The Inhibitory Activity Studies Of Thiophene [2,3-d] Pyrimidine Compound On SHP2

Objective:SHP2 is a crucial node for integration of signals from different cell surface receptors with all the major cellular signaling pathways within the cells.Therefore,while aberrant activation of SHP2 triggers activation of signaling pathways leading to the development of hematological malignancies;it also provides a vulnerable therapeutic target.However,given its ubiquitous expression and functional role in different tissues and organs further studies are needed before SHP2targeted therapeutics enter the clinic.The purpose of this study was to obtain the SHP2 inhibitors by determining the SHP2-PTPase activity of the thiophene[2,3-d]pyrimidine compound obtained from the laboratory.Then,the inhibition was verified at the cellular level.Finally,the interactions between SHP2 and inhibitor were studied by molecular docking and molecular dynamics.This can provide new ideas to find potential inhibitors targeting SHP2.Methods:1.The study of the bioactivity of SHP2 inhibitors:（1）Establishment of SHP2 enzyme activity assay system and activity assay:firstly,SHP2-PTP plasmid was transformed into E.coli BL21;secondly,to obtain sufficient protein,the expression of SHP2-PTP protein was induced by IPTG.The protein was then purified using a Ni-NTA gravity column to obtain a relatively pure SHP2-PTP protein.Finally,the effect of the compounds on SHP2-PTPase activity was determined using pNPP assay.（2）Cellular activity:firstly,the H460 and Hela cell lines were cultured to obtain the sufficient cells at the log phase.Both of the two cells was treated with different concentrations of the compound and incubated for 48 hours.The MTT assay was used to examine the effect of the compound on the two cells.Secondly,the impact of the compound on apoptosis was detected by Annexin V-FITC/PI cell apoptosis double staining kit.Finally,the effect of the compound on SHP2-mediated Ras/Erk signal transduction pathway was further investigated by western blot.2.Binding model of SHP2 and inhibitor:（1）the binding interactions between SHP2and the compound were analyzed by CDOCK docking.（2）the molecular dynamic study of the complex of SHP2 and the compound was applied by Gromacs 4.5.5.Firstly,the stability of this system was verified by RMSD and RMSF.Secondly,the conformation of the protein wasanalyzed by PCA,and the interaction between amino acid and amino acid was analyzed by DCCM and RINs.Finally,the RINs results were verified by binding energy using MM/PBSA.（3）The six parameters of compound were analyzed by ADMET,including aqueous solubility25°C,brain blood barrier（BBB）,cytochrome P450 2D6（CYP2D6）binding,hepatotoxicity,human intestinal absorption（HIA）,and plasma protein binding properties（PPB）.Results:The study of the bioactivity of SHP2 inhibitors:（1）Enzyme level:A molecular level of SHP2-PTP inhibitor was established by expressing His-SHP2-PTP fusion protein and targeting SHP2-PTP with pNPP as substrate.The screening models were built in vitro.By screening our thiophene[2,3-d]pyrimidine compounds,the IC₅₀0 of the Y21 compound to SHP2-PTP was 1.174μΜ.（2）Cell level:Cell proliferation experiments showed that Y21 could significantly inhibit the proliferation of H460 and Hela cells.Apoptosis experiments showed that 8μg/mL Y21 could induce early apoptosis of H460 cells and Hela cells.Western Blot experiments showed that the expression level of SHP2 was down-regulated in Hela cells,and the phosphorylation level of ERK was up-regulated with the treatment of Y21,respectively.It was indicated that Y21 has a certain inhibitory effect on the activity of SHP2 phosphatase.Binding model of SHP2 and inhibitor showed that Y21 can well fit into the binding site of SHP2.Y21 interacted with amino acids of SHP2 binding pocket by electrostatic interaction（ion-dipoleand H-bond）and hydrophobic interaction to inhibit the bioactivity of SHP2.The results of PCA,DCCM,and RINs indicated Y21 inhibited the motions of amino acids in SHP2 binding pocket,and thus,SHP2 was inactive.ADMET results showed that Y21 was a drug-like compound.Conclusion:This study shows that the thiophene[2,3-d]pyrimidine scaffolds SHP2inhibitor can effectively inhibit the proliferation of H460 and Hela cells,and induce early apoptosis,and down-regulate SHP2 phosphate at the cellular level.Computer aided drug design experiments indict that the compounds can fit well with the receptor protein and have drug-like properties.It is a class of compounds that has potential and deserves further development.This study lays a good foundation for further research and provides a new idea for the development of new anticancer drugs targeting SHP2.