Recent advances in the discovery of protein tyrosine phosphatase SHP2 inhibitors

Abstract

Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) is a non-receptor protein tyrosine phosphatase encoded by the Ptpn11 gene, which regulates cell growth, differentiation and apoptosis via modulating various signaling pathways, such as the RAS/ERK signaling pathway, and participates in the PD-1/PD-L1 pathway governing immune surveillance. It has been recognized as a breakthrough antitumor therapeutic target. Besides, numerous studies have shown that SHP2 plays an important role in the regulation of inflammatory diseases. However, inhibitors targeting the active site of SHP2 lack drug-likeness due to their low selectivity and poor bioavailability, thus none has advanced to clinical development. Recently, allosteric inhibitors that stabilize the inactive conformation of SHP2 have achieved breakthrough progress, providing the clinical proof for the druggability of SHP2 as an antitumor drug target. This paper reviews the recently reported design and discovery of SHP2 small molecule inhibitors, focused on the structure–activity relationship (SAR) analysis of several representative SHP2 inhibitors, outlining the evolution and therapeutic potential of the small molecule inhibitors targeting SHP2.

Src homology 2 domain-containing protein tyrosine phosphatase (SHP2) has emerged as a breakthrough antitumor therapeutic target. Inhibitors targeting SHP2 active site or allosteric site confer new treatments for tumor and inflammatory diseases.

1. Introduction

The cellular equilibrium of protein tyrosine phosphorylation is achieved through the concerted actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Abnormal tyrosine phosphorylation resulting from the disorders of PTK and PTP balances can cause a variety of human diseases such as cancer, diabetes, and autoimmune disorders.^1,2 Given that many small molecule inhibitors against PTKs have been approved as clinical drugs by the FDA,³ PTPs have been suggested as next generation drug targets.^4,5

The PTP family can be divided into 4 groups based on their protein sequences and functions.⁶ The major group is class I PTPs, which are further divided into classic tyrosine-specific PTPs and tyrosine and serine/threonine dual-specific phosphatases (DUSPs).⁷ The former, tyrosine-specific PTPs, contains 17 cytosolic PTPs and 21 trans-membrane receptor-like PTPs.⁸ Among them, SHP2 is a cytosolic PTP encoded by the PTPN11 gene, importantly, it was the first reported oncogenic tyrosine phosphatase.^9–11 SHP2 has become an attractive target for therapeutic intervention in cancer, mainly manifested in 3 aspects as follows: (1) SHP2 is an essential convergent node that activates multiple RAS signaling pathways.^12–14 Almost all receptor tyrosine kinases (RTKs) activate SHP2 to activate the RAS signaling pathway.^15,16 Therefore, a suitable SHP2 inhibitor can be applied to RTK mutations and has the potential to become a broad-spectrum anticancer drug. (2) Due to some of the protein tyrosine kinases and SHP2 acting on the same signaling pathway, combining an SHP2 inhibitor with a PTK inhibitor leads to dual inhibition of signaling pathways, which may be synergistic thus more effective and less drug resistant.^17,18 (3) SHP2 is associated with T cell receptor (TCR) signaling and immune checkpoint receptor programmed death-1 (PD-1).^19–21 In view of the current successful clinical application of anti-PD-1/PD-L1 tumor immunotherapy,^22–24 SHP2 small molecule inhibitors are an important supplement to PD-1/PD-L1 antibody drugs. Taken together, SHP2 inhibitors have drawn great attention and tremendous interest from academia and industry to be developed into a novel class of anticancer drugs.

Two broad classes of SHP2 inhibitors have been widely investigated as potential therapeutics: catalytic site and allosteric inhibitors. The positive-charged environment of the PTP catalytic pocket presents unique drug discovery challenges.^25,26 Most catalytic site-directed inhibitors require multiple ionizable functional groups to gain potent binding with the enzyme thus effectively blocking the catalytic function. These functional groups, in turn, complicate the drug discovery and development due to poor cell permeability and bioavailability.²⁷ In recent years, SHP2 allosteric inhibitors have been identified by high-throughput screening specifically designed to bind to the non-conservative allosteric site of SHP2 to stabilize the inactive conformation of the enzyme and thereby inhibit the catalytic function of SHP2. Unlike the conserved catalytic domain in PTPs, targeting these non-conserved allosteric sites tends to be more specific for SHP2 over other PTPs. However, allosteric inhibitor drug discovery comes with trade-offs and is relatively complicated.²⁸ In the following review, we will introduce the structure and function of SHP2 and recent advances in the development of potent SHP2 inhibitors based on the binding modes, providing an evolution and outlook in the therapeutic potential of drug-like inhibitors targeting SHP2.

