Primary Studies On Ligands Screening For Three Functional Proteins

In the past 20 years,fragment-based drug discovery(FBDD)has gradually became one of the mainstream methods for prodrug discovery and development.Compared with traditional high-throughput screening,FBDD has the advantages of high specificity and high drug-like properties.In this dissertation,human dihydrofolate reductase(h DHFR),6-hydroxymethyl-7,8-dihydrotrexate pyrophosphate kinase(HPPK)and RBR-type E3 ubiquitin ligase RNF31 were selected as the receptor targets for fragment screening,and the active fragments were screened by orthogonal screening method consisting of Differential Scanning Fluorescence(DSF)and Saturation Transfer Differential-NMR(STD-NMR),the interactions of hit fragments with target protein were further verified by computational simulation docking method.Human dihydrofolate reductase(h DHFR)is an important catalytic enzyme in folic acid pathway,which is closely associated with the development of cancers.By targeting h DHFR to block the catalytic reaction can block the folic acid pathway in cancer cells and achieve the effect of cancer treatment.In this dissertation,the E.coli system was used for the expression of h DHFR,and the protein was purified by affinity chromatography and molecular sieve chromatography.The affinity fragments were screened from a library containing 1201 fragments by differential scanning fluorescence and saturated transfer differential spectroscopy.24 affinity fragments were screened by DSF assay,and 11 of them were confirmed to be positive hits by the secondary screening of STD-NMR.And the interaction of the 11 hit fragments with the target protein was further verified by computational simulation docking method.7,8-dihydro-6-hydroxymethylpterin pyrophosphokinase(HPPK)is the first catalytic enzyme in the microbial folic acid biosynthesis pathway.It catalyzes the transfer of pyrophosphate group from ATP donor to6-hydroxymethyl-7,8-dihydrotrexate(HP).The targeted screening of inhibitors of HPPK can block the biosynthesis of folic acid,to achieve the purpose of inhibiting microbial growth.Therefore,HPPK is an ideal target for new antibacterial drugs.A hit fragment was screened by DSF experiment,and orthogonal verification was carried out by STD-NMR experiment to confirm its affinity with HPPK.RBR E3 ubiquitin ligase RNF31 is a proto-oncogene that induces disease progression through the NF-κB pathway and regulates key nuclear receptors/transcription factors,which are closely associated with human lymphoma and breast cancer.Screening fragment inhibitors that target RNF31 is an important means to develop targeted drugs for lymphoma and breast cancer.Two positive fragments were screened by the DSF experiment,and one of them was confirmed as an orthogonal hit by the STD-NMR experiment.In this dissertation,three kinds of functional proteins h DHFR,HPPK and RNF31 were expressed and purified by genetic engineering.Then,differential scanning fluorescence(DSF)and saturation transfer based on NMR(STD-NMR)were used to screen the active compounds,and the computer simulations were used to analyse the interaction interface,which laid a foundation for molecular structure optimization of fragments and further study of target protein inhibitors screening.