Discovery Of Novel SHP2 Inhibitors

Phosphorylation and dephosphorylation are important components of post-translational modifications of proteins,which are involved in the regulation of physiological and pathological activities such as cell growth,proliferation,differentiation,metastasis,and tumor development,and which are mainly executed by phosphatases and kinases.SHP2,Src homology-2 domain-containing protein tyrosine phosphatase-2,is an important tyrosine phosphatase encoded by PTPN11,and it is associated with the regulation of various cancer-and inflammation-related signaling cascades.SHP2 mutations can cause numerous diseases such as hematopoietic malignancy,solid tumor,noonan syndrome,and leopard syndrome.Accordingly,development of SHP2 inhibitors is of great significance for the treatment of SHP2-related diseases,especially for cancer therapy.E76K mutagenesis of SHP2 is the most common mutation observed in juvenile myelomonocytic leukemia,and the inhibitory activity of known SHP2 inhibitors against SHP2＿E76K is usually lower than wild-type SHP2.In addition,the amino acid sequence is highly conserved between SHP2 and SHP1,the closest homologue of SHP2.Therefore,the selectivity of inhibitors is critically important.In this study,we mainly established an inhibitor screening platform for SHP2＿E76K and the selectivity test of the compounds toward SHP2 and SHP1,aiming to provide new hits for the discovery of SHP2 inhibitors with high potentcy and selectivity.First,we constructed multiple plasmids for SHP2 and SHP1 protein expression,which includes the wild-type,full-length SHP2（SHP2＿WT）,full-length SHP2 with E76K mutat（SHP2＿E76K）,the catalytic domain of SHP2（SHP2＿PTP）,the wild-type,full-length SHP1（SHP1＿WT）,full-length SHP1 with E74K mutant（SHP1＿E74K）,and the catalytic domain of SHP1（SHP1＿PTP）,and purified these protein samples for subsequent experiments.Then,we established the enzymatic assay for both SHP2 and SHP1,and utilized the assay to screen inhibitors of SHP2＿E76K from 2560 drugs.Ten compounds were found to have inhibitory activity against SHP2＿E76K,among which,eltrombopag olamine and anacardic acid have good inhibitory activity against SHP2＿E76K,with IC₅₀ values of 4.5μM and 6.5μM,respectively.Anacardic acid also showed moderate selectivity toward SHP1,with an IC₅₀ value of 2.2μM for SHP2＿WT and 10.1μM for SHP1＿WT,respectively.Then,we applied the thermal shift assay to verify the binding of the compounds to SHP2.Molecular docking was used to explore the possible binding modes of small-molecule inhibitors with SHP2,revealing that these small molecules may interact with the key amino acids K364,K366,C459,S460,and R465 in the substrate binding pocket by forming hydrogen bond and salt bridge.Finally,we obtained SHP2 crystals by screening 960 crystal conditions,and the X-ray diffraction data were collected with an attempt to solve the complex structure of SHP2boud with our inhibitors.However,the electron density map of small molecule was not observed,and only the 3D crystal structure of SHP2＿PTP and SHP2＿WT were determined at a resolution of 1.7?and 2.5?,respectively.In summary,we established a SHP2 inhibitor screening platform and obtained ten compounds with micromolar potency.The binding mechanisms of these compounds with SHP2 were also investigated.Moreover,we solved the high-resolution crystal structures of SHP2＿PTP and SHP2＿WT,which provided a basis for the optimization of SHP2 inhibitors and the determination of SHP2-inhibitor complex structures.