activity relationships(SAR)of DC_H31 and its derivatives based on the results of biochemistry assay to validate the scaffold authenticity of DC_H31.In cellular level,the cell proliferation assay and the 2-HG assay was carried out to evaluate the cellular activity of DC_H31.By studying the effects on downstream genes to determinethe ability of inducing differentiation,further confirming the inhibitory activity of DC_H31 with on-target behavior.Overall,we discovered and identified a novel inhibitor which was promising to become drug candidate after further optimization,and was promising to be a probing small molecule to investigate the mechanism of cancer iniatiation and progression.