Fig 2: Effects of SHP099-resistance and pY binding-site mutations on pY abundance at protected sites on GAB1 and GAB2.(A) Schematic illustrating wild-type and SHP099-resistant forms of SHP2. (B) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or T253M/Q257L SHP2 proteins were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. (C) Schematic illustrating wild-type and mutated forms of SH2-only tandem fragments of SHP2. (D) Parental MDA-MB-468 cells or MDA-MB-468 cells stably expressing wild-type or mutated tandem SH2 fragments of SHP2 (SH2) as indicated (R32M, R138M, and R32M/R138M, denoted DM here) were pre-treated with SHP099 (10 µM). The cells were then mock treated or stimulated with EGF (10 nM) for 10 min and immunoblotted after lysis using the specified antibodies. Western blot results are representative of at least n = 2 independent biological replicates.

Fig 3: Evaluation of occludin, ARHGAP35, and PLC?2 as SHP2 substrates.MDA-MB-468 cells pre-treated with DMSO carrier or SHP099 (10 µM) for 2 hr were mock treated or stimulated with EGF (10 nM) for 10 min (A) or 30 min (B, C, and E). Total cell lysates were immunoprecipitated with occludin (A), PLC?2 (B), or ARHGAP35 (C, E) antibodies and the eluted samples were subjected to Western blotting with the phosphotyrosine antibody pY1000 (A–C), pY1105-ARHGAP35 (E), and anti-protein antibodies as indicated (A–C, E). Input samples represent 1% of total cell lysate. Non-specific IgG was used as a negative control. (D) Plot of pY abundance as a function of treatment condition for Y1105 of ARHGAP35. (F) Dephosphorylation activity of full-length wild-type SHP2, activated with 6 µM bisphosphorylated IRS-1 peptide [SLNY(p)IDLDLVKdPEG8-LSTY(p)ASINFQK], toward synthetic phosphopeptides (OCLN_pY443, PLCG2_pY818, GRB2_pY209, ARHGAP35_1105, TMEM134_pY57, CSF-1R_pY708). Immunoprecipitation – Western blot assays are representative of at least two independent biological replicates.Figure 3—source data 1.Table showing TMT relative abundance values for Y1105 of ARHGAP35.Figure 3—source data 2.Table showing micromoles of phosphate released per minute for all phosphopeptides tested for dephosphorylation by IRS1-activated wild-type SHP2.

Fig 4: SHP2 regulation, inhibition, and oncogenic mutations.A, model of SHP2 activation. Receptor tyrosine kinase (RTK) activation leads to SHP2 recruitment by tyrosine phosphorylated motifs via its two SH2 domains, resulting in a conformational switch from the “closed” autoinhibited to the “open” active conformation. B, model of SHP2 inhibition by allosteric inhibitor, such as SHP099. These “molecular glue”–type compounds stabilize the SHP2 inactive closed conformation. C, model of the common mechanism of SHP2 oncogenic variant activation. A single amino-acid mutation at the N-SH2/PTP domain interface prevents the intramolecular binding of the two domains, resulting in a constitutively active SHP2. Allosteric inhibitors such as SHP099 and RMC-4550 are not able to bind the open conformation preferred by the SHP2 gain-of-function mutants. D, crystal structure of WT SHP2 representing the closed and autoinhibited conformation as described in (A) (Protein Data Bank ID: 5EHR). N-SH2 domain, blue; C-SH2 domain, green; PTP domain, orange; the allosteric inhibitor SHP099 (magenta, stick representation) binds to a channel formed by the PTP, N-SH2, and C-SH2 domains and stabilizes the inactive conformation as described in B. E, crystal structure of the SHP2 E76K gain-of-function mutant in the open and active conformation (Protein Data Bank ID: 6CRF). To adopt this conformation, the C-SH2 domain rotates by ~120° and thereby translocates the N-SH2 domain away from the active site. SHP099-like allosteric inhibitors cannot effectively bind this active conformation as described in C. C-SH2, C-terminal SH2; N-SH2, N-terminal SH2 domain; PTP, protein tyrosine phosphatase; SH2, Src-homology 2; SHP2, Src-homology 2 domain–containing phosphatase 2.

Fig 5: Mechanism of action, inhibition, and binding studies of SHP2 inhibitors.A, Michaelis–Menten kinetic studies for the SHP2 inhibitor SBI-4668 with SHP2-E76K. Plots show the initial rates (V) at various substrates (DiFMUP) and inhibitor concentrations fitted to the Michaelis–Menten equation for noncompetitive inhibition. Relative fluorescence units per minute (RFU/min) are represented as mean ± SD (n = 3). B, Eadie–Hofstee plots of the Michaelis–Menten kinetic studies with compound SBI-4668 from A. C, dose–response curves for SBI-2130 with SHP2-E76K after various preincubation times of inhibitor with SHP2. RFU/min are represented as mean ± SD (n = 3). No time-dependent inhibition was observed as demonstrated by the similar potency for the various time points. D, dose–response curves for SBI-2130 with SHP2cat with or without a 10× inhibitor/protein preincubation and jump dilution. Identical IC50 curves indicate that SBI-2130 is a reversible inhibitor. RFU/min are represented as mean ± SD (n = 4). E, dose-dependent binding of SBI-4668 to SHP2cat in a protein thermal shift (PTS) assay. Thermal stabilization of SHP2 by SBI-4668 is shown by the increase in the SHP2 melting temperature (?Tm) compared with vehicle control (DMSO). ?Tm values are represented as mean ± SD (n = 4). DiFMUP, 6,8-difluoro-4-methylumbelliferyl phosphate; DMSO, dimethyl sulfoxide; SHP2, Src-homology 2 domain–containing phosphatase 2; SHP2cat, SHP2 catalytic domain.