A Method For Specific Determination Of Lysine Demethylase Activity And Its Application In Inhibitor Screening

Tumor is a major medical problem that human currently faces.In recent decades,epigenetics has made hub of important breakthroughs around the etiology of tumors caused by abnormal expression of genes.Among them,histone post-translational modification(PTM)is the current hotspot in the world.The fifth amino group(N?)of histone lysine residues often undergoes different types of PTM including acetylation,phosphorylation,ubiquitination and methylation.The abnormal methylation of histone lysine is closely related to the transformation and abnormal proliferation of tumor cells.The modification of histone lysine methylation is regulated by two types of antagonistic enzymes: histone lysine methyltransferases(KMTs)and histone lysine demethylases(KDMs).Furtherly,KDMs play a more important role in the occurrence and development of different tumors,such as promoting the expression of protooncogenes,causing immune system disorders as well as DNA damage repair.Thus,finding effective inhibitors is the key to solving this type of cancer.To date,several different high-throughput screening(HTS)methods have been developed to find inhibitors of KDMs.However,false negatives,false positives and the inability to monitor enzyme activity in real time are the main drawbacks of these methods,so there is an urgent need for a more optimized method for detecting the activity of KDMs.In view of the abovementioned shortcomings of the existing technology,this study aimed at KDMs,using nuclear magnetic resonance technology to create a novel and specific method for detecting enzyme activity and screening inhibitors.First,in a more complex environment such as cell lysate,the substrate and product changes can be detected with high selectivity and specificity,and the enzyme-dependent reaction process is determined,and then the substrate and product changes and product Absolute quantification,design of enzyme activity calculation formula,that is,realtime monitoring of KDMs enzyme activity and quantitative changes of substrates and products,and we also detected that KDMs inhibitors have obvious effects in this system.Finally,we use the natural small molecule compound library for screening.With the advantages of less false negatives,false positives,time saving and high specificity,this method screens out smallmolecule compounds that inhibit the enzyme activity of KDMs.In the end,it will help clinical treatment.Compared with previous methodological studies,this method has the advantages of higher specificity,safety,and real-time performance.Combined with biomolecular technology,it can more accurately qualitatively and quantitatively react molecules.In the future,using this technology can also directly or indirectly explore the occurrence and development of tumor biology and better achieve precise treatment.