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The Src-homology 2 domain containing phosphatase 2 (SHP2) plays a critical role in crucial signaling pathways and is involved in oncogenesis and in developmental disorders. Its structure includes two SH2 domains (N-SH2 and C-SH2), and a protein tyrosine phosphatase (PTP) domain. Under basal conditions, SHP2 is auto-inhibited, with the N-SH2 domain blocking the PTP active site. Activation involves a rearrangement of the domains that makes the catalytic site accessible, coupled to the association between the SH2 domains and cognate proteins containing phosphotyrosines. Several aspects of this transition are debated and competing mechanistic models have been proposed. A crystallographic structure of SHP2 in an active state has been reported (PDB code 6crf), but several lines of evidence suggests that it is not fully representative of the conformations populated in solution. To clarify the structural rearrangements involved in SHP2 activation, enhanced sampling simulations of the autoinhibited and active states have been performed, for wild type SHP2 and its pathogenic E76K variant. Our results demonstrate that the crystallographic conformation of the active state is unstable in solution, and multiple interdomain arrangements are populated, thus allowing association to bisphosphorylated sequences. Contrary to a recent proposal, activation is coupled to the conformational changes of the N-SH2 binding site, which is significantly more accessible in the active sate, rather than to the structure of the central β-sheet of the domain. In this coupling, a previously undescribed role for the N-SH2 BG loop emerged., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

The protein tyrosine phosphatase Src homology phosphotyrosyl phosphatase 2 (SHP2) is implicated in various cancers, and targeting SHP2 has become a promising therapeutic approach. We herein described a robust cross-validation high-throughput screening protocol that combined the fluorescence-based enzyme assay and the conformation-dependent thermal shift assay for the discovery of SHP2 inhibitors. The established method can effectively exclude the false positive SHP2 inhibitors with fluorescence interference and was also successfully employed to identify new protein tyrosine phosphatase domain of SHP2 (SHP2-PTP) and allosteric inhibitors. Of note, this protocol showed potential for identifying SHP2 inhibitors against cancer-associated SHP2 mutation SHP2-E76A. After initial screening of our in-house compound library (∼2300 compounds), we identified 4 new SHP2-PTP inhibitors (0.17% hit rate) and 28 novel allosteric SHP2 inhibitors (1.22% hit rate), of which SYK-85 and WS-635 effectively inhibited SHP2-PTP (SYK-85: IC 50  = 0.32 μmol/L; WS-635: IC 50  = 4.13 μmol/L) and thus represent novel scaffolds for designing new SHP2-PTP inhibitors. TK-147, an allosteric inhibitor, inhibited SHP2 potently (IC 50  = 0.25 μmol/L). In structure, TK-147 could be regarded as a bioisostere of the well characterized SHP2 inhibitor SHP-099, highlighting the essential structural elements for allosteric inhibition of SHP2. The principle underlying the cross-validation protocol is potentially feasible to identify allosteric inhibitors or those inactivating mutants of other proteins., Competing Interests: The authors have no conflicts of interest to declare., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

Noonan syndrome (NS) is a common autosomal dominant congenital disorder which could cause the congenital cardiopathy and cancer predisposition. Previous studies reported that the knock-in mouse models of the mutant D61G of SHP2 exhibited the major features of NS, which demonstrated that the mutation D61G of SHP2 could cause NS. To explore the effect of D61G mutation on SHP2 and explain the high activity of the mutant, molecular dynamic simulations were performed on wild type (WT) of SHP2 and the mutated SHP2-D61G, respectively. The principal component analysis and dynamic cross-correlation mapping, associated with secondary structure, showed that the D61G mutation affected the motions of two regions (residues Asn 58-Thr 59 and Val 460-His 462) in SHP2 from β to turn. Moreover, the residue interaction networks analysis, the hydrogen bond occupancy analysis and the binding free energies were calculated to gain detailed insight into the influence of the mutant D61G on the two regions, revealing that the major differences between SHP2-WT and SHP2-D61G were the different interactions between Gly 61 and Gly 462, Gly 61 and Ala 461, Gln 506 and Ile 463, Gly 61 and Asn 58, Ile 463 and Thr 466, Gly 462 and Cys 459. Consequently, our findings here may provide knowledge to understand the increased activity of SHP2 caused by the mutant D61G.