2. Structure and signaling of SHP2

SHP2 is a 593-amino acid classical non-receptor protein tyrosine phosphatase. It contains two N-terminal SH2 domains (N-SH2 and C-SH2), a PTP catalytic domain, a C-terminal tail with four tyrosine phosphorylation sites (Tyr542, Tyr546, Tyr580 and Tyr584) and a proline rich region.^29,30 Functional SH2 and PTP domains are required for all known SHP2 biological behaviors and almost all reported SHP2 biochemical effects.^29,31 The C-terminal tyrosines are phosphorylated upon activation of some, but not all receptors, and regulates those signaling pathways.³² Cys459 is a highly reactive cysteine in the conserved characteristic motif of the catalytic domain of PTP, and has important catalytic functions.³³ In 1998, the crystal structure of SHP2 was resolved.³⁴ The crystal structure (PDB: 2SHP, including residues 1-527) shows that in the inactive state, the N-SH2 domain of the SHP2 protein binds to the PTP domain and blocks its substrate from entering the catalytic site, resulting in the inhibition of SHP2 activity. However, upon stimulation, binding of tyrosine-phosphorylated proteins, such as RTKs or scaffold proteins with the SH2 domain disrupts its autoinhibitory interactions and triggers the release of the PTP domain and thus activation of the enzyme (Fig. 1).^35,36 Simultaneously, the binding of the phosphorylated tyrosine motif to the SH2 domain anchors SHP2 to its physiological substrate, leading to the dephosphorylation of the substrate. On the other hand, certain pathogenic mutations of PTPN11 often occur at the interface between the N-SH2 domain and the PTP domain, which will weaken the auto-inhibition between the SH2 domain and PTP domain, and thus activate the phosphatase abnormally.

Fig. 1. Schematic illustration of the activation of SHP2 by the binding of SH2 domains with the tyrosine phosphorylated proteins or mutations in N-SH2 and PTP domains.

SHP2 has emerged as a key regulator of RTKs and cytokine receptor signaling, such as RAS/ERK/MAPK, phosphatidylinositol-3-kinases (PI3K)/protein kinase B (PKB or AKT) and the Janus kinase/signal transducer and activator of trans-ions (JAK/STAT), etc.^37–41 The RAS/ERK/MAPK pathway is a major pathway downstream of RTKs and cytokine signaling that is modulated by SHP2.⁴² SHP2 activates RAS/ERK/MAPK through different mechanisms, such as dephosphorylation of RASGAP binding sites on specific receptors and adapter proteins or dephosphorylation of the negative RAS/ERK modulator sprouty.^43–46 Alternatively, SHP2 induced activation of RAS is through the activation of Src family kinases. It dephosphorylates Csk binding protein, resulting in the dissociation of Csk that negatively regulates Src family kinases.^13,47,48

In addition to the positive regulator of RAS/ERK/MAPK signaling, SHP2 is also a key downstream effector of PD-1 in T cells. Once stimulated by its natural ligand PD-L1 or PD-L2, PD-1 is phosphorylated by Src kinase at two tyrosine-containing motifs located at the tail of its cytoplasm.¹⁹ These two motifs, the immunoreceptor tyrosine-based inhibitory motif (ITIM) and the immune receptor tyrosine-based switch motif (ITSM), recruit and activate SHP2, which then removes phosphate groups from nearby effector proteins.^21,49,50 Ultimately, this leads to a reduction in cytokine production and T cell proliferation, thereby suppressing T cell-mediated immune responses (Fig. 2). Therefore, inhibitors that can block the protein–protein interaction between PD-1 and SHP2 are expected to become small molecule tumor immunotherapeutics.

Fig. 2. Schematic SHP2-mediated main signaling pathways: 1) SHP2-associated growth factor dependent signalling pathways, including RAS/Raf/ERK and PI3K/AKT pathway, and 2) SHP2 inhibiting T cell activation through PD-L1/PD-1 mediated ITIM and ITSM signalling.

