Lead Compounds Discovery And Activity Regulation Mechanism Of Nicotinamide Adenine Dinucleotide Kinase

Nicotinamide adenine dinucleotide kinase（NADK）catalyzes the phosphorylation of NAD⁺to NADP⁺and controls intracellular NADP⁺/NADPH content.Compared with normal cells,highly proliferating tumor cells require large amounts of NADP⁺/NADPH for rapid synthesis of nucleic acids,proteins,lipids,and other molecules as well as for neutralizing excessive reactive oxygen species（ROS）.KRAS activation mutations are the most widely present activated mutations in tumors,especially in lung cancer,pancreatic cancer,and colorectal cancer.Studies have shown that knockdown of NADK did not affect the growth of normal cells or the transformational growth of normal KRAS tumor cells.However,knockdown of NADK expression or inhibition of its enzyme activity can significantly reduce the transformational growth of KRAS mutant tumor cells,suggesting that NADK is involved in the KRAS-mediated tumor growth process.Developing small molecule inhibitors targeting NADK may specifically kill tumor cells with KRAS-activated mutations.However,the molecular mechanism of KRAS regulating NADK enzyme activity and the small molecule inhibitors targeting NADK have not been reported.In this study,we proved that the key kinase ERK1/2downstream of KRAS can directly bind NADK protein which was supported by several assays including protein mass spectrometry,protein immunoprecipitation,and in vitro Pull down assay.By protein modification mass spectrometry,in vitro phosphorylation and intracellular phosphorylation experiments,we found that ERK1/2 kinase can phosphorylate NADK-S48/50 amino acids.Further NADK enzyme activity assay in vitro and in cells revealed that phosphorylation of NADK-S48/50 significantly activated NADK activity,revealing a key mechanism by which KRAS directly regulates NADK activity and cellular NADP⁺/NADPH synthesis.In this study,a high-throughput screening assay was established and optimized with the Z factor of 0.71,and we found the first non-substrate analog inhibitor（-）-epigallocatechin gallate（EGCG）,whose IC₅₀ is 1.3±1.1μM.One-dimensional NMR showed that EGCG could directly bind to NADK.Surface plasmon resonance experiments detected the binding affinity of EGCG to NADK with K_d=1.78±1.15μM.EGCG was found to be a non-competitive inhibitor by competitive experiments with K_i=3.28±0.32μM.Hydrogen-deuterium exchange mass spectrometry experiment showed that EGCG was bound at the non-substrate binding site of NADK.Besides,binding mode prediction and derivative activity analysis revealed a potential structure-activity relationship between EGCG and NADK.Furthermore,EGCG can specifically inhibit the proliferation of KRAS-mutated lung cancer cell lines without affecting KRAS wild-type lung cancer cell lines.To sum up,this project took NADK as the research object and found that NADK is a substrate protein of ERK1/2,which activates the activity of NADK through phosphorylation.Through screening,we found that the natural product EGCG is a non-competitive inhibitor of NADK,which can specifically inhibit KRAS-mutated lung cancer cell lines,laying a good foundation for the subsequent development of NADK-targeted drugs.