The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

Activating mutations in PTPN11, encoding the cytosolic protein tyrosine phosphatase SHP2, result in developmental disorders and act as oncogenic drivers in patients with hematologic cancers. The allosteric inhibitor SHP099 stabilizes the wild-type SHP2 enzyme in an autoinhibited conformation that is itself destabilized by oncogenic mutations. Here, we report the impact of the highly activated and most frequently observed mutation, E76K, on the structure of SHP2, and investigate the effect of E76K and other oncogenic mutations on allosteric inhibition by SHP099. SHP2E76K adopts an open conformation but can be restored to the closed, autoinhibited conformation, near-identical to the unoccupied wild-type enzyme, when complexed with SHP099. SHP099 inhibitory activity against oncogenic SHP2 variants in vitro and in cells scales inversely with the activating strength of the mutation, indicating that either oncoselective or vastly more potent inhibitors will be necessary to suppress oncogenic signaling by the most strongly activating SHP2 mutations in cancer.

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.

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.

The proto-oncogene PTPN11 encodes a cytoplasmic protein tyrosine phosphatase, SHP2, which is required for normal development and sustained activation of the Ras-MAPK signaling pathway. Germline mutations in SHP2 cause developmental disorders, and somatic mutations have been identified in childhood and adult cancers and drive leukemia in mice. Despite our knowledge of the PTPN11 variations associated with pathology, the structural and functional consequences of many disease-associated mutants remain poorly understood. Here, we combine X-ray crystallography, small angle X-ray scattering and biochemistry to elucidate structural and mechanistic features of three cancer-associated SHP2 variants harboring single point-mutations within the N-SH2:PTP interdomain autoinhibitory interface. Our findings directly compare the impact of each mutation on autoinhibition of the phosphatase and advance the development of structure-guided and mutation-specific SHP2 therapies.

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 nonreceptor protein tyrosine phosphatase within the mitogen-activated protein kinase (MAPK) pathway controlling cell growth, differentiation, and oncogenic transformation. SHP2 also participates in the programed cell death pathway (PD-1/PD-L1) governing immune surveillance. Small-molecule inhibition of SHP2 has been widely investigated, including in our previous reports describing SHP099 (2), which binds to a tunnel-like allosteric binding site. To broaden our approach to allosteric inhibition of SHP2, we conducted additional hit finding, evaluation, and structure-based scaffold morphing. These studies, reported here in the first of two papers, led to the identification of multiple 5,6-fused bicyclic scaffolds that bind to the same allosteric tunnel as 2. We demonstrate the structural diversity permitted by the tunnel pharmacophore and culminated in the identification of pyrazolopyrimidinones (e.g., SHP389, 1) that modulate MAPK signaling in vivo. These studies also served as the basis for further scaffold morphing and optimization, detailed in the following manuscript.

The TMPRSS2:ERG gene fusion is common in androgen receptor (AR) positive prostate cancers, yet its function remains poorly understood. From a screen for functionally relevant ERG interactors, we identify the arginine methyltransferase PRMT5. ERG recruits PRMT5 to AR-target genes, where PRMT5 methylates AR on arginine 761. This attenuates AR recruitment and transcription of genes expressed in differentiated prostate epithelium. The AR-inhibitory function of PRMT5 is restricted to TMPRSS2:ERG-positive prostate cancer cells. Mutation of this methylation site on AR results in a transcriptionally hyperactive AR, suggesting that the proliferative effects of ERG and PRMT5 are mediated through attenuating AR’s ability to induce genes normally involved in lineage differentiation. This provides a rationale for targeting PRMT5 in TMPRSS2:ERG positive prostate cancers. Moreover, methylation of AR at arginine 761 highlights a mechanism for how the ERG oncogene may coax AR towards inducing proliferation versus differentiation.DOI: http://dx.doi.org/10.7554/eLife.13964.001