3. SHP2 mutations in human diseases

SHP2 mutation has been detected in several diseases, such as Noonan syndrome (NS),^51–53 and Leopard syndrome (LS),^54–56 as well as hematological malignancies [juvenile myelomonocytic leukemia (JMML), childhood myelodysplastic syndrome (MDS), childhood B acute lymphoblastic leukemia (B-ALL), and childhood acute myelogenous leukemia (AML)],^57–60 and solid tumors.^7,61

In human diseases (including NS, LS, and leukemia), PTPN11 mutation has following multiple regulatory functions. (1) The gain-of-function (GOF) SHP2 mutations (e.g. A72V, E76K, I282V, and N308D) mostly located in or close to the N-SH2 and PTP domain interaction surface can enhance the catalytic activity of PTP and activate SHP2 by disrupting its autoinhibitory conformation, thereby activating the RAS/MAPK pathway, which promotes the occurrence of NS and leukemia.^62–65 (2) Loss-of-function (LOF) mutations (e.g. Y279C and T468M) can severely impair the catalytic activity of PTP, but increase its open and active conformation, resulting in continuous activation of key pathways.^66,67 Although they strongly inhibit growth factor-induced MAPK signaling, LOF mutations do not affect the basic MAPK activation necessary for NS-related phenotypes.^68,69 However, the LOF mutation enhances PI3K/AKT/mTOR signaling through an unknown mechanism and eventually contributes to LS, resulting in a GOF phenotype.^70–72 (3) Another type of PTPN11 mutation enhances or decreases its affinity to the phosphotyrosine (pTyr) motif in the scaffold protein and further changes the response of SHP2 to ligand stimulation by causing extensive structural rearrangement and severely disrupting the domain.^67,73 This mechanism may occur in NS, leukemias, and LS, and is accompanied by GOF and LOF mutations. (4) Recent research shows that both NS/NS-ML and cancer-associated SHP2 mutants gain acquired capability of liquid–liquid phase separation (LLPS), which promotes its PTP enzymatic activity. The LLPS of SHP2 is driven by the PTP domain through intermolecular electrostatic interaction and is regulated by intramolecular conformation. Disease-related SHP2 mutants can recruit wild-type (WT) SHP2 in separate aggregates and promote the downstream MAPK pathway in NS-related syndrome. In addition, SHP2 allosteric inhibitors can attenuate the LLPS of SHP2 mutants.⁷⁴ The close association between SHP2 abnormality and human diseases strongly indicates that small molecule SHP2 inhibitors may have important therapeutic applications.

4. SHP2 inhibitors targeting the catalytic pocket

The substrates of the SHP2 enzyme are tyrosine phosphates, and most of its catalytic site inhibitors contain ionic functional groups that mimic pTyr. As pTyr mimetics, these ionic functional groups can be bound to the substrate catalytic pocket of SHP2, thereby inhibiting the function of SHP2. Based on the substitution pattern of specific ionic functional groups, the catalytic site inhibitors of SHP2 can be divided into sulfonic acid group- and salicylic acid group-containing inhibitors; in addition, active site-directed SHP2 inhibitors have also been identified from natural products, such as cryptotanshinone derivatives (selected examples are shown in Fig. 3).

Fig. 3. SHP2 inhibitors targeting the catalytic pocket of the enzyme.

4.1. SHP2 catalytic site inhibitors containing sulfonic acid groups

NSC-87877 (1, Fig. 3A) was identified as a potent SHP2 inhibitor with an IC₅₀ of 0.318 μM by screening the National Cancer Institute (NCI) Diversity Set chemical library which contains 1981 compounds.⁷⁵ However, NSC-87877 seemed to have no selectivity between human SHP2 and SHP1 in vitro. Docking studies showed that both sulfonic acid moieties in NSC-87877 formed hydrogen bonds with amino acid residues in the SHP2 PTP catalytic domain. This was further confirmed by in silico point mutation test. NSC-87877 inhibited the SHP2 E76K mutant with an IC₅₀ of 0.295 μM that was similar to that for the SHP2 PTP domain construct, suggesting that NSC-87877 preferentially inhibited the activated SHP2. Experiments in HEK293 cells (expressing SHP2 but not SHP1) and in MDA-MB-468 cells (expressing SHP1/2) showed that NSC-87877 significantly inhibited epidermal growth factor (EGF) induced SHP2 activation and affected the interaction between SHP2 and Gab1, thereby suppressing ERK activation. But in MDA-MB-468 cells, cotreatment of EGF and NSC-87877 did not suppress SHP1 activation. NSC-87877 can significantly reduce MDA-MB-468 cell viability and proliferation, especially in combination with a PI3K inhibitor. This important study discovered the first PTP inhibitor capable of inhibiting SHP2 PTP in cell cultures, thus opened a new avenue in SHP2 research by providing a novel tool for chemical biology exploration of SHP2 functions and signaling mechanisms.

