Screening Of Glycolysis Inhibitors And Evaluation Of Its Antitumor Efficacy

Objective:Under the condition of complete mitochondrial function and sufficient oxygen,tumor cells still use glycolysis as the main energy source,which is called Warburg Effect.GAPDH is an important enzyme located in the middle step of glycolysis,and targeted inhibition of its activity is of great significance for tumor treatment.This study intends to use a virtual screening method to screen out GAPDH-targeted inhibitors,observe its effect on tumor cells,explore its mechanism,and evaluate its efficacy by ¹⁸F-FDG PET/CT.It lays the foundation for clinical transformation and efficacy monitoring of new tumor glycolysis anti-tumor drugs.Methods:1.The candidate small molecule compounds were screened out by the virtual screening based on molecular docking.A molecular-level GAPDH inhibitor screening method was constructed,and the activity of the candidate small molecule compounds was verified to obtain lead compounds.2.Validate the inhibitory effect of the lead compound on GAPDH enzyme activity at the molecular level,explore interaction between the lead compound and GAPDH,detect the inhibitory effect of the lead compound analogs on GAPDH enzyme activity,and perform structure-activity analysis.3.Detect the effect of the lead compound on intracellular GAPDH enzyme activity.4.CCK-8 test was used to detect the effect of the lead compound on tumor cell proliferation.5.Detect the effect of the lead compound on glucose consumption and lactic acid production levels,and explore the effect of the lead compound on tumor cell uptake of ¹⁸F-FDG through ¹⁸F-FDG uptake experiment and ¹⁸F-FDG micro PET/CT imaging.6.Detect the effect of the lead compound on apoptosis by flow cytometry.7.Observe the effect of the lead compound on tumor growth in nude mice.8.¹⁸F-FDG micro PET/CT nude mouse imaging was used to evaluate the efficacy of the lead compound and its effect on glycolysis.9.HE staining was used to observe tumor necrosis,and immunohistochemical staining was used to observe the expression of the proliferative protein Ki-67 and apoptotic protein Caspase-3.Results:1.141 candidate small molecule compounds were obtained through virtual screening.2.A molecular-level GAPDH inhibitor screening method was successfully constructed.3.The lead compound DC-5163 was screened out by candidate activity verification;DC-5163 can interact with GAPDH and inhibit GAPDH enzyme activity with the IC₅₀ of 176.3 n M.4.Docking analysis predicts that DC-5163 may bind to the NAD⁺pocket of GAPDH protein.5.DC-5163 can inhibit GAPDH enzyme activity in a variety of tumor cells.6.DC-5163 can inhibit the proliferation of MDA-MB-231 cells,which is tolerated by human normal breast epithelial cells MCF-10A.7.DC-5163 can partially block the glycolytic pathway and induce apoptosis of MDA-MB-231 cells.At the same time,DC-5163 can reduce the uptake of ¹⁸F-FDG by MDA-MB-231 cells.8.Compound DC-5163can inhibit tumor growth in nude mice,cause tumor tissue necrosis,decrease Ki-67expression and increase Caspase-3 expression.¹⁸F-FDG micro PET/CT showed that both SUVmax and SUVmean of nude mice after treating in DC-5163 group are smaller than that in the control group.Conclusion:Docking-based virtual screening is an efficient method for obtaining lead compounds.Through virtual screening,we obtained GAPDH enzyme activity inhibitor DC-5163;DC-5163 can inhibit tumor cell proliferation,induce apoptosis,partially block the glycolytic pathway,decrease ¹⁸F-FDG uptake,and normal cells tolerate it;meanwhile DC-5163 can inhibit tumor growth in vivo,cause tumor tissue necrosis,reduce Ki-67 and increase Caspase-3 expression,¹⁸F-FDG micro PET/CT showed that DC-5163 could reduce SUVmax and SUVmean of tumors in nude mice.The research lays the foundation for the development of new drugs targeted tumor glycolysis with independent intellectual property rights in China and the evaluation of their efficacy.