The Drug Design And Mechanism Study On Lipid Kinase PI4KⅡα And Protein Kinase PDK1

Kinases participate in most aspects of cell life and have become one of the most attractive groups of drug targets.The strategy of drug design and computational chemistry methods play important roles in the study of kinase inhibitors.The core issues in kinase inhibitors design include solving the selectivity and resistance problems,discovering new skeleton leads,explaining the related mechanisms and so on.In chapter one of this thesis we will introduce the drug design methods targeting kinases.The lipid kinase PI4KⅡα plays an important role in a number of human diseases and has been found to be a potential target for anti-tumor therapy.However,the clinical applications of PI4KⅡα are limited by lack of specific inhibitors.Here,in chapter two we report the first human PI4KⅡα substrate(PI)competitive inhibitor,PI-273.A ligand based following structure based virtual screening strategy was first employed to identify promising hits,and then biochemical experiments of validation on PI4KⅡα inhibitory effect both in molecular and cellular levels were performed.PI-273 exhibited inhibitory effect both on PI4KⅡα kinase activity with an IC50 of 0.47 μM,and on cell proliferation suppression in breast cancer cell lines(The IC50 of PI-273 to MCF-7 cell viability was 3.51μM).PI-273 profoundly suppressed the growth of MCF-7 xenografts by intraperitoneal injection with PI-273 25 mg/kg/day.Further study revealed that PI-273 can significantly inhibit MCF-7 cell-induced breast tumor growth without toxicity.As PI-273 is the first substrate-competitive and subtype-specific inhibitor of PI4KⅡα,it will greatly facilitate basic scientific research and application of PI4KⅡα.Protein kinase PDK is an important target in regulation of cancer cells metabolism.Developing inhibitors of PDK1 is expected to get promising antitumor effect in cancer treatment.Herein,in chapter three,through combination of computational methods of molecular docking and molecular dynamics simulation with biological assays we investigated on mechanism of JX06 inhibition on PDK1,the selective covalent inhibitor of PDK1.Further on,based on the PDK1/JX06 binding mode and the mechanism study,we identified a first generation of disulfide-based subtype-selective and potent covalent PDK1 Inhibitors represented by 3a.3a could effectively inhibit PDK1 both at the molecular(kinact/Ki = 4.17×103M-1s-1)and the cellular(IC50 down to 0.1 μM)level.3a is a potent and subtype-selective inhibitor of PDK1 with > 40-fold selectivity for PDK2-4.Treatment of 3a could considerably reduce tumor volume in A549 subcutaneous xenograft mice models(TGI = 47.8% at 80 mg/kg/day)without mice bodyweight loss.3a may be an excellent lead compound for the treatment of cancer as a first-generation subtype-selective and covalent PDK1 inhibitor.And also our work will greatly facilitate the functional study of PDK1 and related cancer metabolism research.As an allosteric inhibitor of PDK1,DCA inhibits activity of PDK1 through binding with the N-terminal helixes bundle structure.Here,in chapter four we studied on the molecular mechanism of DCA on PDK1 inhibition by computational chemistry and computational biology methods including normal mode analysis,molecular dynamics simulation and principal component analysis.We figured out that DCA bound with α6 and α7 helixes and decreased their structural stability,which could be further transported to both the ATP binding pocket and the substrate binding pocket and affect the affinity between PDK1 with ATP and the substrate protein,triggering the loss of activity of PDK1.This study not only helps uncover the molecular mechanism of the allosteric inhibitor DCA on PDK1 inhibition,but also offers a new idea for kinase targeted drug design.