Skip to main content
. Author manuscript; available in PMC: 2019 Feb 13.
Published in final edited form as: Nat Cell Biol. 2018 Aug 13;20(9):1064–1073. doi: 10.1038/s41556-018-0169-1

Figure 1. RMC-4550 is a potent, selective, allosteric inhibitor of SHP2.

Figure 1.

(a) Structure of RMC-4550. (b) Effect of RMC-4550 and SHP099 on enzyme activity of SHP2 full length or SHP2 catalytic domain. Purified full-length SHP2 was incubated with di-phosphotyrosine peptide and inhibitor for 30 min prior to determining the initial rate of substrate (DiFMUP) hydrolysis. Assay conditions for SHP2 catalytic domain were identical but omitted peptide. RMC-4550 and SHP099 produced a concentration-dependent reduction in activity of full-length SHP2 but had no effect on the catalytic domain up to the maximum test concentrations of 3 μM (RMC-4550) and 10 μM (SHP099). Geometric mean IC50 values for RMC-4550 and SHP099 were 0.583 nM and 53.7 nM (RMC-4550 n = 8 independent experiments SHP099 n = 6 independent experiments; figures show mean +/− S.D.) (c) Effect of RMC-4550 on wild-type, full-length SHP2, SHP2E76K, or SHP2T253M/Q257L. The phosphatase activity of all enzymes was assayed as described in (b). RMC-4550 produced a concentration-dependent reduction in activity of wild type SHP2 but had greatly reduced effect in E76K or T253M/Q257L mutants (SHP2WT n = 8, SHP2E76K n = 3, SHP2T253M/Q257L n = 3; figures show mean +/− S.D.) independent experiments (d) PC9 cells were grown in 2D culture and incubated with increasing concentrations of RMC-4550 or erlotinib for one hour prior to determining pERK levels. RMC-4550 produced a concentration-dependent reduction in pERK with a geometric mean IC50 value of 31 nM (n = 4 independent experiments; figures show mean +/− S.D.) e) Effect of RMC-4550 on HEK293 cells expressing wild type SHP2, SHP2E76K, or SHP2T253M/Q257L. Isogenic HEK293 cell lines expressing each SHP2 variant were incubated with RMC-4550 for 1 hour, stimulated with EGF for 5 minutes, lysed, and the levels of pERK were determined. RMC-4550 produced a concentration-dependent reduction in the level of pERK in cells expressing wild-type SHP2 with a geometric mean IC50 value of 49.2 nM (n = 4 independent experiments; figures show mean +/− S.D.) No inhibitory activity was observed in cells expressing E76K or T253M/Q257L mutants up to the maximum test concentration of 10 μM. Source data is provided in Supplementary Table 9.