Novel Drug Target Discovery And Drug Screening For Pancreatic Cancer Based On Allosteric Knowledge

Allostery is a rapid and effective way to regulate protein function.Allosteric modulators induce conformational changes in proteins by binding to allosteric sites,resulting in changes in protein function.Small allosteric molecular drugs have the advantages of high selectivity,low toxicity due to its combination in allosteric sites,they have become a research hotspot in pharmaceutical field.Macromolecular allosteric modulators,such as proteins,play an important role in cellular metabolism and signaling pathways through allosteric protein-protein interactions.The first part of this thesis builds the third edition of Allosteric Database.we gain deep understanding of the concept of allostery and allosteric mechanism.The second part explores a novel drug target in pancreatic cancer and design allosteric drugs screening system for allosteric protein SIRT3.Pancreatic is an adenocarcinomasisa malignant tumor with high malignancy,difficult diagnosis and noneffective treatment,Pancreatic ductal adenocarcinoma(PDAC)account for 90% of this deadly disease.Studies have shown that Kras oncogene mutation is associated with over 95% of invasive PDAC.Moreover,experimental and clinical data further revealed that Phosphatidylinositol-4,5-bisphosphate3-kinase(PI3K)and calmodulin(CaM)regulate KRas,which plays an important role in the formation of PDAC.In addition,SIRT3,an allosteric mitochondrial sirtuin,plays an inhibitory role in pancreatic cancer.In the second chaper,based on these facts,we combined structural biology,computational biology and bioinformatics methods,to precisely reveal KRas /PI3K?/ CaM ternary complexes,and the mechanism of PI3K?/CaM coordinated Kras in the forming of PDAC.CaM directly interacts with the dual specificity kinase PI3K? through the SH2 domains of the p85 regulatory subunit.In adenocarcinomas,the CaM interaction removes the autoinhibition of the p110 catalytic subunit of PI3K?,leading to activation of PI3K? and promoting cell proliferation,survival,and migration.Here we demonstrate that the cSH2 domain of p85? engages its two CaM binding motifs in the interaction with the N-and C-lobes of CaM as well as the flexible central linker and our NMR experiments provides tructural details.Due to the flexible nature of both CaM and cSH2,multiple allosteric binding modes of the interactions are possible.In the third chapter,according to the function of SIRT3,a stable and efficient SIRT3 in vitro deacetylase activity assay was established for allosteric modulators screening.The concentration of substrate,coenzyme,protein conditions were explored,and the endogenous SIRT3 inhibitors nicotinamide and allosteric resveratrol was used ot test stability and efficiency of this system.