Cdk9 Small Molecule Inhibitor Design And Discovery

Cell cycle in an orderly way is the basic process of life of normal cell,which depends on a set of sophisticated molecular regulatory mechanism forming in their evolution.One of the most basic features of tumor cells is the performance of their cell cycle for the out-of-control growth and. cell disorders. With the further development of molecular biology, the mechanisms of regulation and control for cell cycle gradually revealed to humanity,at the core of which is known as a protein family named cyclin-dependent kinase (CDKs), which is a serine/threonine kinase family. Members of the family have been found 13 (CDK1-CDK13). According to their different intracellular functions, the members were divided into two categories: control of cell cycle CDKs and control of transcription CDKs. CDK9 we studied belong to the latter, it combines to the corresponding cyclin(s) forming compounds called positive transcription elongation factor b (P-TEFb), which can phosphorylated RNA polymeraseⅡ(RNA polymeraseⅡ) and a number of negative transcription elongation factor (NELF and N-TEFs) so that transcription can be extended. With a negative role in CDK regulation, CDKIs divided into two main categories, most of them can inhibit CDK.As the key roles in cell cycle, the inhibition of CDK in the purpose of anti-tumor has good application prospects. In order to get its three-dimensional structure, we used homology modeling approach, and then use DOCK (molecular docking) program for small molecules. From the top 1000 compounds with the best DOCK energy score, 27 compounds were selected for biological assay based on the diversity of chemical structure and functional group. MTT method was used in inhibition of tumor cell growth in vitro, while western blotting was used for further study of molecular mechanisms. 12 of 27 selected compounds show significantly inhibition activity on tumor cell proliferation, and only one compound in 12 with half-maximum inhibition concentration (IC₅₀)values less than 20μmol·L^-1 named C-21 was selected for further molecular mechanism study. The western blotting data showed C-21 compound can effectively inhibit CDK9 from phosphorylating large subunit C-terminal of RNA polymeraseⅡin a dose-dependent manner.The further study results showed that such types of compounds can effectively inhibit the proliferation of all tumor cell lines we have.The results in molecular mechanisms study of such compounds showed it can effectively inhibited activity of CDK9 in a dose-dependent manner according to the phosphorylation change of the downstream substrate.The conclusions is through homology modeling, virtual screening by computer, determination of biological activity and experimental studies of molecular mechanism, a new promising leading compound targeted CDK9 was found and confirmed.