Identification Of Small Molecule Inhibitors Of Tyrosine Phosphatase SHP2 And Their Inhibitory Mechanism On Cancer Cell Proliferation

The nonreceptor PTP Shp-2, widely expressed, encoded by the gene Ptpn11,is a member of a subfamily of protein tyrosine phosphatases PTPs that possess two src-homology 2 (SH2)domains at the N-terminus. The SHP2 is required for normal development and apprarently participates in signaling events downstream of receptors for growth factors, cytokines,hormones, antigens, and extracellular matrixes in control of cell growth, differentiation,migration, and deadth. Mutations in SHP2 PTP and/or altered expression can contribute to diseases, incluing cancer, autoimmune disorders, inflammation, and/or developmental defects.Genetic mutations in tyrosine phosphatase Ptpn11(SHP2) that cause hyperactivation of its catalytic activity play a causal role in the pathogenesis of Noonan syndrome and various childhood leukemias. The direct connection between hyperactivation of SHP2 and these diseases indicates that SHP2 may be a useful target for mechanism-based therapeutics. It is very important to identify SHP2 specific inhibitors. SHP2 selective inhibitors could lead to the development of new drugs that would ultimately serve as treatments for SHP2-associated malignancies. In this study, virtual screening methods combined with experimental assays were used to identify SHP2 inhibitors from natural chemical products that have great advantage over novel compounds for drug discovery.2 of computationally selected compounds (43 natural compounds and 28 derivatives)were shown to effectively inhibit SHP2 activity in vitro with IC50 of 1.39μM(lyl),2.2μM( NCz). These two compounds were further verified to inhibit the proliferation of Ba/F3 response to IL-3 with IC50 of 3.1μM(lyl), 1.5μM( NCz) respectively, the proliferation of U937( an human leukemia lymphoma cell line with active mutation (G60R) in SHP2) with IC50 of 3.2μM(lyl), 1.2μM(NCz) respectively. MEF with activating mutation E76k in SHP2 were more sensitive to the active compound of lyl than to wild type cells with IC50 of 0.66pM,2.6μM respectively. The inhibition of the active compound of NCz to MEF with activating mutation E76k in SHP2 equals to the wild type MEF with IC50 of 1.4pμM(lyl), 1.8μM(NCz).H661 lung cancer cell( a kind of non-small cell lung cancer cells) with activating mutant E76k in SHP2 was more sensitive to these two compounds than H596 lung caner( a kind of non-small cell lung cancer cells) without mutant in SHP2. The two compounds effectively inhibit cell proliferation of SHP2 conditional knock-out MEF, which were more sensitive to the active compounds than the wild type cells. They also suppressed the growth of the pation(JMML) bone marrow with activating mutation G60R in SHP2 more effectively than the normal bone marrow. They suppress the colony formation of mouse bone marrow derived from mouse (Ptpn11E76k) more effectly than the normal bone marrow. They suppress the colony formation of mouse bone marrow derived from mouse (Ptpn11E76k) more effectively than the normal bone marrow. They also suppressed the colony formation of the pation( JMML) bone marrow with activating mutation G60R in SHP2 more effectively than the normal bone marrow.These two active compounds (lyl, NZc) was further verified for its ability to inhibit SHP2-mediated cell signaling pathway (Ras/Erk, PI3/Akt, and JAK2/stat5 activation). They also inhibit the bind between SHP2 and Gab2. They inhibit the phosphorylation level of downstream signaling factors. The compound of lyl shows the activity of inducing apoptosis and the compound of NCz induced cell cycle arrest in G0/G1 phase.