Design,Synthesis And Anti-Tumour Activity Of Novel Small Molecule Inhibitors Targeting K-Ras Protein-Effector Interaction

The oncogene K-Ras mutation is the most common molecular mechanism in more than one-quarter of non-small cell lung cancer(NSCLC).In recent years,with the increasing living standards of the people and the tremendous changes in the surrounding living environment,the frequency of K-Ras mutations has increased in malignant tumors and smokers.And the overall effect of chemotherapy and targeting therapies for patients with K-Ras mutations treatment is poor,so this is urgent for the effective treatment of patients with K-Ras mutant malignant tumors.To date,a number of chemical agents have been developed for patients with K-Ras mutant NSCLC,but most inhibitors are for Ras protein downstream effector proteins or kinases,such as the Raf kinase inhibitor Sorafinib,by blocking Raf-MEK-MRK signaling.The pathway,in turn,directly inhibits tumor cell proliferation and growth,and the like.Since Ras proteins are extremely important in the cell signaling pathway,the development of small molecule compounds that directly target Ras proteins is of great significance.However,it has not yet been developed to target Ras protein antitumor drugs that can be applied to the clinic.In this thesis,the small molecule compound Kobe0065 reported in the literature was used as a lead scaffold.The compounds’ design was based on the K-Ras protein receptor structure,and the related groups are replaced by bioisosteres to improve the affinity and selection of small molecule ligands.Compounds TKR01-TKR21 were prepared by high-efficiency organic synthesis strategies,and the structure of the compounds were confirmed by NMR spectrometer and LC-MS.Then,the biological activity of this series of compounds was evaluated by CCK-8 method,and it was found that the small molecule compound TKR15 can significantly inhibit the growth and proliferation of A549 cells(IC50=0.21μM).Subsequently,a preliminary mechanism verification and investigation of the small molecule TKR15 was carried out.Firstly,the binding mode of the compound to the active site was analyzed by the molecular docking software Autodock Vina.It was found that the small molecule ligand can markedly bind to the hydrophobic cavity through a strong hydrophobic interaction and form a hydrogen bond with the Glu-37 residue,and leading the inhibition of K-Ras protein interaction with effector proteins;secondly,it was confirmed by flow-through apoptosis assay that the small molecule compound can significantly induce A549 apoptosis through the apoptotic pathway;finally,in the process of immunofluorescence staining experiment for K-Ras protein and Raf-1 protein,after the treating of small molecule TKR15 to A549 cells,the red-colored K-Ras protein was clearly observed in the experimental group under the laser confocal microscope by comparison with the blank control group.This directly confirmed that the small molecule compound TKR15 can effectively inhibit the interaction of the K-Ras protein with the downstream Raf-1 protein.Through the design and optimization of small molecule compounds,activity screening and preliminary mechanism research,finally this project found a small molecule compound TKR15 with excellent anti-tumor activity and capable of targeting K-Ras protein,which laid a solid foundation for future research.