The phenylhydrazonopyrazolone sulfonate compound PHPS1 (2, Fig. 3A) was identified from high-throughput in silico screening.⁷⁶ Computer docking predicted that PHPS1 interacted with the active site of homology-modeled SHP2 more strongly than with the corresponding sites of the closely related PTPs, SHP1 and PTP1B. The phenyl sulfonate group of PHPS1 acted as a pTyr mimetic and penetrated into the substrate-binding pocket of SHP2, whereas the pyrazolone core of PHPS1 and its substituents made contacts with residues at the periphery of this cleft, where residues Lys280, Asn281, Arg362 and His426 were important for selectivity. The dissociation constant (K_i) of PHPS1 for inhibition of SHP2 was 0.73 μM and 8- and 15-fold lower toward the closest PTP1B and SHP1, respectively. In MDCK cells, PHPS1 inhibited SHP2-dependent cellular events such as hepatocyte growth factor/scatter factor (HGF/SF)-induced epithelial cell scattering and branching morphogenesis. PHPS1 also blocked SHP2-dependent downstream signaling, namely HGF/SF-induced sustained phosphorylation of ERK1/2 MAP kinases and dephosphorylation of paxillin. Furthermore, PHPS1 efficiently inhibited activation of ERK1/2 by the leukemia-associated SHP2 mutant, SHP2 E76K, and blocked the anchorage-independent growth of a variety of human tumor cell lines. In short, PHPS1 was the first compound that specifically inhibited SHP2 over the closely related phosphatases SHP1 and PTP1B, laying the foundation for further development of novel therapeutics for the treatment of SHP2-dependent human malignancies and other diseases.

To address the bioavailability issue, the Zhang group explored the existing drug space for previously unknown PTP inhibitory scaffolds.⁷⁷ Therefore they screened the Johns Hopkins Drug Library and identified a third generation β-lactam antibiotic – cefsulodin (3, Fig. 3A) – as a reversible and competitive SHP2 inhibitor with an IC₅₀ value of 16.8 μM. The crystal structure of SHP2 in complex with compound 4 (PDB: 4RDD), but not cefsulodin, showed that the sulfonic acid was in close proximity to the catalytic P-loop and formed multiple hydrogen bonds with the backbone amides. The sulfophenyl acetic amide (SPAA) served as a unique pTyr mimetic. To introduce structural diversity, the SPAA fragment-based focused libraries using appropriately functionalized linkers were prepared, which led to the discovery of compounds 5 to 7 (Fig. 3A) exhibiting IC₅₀'s in the range of 1.4–2.3 μM, which were 7–12 fold more potent than that of cefsulodin. In addition, compounds 5 to 7 have either a biaryl or single aryl group with bulky substituents at the terminal position, highlighting a clear preference for lipophilic moieties. In H1975 cells, compound 5 effectively blocked EGF-induced ERK1/2 activation and SHP2-mediated dephosphorylation of paxillin, and had no effect on phorbol 12-myristate 13-acetate (PMA)-induced ERK1/2 activation (SHP2 independent). Notably, compound 5 could specifically inhibit the growth of H1975 lung cancer cells, MDA-MB-231, and ErbB2 positive SKBR3 breast cancer cells. In a word, this study demonstrated the utility of exploring the existing drug space for the development of new scaffold PTP inhibitors with favorable pharmacological properties.

4.2. SHP2 catalytic site inhibitors containing salicylic acid groups

To target both the active site and an adjacent, peripheral secondary binding site in SHP2, the Zhang group constructed a salicylic acid-based combinatorial library, where the salicylic acid core (containing an alkynyl group) was tethered to different azide based units through the click reaction. By this approach the hit compound 8 (Fig. 3B) was generated, which appeared to be the most potent for SHP2 with an IC₅₀ of 5.5 μM in this series and exhibited at least several-fold selectivity for SHP2 over all PTPs examined.⁷⁸ In HEK293 and NIH3T3 cells, 8 strongly inhibited SHP2 dependent EGF-induced ERK1/2 activation. Furthermore, 8 inhibited SHP2 in intact cells with similar potency to that toward the isolated enzyme, which demonstrated that 8 was cell permeable. In SHP2 E76K-, and SHP2 D61Y-expressing hematopoietic progenitors from JMML patients, 8 was effective at significantly reducing granulocyte macrophage colony stimulating factor (GM-CSF)-stimulated hyperproliferation. X-ray crystallographic analysis of the compound 8-bound SHP2 structure (PDB: 3JRL) revealed that the salicylic acid core occupied the PTP active site, while the distal biphenyl ring made hydrophobic contacts with a region highly divergent among the PTPs. The molecular basis of SHP2 inhibition by compound 8 can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

