Extended Data Fig. 3: Characterization of SHP1 binding and phosphatase activation kinetics by SCA1 and analogs in vitro.

a, Percent SHP1 engagement of human recombinant SHP1 (2 μM) incubated with the full breadth of SCA1 analogs at 5 molar equivalents (10 μM) for 24 h (4 °C) and measured by intact protein MS. Percent SHP1 engagement represents relative abundance of small molecule engaged SHP1 to relative abundance of unlabeled SHP1. Data are mean ± s.d. of two independent experiments. b, Full list of structures of SCA1 analogs at the indicated molar equivalents and their respective percent SHP1 engagement as measured by intact SHP1 protein (2 μM) MS are shown. c, Differential backbone amine proton solvent accessible surface area (BB NH proton differential SASA) between SCA9 bound and apo SHP1 calculated from molecular dynamics simulations. Highlighted peptide regions that showed extensive uptake of deuterium were also more solvent exposed upon SCA9 binding during molecular simulations. For full list of molecular simulations peptides, refer to Supplementary Table 6. d, Recombinant human SHP1 phosphatase enzymatic activity following 30 min pre-incubation (room temperature) with DMSO or SCA1 (10 μM) measured at 37 °C and represented as phosphate equivalents released over a time course of 2 h (n = 4). e, Intact protein MS of human recombinant SHP1 (2 μM) incubated with 10, 15, 20, 25, 30, 35, 40, 45, or 50 molar equivalents of SCA1 for 48 h (4 °C). Percent SHP1 labeling represents relative abundance of SCA1 engaged SHP1 to relative abundance of unlabeled SHP1. Data are mean ± s.e.m. P values calculated using two-tailed Student’s t-tests for unpaired comparisons.

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