Figure 3.

Compound 12 is a C333-targeted, time-dependent SHP2 inhibitor. (A) Chemical structure of 12. (B) Dose dependence of inhibition by 12. PTP activities of the SHP2 and C333P SHP2 catalytic domains (250 nM) were measured with pNPP (5 mM) after preincubation (2 h, 37 °C) with vehicle or 12 at the indicated concentrations. (C) Time dependence of inhibition by 12. PTP activities of the SHP2 and C333P SHP2 catalytic domains (250 nM) were measured with pNPP (5 mM) after preincubation with vehicle or 12 (150 μM) for the indicated times at 37 °C. (D) Kinetic analysis of SHP2 inhibition. Time-dependent inhibition assays were carried out essentially as described for panel (C) to determine the half-life (t_1/2) of PTP activity at various concentrations of 12. The linear relationship between t_1/2 and the reciprocal of inhibitor concentration (1/[12]) was analyzed to estimate the indicated kinetic constants as previously described.^34,35