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.

SHP2 is a nonreceptor protein tyrosine phosphatase encoded by the PTPN11 gene and is involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also plays an important role in the programed cell death pathway (PD-1/PD-L1). As an oncoprotein as well as a potential immunomodulator, controlling SHP2 activity is of high therapeutic interest. As part of our comprehensive program targeting SHP2, we identified multiple allosteric binding modes of inhibition and optimized numerous chemical scaffolds in parallel. In this drug annotation report, we detail the identification and optimization of the pyrazine class of allosteric SHP2 inhibitors. Structure and property based drug design enabled the identification of protein–ligand interactions, potent cellular inhibition, control of physicochemical, pharmaceutical and selectivity properties, and potent in vivo antitumor activity. These studies culminated in the discovery of TNO155, (3S,4S)-8-(6-amino-5-((2-amino-3-chloropyridin-4-yl)­thio)­pyrazin-2-yl)-3-methyl-2-oxa-8-azaspiro­[4.5]­decan-4-amine (1), a highly potent, selective, orally efficacious, and first-in-class SHP2 inhibitor currently in clinical trials for cancer.

SHP2 is a cytoplasmic non-receptor tyrosine phosphatase involved in the propagation of extracellular signaling through receptor tyrosine kinases. Aberrant SHP2 activity has been identified as a driver in multiple cancers and SHP2 has also been implicated in the PD-1/PD-L1-mediated exhaustion of effector T-cells, leading to immune system evasion of tumors. Recently, we reported the identification of SHP099, an allosteric inhibitor of SHP2 with in in vivo efficacy against multiple RTK-driven tumor xenograft models. Here we report the use of alternate screening paradigms to identify a novel allosteric inhibitor which binds to a previously uncharacterized pocket on SHP2. Like SHP099, the second allosteric inhibitor stabilizes a closed conformation of SHP2, which blocks access to the phosphatase active site. Structure based drug design led to improvements in potency, and combination studies in biochemical, biophysical and cellular assays confirm dual occupation of SHP099 and the second allosteric molecule, resulting in improved potency. This work highlights a rare opportunity for dual occupation of inhibitors for a single target and provides additional tools for the exploration of SHP2 biology. Citation Format: Michelle Fodor, Edmund Price, Ping Wang, Hengyu Lu, Andreea Argintaru, Zhouliang Chen, Meir Glick, Huai-Xiang Hao, Mitsunori Kato, Robert Koenig, Jonathan R. LaRochelle, Gang Liu, Eric McNeill, Dyuti Majumdar, Gisele Nishiguchi, Lawrence Perez, Greg Paris, Christopher Quinn, Timothy Ramsey, Martin Sendzik, Michael Shultz, Sarah Williams, Travis Stams, Stephen C. Blacklow, Matthew J. LaMarche, Michael G. Acker. Simultaneous inhibition of SHP2 phosphatase at two allosteric sites [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2808.