Dual Allosteric Inhibition of SHP2 Phosphatase

Abstract

SHP2 is a cytoplasmic protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell proliferation, differentiation, and survival. Recently, we reported an allosteric mechanism of inhibition that stabilizes the auto-inhibited conformation of SHP2. SHP099 (1) was identified and characterized as a moderately potent, orally bioavailable, allosteric small molecule inhibitor, which binds to a tunnel-like pocket formed by the confluence of three domains of SHP2. In this report, we describe further screening strategies that enabled the identification of a second, distinct small molecule allosteric site. SHP244 (2) was identified as a weak inhibitor of SHP2 with modest thermal stabilization of the enzyme. X-ray crystallography revealed that 2 binds and stabilizes the inactive, closed conformation of SHP2, at a distinct, previously unexplored binding site-a cleft formed at the interface of the N-terminal SH2 and PTP domains. Derivatization of 2 using structure-based design resulted in an increase in SHP2 thermal stabilization, biochemical inhibition, and subsequent MAPK pathway modulation. Downregulation of DUSP6 mRNA, a downstream MAPK pathway marker, was observed in KYSE-520 cancer cells. Remarkably, simultaneous occupation of both allosteric sites by 1 and 2 was possible, as characterized by cooperative biochemical inhibition experiments and X-ray crystallography. Combining an allosteric site 1 inhibitor with an allosteric site 2 inhibitor led to enhanced pharmacological pathway inhibition in cells. This work illustrates a rare example of dual allosteric targeted protein inhibition, demonstrates screening methodology and tactics to identify allosteric inhibitors, and enables further interrogation of SHP2 in cancer and related pathologies.

Resistance to allosteric SHP2 inhibition in FGFR-driven cancers through rapid feedback activation of FGFR

“…The above referenced allosteric SHP2 inhibitors such as SHP099, SHP394, and RMC-4550 bind to the tunnel-like pocket formed by the confluence of three domains of SHP2 (site 1 inhibitor) and stabilize the closed auto-inhibited state of SHP2. We also reported a series of site 2 allosteric inhibitors such as SHP244 that bind to a distinct latch-like allosteric site in a cleft at the interface of the N-terminal SH2 and PTP domains [29]. We also demonstrated that site 1 and site 2 inhibitors can simultaneously bind to SHP2 and cooperatively stabilize the closed inactive conformation of SHP2 [29].…”

Targeting SHP-1, 2 and SHIP Pathways: A Novel Strategy for Cancer Treatment?

“…Most recently, the development of allosteric SHP-2 inhibitors has been employed to circumvent these problems using computer-aided drug designs to discover SHP-2 inhibitors [11,19,20,56]. Recently, the medical chemistry of the SHP-2 inhibitor SHP099 has been reported [20].…”

Exploring the effect of E76K mutation on SHP2 cause gain‐of‐function activity by a molecular dynamics study

“…The in silico mutant structure was used as the reference structure during the 3-50 ns MD simulations. It was worth noting that the region Gln 255-Arg 265 was an allosteric pocket that affected the opening and closing of the SHP2 switch, 52,53 which was the focus in this study. The effects of translation and rotation were eliminated during the simulation progress.…”

“…These results showed that the mutant protein had higher flexibility than the wild-type protein. It was worth noting that the region Gln 255-Arg 265 was an allosteric pocket that affected the opening and closing of the SHP2 switch, 52,53 which was the focus in this study. Moreover, the RMSF values of the loop region Ile 310-Asn 320 were 0.41 nm and 0.35 nm in the SHP2-E76K system and the SHP2-WT system, respectively.…”