In order to increase the binding affinity and selectivity for SHP2, the Zhang group took a structure-guided approach to target the SHP2 active site with hydroxyindole carboxylic acid. In combination with a fragment-based library strategy to introduce additional functionalities to interact with peripheral sites proximal to the catalytic pocket, they found a novel hydroxyindole carboxylic acid-based SHP2 inhibitor compound 9 (Fig. 3B), with an IC₅₀ value of 200 nM and a 7- and 11-fold preference for SHP2 over its closely related homologue SHP1 and PTP1B, respectively.⁷⁹ Structural analysis and molecular modeling revealed that the hydroxyindole carboxylic acid anchored the inhibitor to the SHP2 active site, while interactions of the oxalamide linker and the phenylthiophene tail with residues in the β5–β6 loop contributed to the binding potency and selectivity of 9. In H1975 cells, 9 effectively reduced the EGF-induced ERK1/2 phosphorylation and increased paxillin phosphorylation on Y118. Moreover, 9 exhibited excellent antiproliferative activity not only in H1975 cells but also in ErbB2 positive breast cancer cell line SKBR3, and oncogenic KITD814V expressing 32D myeloid cells and primary low-density bone marrow cells. As a cellular potent SHP2 catalytic site inhibitor, 9 can serve as a useful tool to interrogate the function of SHP2 in normal physiology.

4.3. SHP2 catalytic site inhibitors derived from natural products

Using computer-aided drug design (CADD) screening based on the 3D structure of the SHP2 protein, a database of natural products was screened, this effort led to the identification of cryptotanshinone (10, Fig. 3C) as an inhibitor of SHP2.⁸⁰ Cryptotanshinone inhibited the SHP2-catalyzed hydrolysis of a phosphopeptide substrate with an IC₅₀ of 22.50 μM, which was approximately 1.76-fold selective for SHP2 over its homologue SHP1. This hit compound inhibited the SHP2 E76K mutant with an IC₅₀ of 23.90 μM that was equal to the IC₅₀ value for the SHP2 PTP domain (22.50 μM). In IL-3-dependent Ba/F3 cells, this compound displayed a significant growth inhibitory effect with an IC₅₀ of 17.22 μM, and IL-3 signaling processes including RAS/ERK, PI3K/AKT, and JAK2/STAT5 pathways were down-regulated following the treatment with cryptotanshinone. Colony formation of myeloid blasts from JMML patients with the PTPN11^E76K/+ mutation was exquisitely sensitive to cryptotanshinone, additionally, U973 human lymphoma cells harboring an activating mutation (G60R) of PTPN11 and of H661 human lung cancer cells carrying another activating mutation (N58S) of PTPN11 with the compound decreased cell proliferation in a dose-dependent manner. Because cryptotanshinone functioned much more effectively in primary leukemic cells from human patients even at the concentrations lower than its in vitro IC₅₀, the cryptotanshinone derivatives (Fig. 3C), i.e., tanshinone I (11), dihydrotanshinone (12), and tanshinone IIA (13) on SHP2 activity were tested. And indeed these three derivatives potently inhibited SHP2. The IC₅₀ of tanshinone I, dihydrotanshinone, and tanshinone IIA was 8.75-, 5.71-, and 8.69-fold lower than that of cryptotanshinone, respectively, even though these compounds showed no selectivity between SHP2 and SHP1. Computer docking predicted that cryptotanshinone bonds to a specific site where two carbonyl oxygen atoms formed hydrogen bonds with Lys364 and Lys366 at the periphery of the catalytic cleft. Since cryptotanshinone is used to treat cardiovascular diseases in Asian countries, this study provided the first evidence that cryptotanshinone was an inhibitor of SHP2 and thus has potential to be used directly or to be further developed to treat PTPN11-associated malignancies.

