Ligand Fragments Screening For Three Proteases Based On Biophysical Methods

Fragment-based drug discovery(FBDD)has matured over the past decade,and its structure-oriented optimization or rational combination of active fragments has greatly promoted the transformation of drug discovery strategies from serendipitous discovery to targeted selection and rational design.Compared with other drug discovery methods,FBDD has the advantage of small molecular screening scale but very wide chemical space coverage.In addition,the extensibility of hit fragment structure provides novel and powerful molecular drug discovery methods.Currently,four fragment derived drugs have been approved by the FDA.Appropriate receptor targets can give the potential to optimize the structure of hit molecules,and the biophysical method is the core of hit discovery and verification in FBDD process.In this dissertation,Dihydrofolate reductase(DHFR),RING-type E3 ligases RNF55 and RNF56 were selected as receptor targets for fragment screening.Fragments were sequentially screened by the orthogonal screening strategy composed of Differential Scanning Fluorescence(DSF),Saturation Transfer Differential spectrum(STD)and Fluorescence Polarization(FP)was used to screen the fragments in order to obtain the fragment molecules with affinity to the receptor protein.The screening process is as follows:1.Dihydrofolate reductase is a key enzyme in the biological folate synthesis pathway.Targeting the folate binding pocket of dihydrofolate reductase can affect the normal base pair synthesis of cells.Moreover,studies have shown that folate pathway is closely related to the metabolism of tumor cells,and targeting it can achieve the treatment of some diseases such as breast cancer,lung cancer and digestive tract cancer.Therefore,DHFR was selected as the screening target,and the expression and purification of DHFR was completed by using E.coli system.The fragment library containing 1201 molecules was screened by differential scanning fluorescence and saturated transfer differential spectroscopy.16 positive molecules were preliminarily identified from the fragment library based on differential scanning fluorescence assay,and 7 of them were confirmed as orthogonal hit by STD,but the affinity effect was weak.These hit fragments are structurally similar to folic acid molecules and contain basic groups such as nitrobenzene and amines.And the interaction of the 5 hit fragments with the target protein DHFR was further verified by computational simulation docking method in folate binding pocket.2.RING type E3 ligase RNF55 is a potential therapeutic target for prostate cancer and myeloproliferative diseases,which can mediate the ubiquitination degradation of protein tyrosine kinases(PTKs),thus playing an important role in the regulation of signal transduction pathways.RNF55 binds to the substrate through its conserved TKB domain,and can screen for small molecule inhibitors by targeting its phosphotyrosinase binding site in the TKB domain.In the experiment,DSF,STD and FP were used to screen the fragment targeting RNF55.11 fragments were screened by differential scanning fluorescence,and one orthogonal hit which contains thioamide and benzene ring groups was obtained by STD.According to the results of fluorescence polarization experiments,the hit was not the phosphotyrosine binding site in the Tk B domain,indicating that the compound may target to other sites of RNF55.3.RING type E3 ligase RNF56 has a similar structure to RNF55,and also plays a regulatory role of ubiquitination through the phosphotyrosinase binding site in its Tk B domain.In the screening experiment,8 positive fragments were obtained by DSF,and 3 affinity fragments were determined by NMR.These binding fragments targeted to the receptor protein are relate to chlorphenyl,morpholine acetonitrile,pyrimidine and acetic acid.This thesis provides the experimental basis for further screening the corresponding inhibitors or ligands for DHFR,RNF55 and RNF56 proteins which with important biological functions.Three protein targets were expressed,purified and enriched by means of genetic engineering.Then,the biophysical methods such as differential scanning fluorescence(DSF)saturation transfer differential spectroscopy(STD)based on NMR and fluorescence polarization(FP)and computational simulation docking were used to screen several fragment molecules binding to target proteins from fragment libraries.serveral hits and its structural characteristics were obtained.It has certain theoretical and applied values.