5. SHP2 inhibitors targeting the allosteric sites

After more than a decade of development, SHP2 catalytic site inhibitors have not achieved the desired in vivo efficacy yet. Compound 9, the most active catalytic site inhibitor, showed promising efficacy in melanoma models.⁸¹ However, another recent study has called into question the specificity of such active site-directed inhibitors in the cellular context.⁸² As a result, scientists turned their research focus to the allosteric pattern of SHP2, which paved a new avenue for SHP2-targeting therapeutics research.

In 2016, the first SHP2 allosteric inhibitor from Novartis was reported (the selected examples 14–18, Fig. 4A).^83,84 The team designed a novel drug screening paradigm: using DIFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) as a substrate⁸⁵ and bisphosphotyrosyl peptide (2P-IRS-1) to activate SHP2, compounds that can inhibit the full-length SHP2 protein (residues 1-525) were screened from the Novartis compound library, and active site inhibitors were removed on the basis of their activity in the phosphatase activity assay utilizing the SHP2 PTP domain (residues 237-529). This process led to the identification of compound SHP836 (14, Fig. 4A), which had an IC₅₀ = 12 μM against the full-length enzyme and an IC₅₀ > 100 μM against the PTP domain. The X-ray cocrystal structure for SHP836 with SHP2 (PDB: 5EHP) revealed that it did not bind to the PTP catalytic site but to a tunnel-like region formed between the C-terminal SH2, N-terminal SH2, and PTP domains, and stabilized the inactive conformation for SHP2 where the catalytic site was blocked and no longer accessible to the substrate. Then, a SAR study was performed, morphing the piperazine ring to a 4-methyl-4-aminopiperidine motif, and the central pyrimidine ring was replaced with the pyrazine ring, which led to the generation of a promising molecule SHP099 (15), with a >70-fold improvement in biochemical potency to IC₅₀ = 0.071 μM. Furthermore, SHP099 showed no detectable activity against a panel of 21 phosphatases and 66 kinases, including SHP1, supporting its high degree of target selectivity. The crystal structure of SHP099 in complex with SHP2 (PDB: 5EHR) revealed that key SHP099 interactions included hydrogen bonds with Arg111 and Phe113 located on the linker between the N-SH2 and C-SH2 domains, as well as Glu250 from the PTP domain. Furthermore, the dichlorophenyl group of SHP099 made extensive hydrophobic interactions with the PTP domain. In SHP2-dependent MDA-MB-468 and KYSE520 cells, SHP099 inhibited p-ERK with an IC₅₀ of ∼0.25 μM. SHP099 also inhibited cell growth in SHP2-dependent haematopoietic cell lines, such as MV-411, MOLM-13 and Kasumi-1. In an in vivo study, SHP099 was administered by oral gavage to nude mice with KYSE520 xenografts and yielded marked tumor growth inhibition. SHP099 is the first example of a potent, selective and orally bioavailable allosteric PTP inhibitor specific to SHP2 that is efficacious and well tolerated in patient-derived tumour xenograft models.

Fig. 4. Representative SHP2 inhibitors targeting the allosteric sites.

Subsequently, Novartis used SiteMap in Maestro revealed three potential small molecule binding sites: (1) the allosteric “tunnel” previously reported for SHP099; (2) the “latch” binding site, located approximately 20 Å from the tunnel; (3) the “groove” site on the opposite side of the protein.⁸⁶ Considering the possibility of multiple allosteric pockets, they used a mutant SHP2 protein (SHP2^T253M/Q257L) to construct a new screening paradigm. A library of approximately 1.5 million compounds was screened as previously reported using DIFMUP as the substrate and 2P-IRS-1 to activate SHP2. Finally, a series of compounds with unknown inhibition modalities were found, including the triazoloquinazolinone, SHP244 (16), a weak SHP2 inhibitor (SHP2^1-525 IC₅₀ = 60 μM). X-ray crystallography (PDB: 6BMR) showed that SHP244 bound to a previously predicted “latch” pocket between the N-SH2 and PTP domains. Based on the derivatization of the structure, compounds SHP844 (17, IC₅₀ = 18.9 μM) and SHP504 (18, IC₅₀ = 21 μM) were designed to evaluate their SHP2 biochemical inhibition. In KYSE-520 cancer cells, 16, 17, and 18 at 30 μM reduced the MAPK pharmacodynamic marker DUSP6 levels by 65%, 43%, and 55%, respectively. Combining allosteric site 1 inhibitors with allosteric site 2 inhibitors was not particularly effective, this may be related to the weak cellular activity of these site 2 inhibitors. So further optimization was required to improve the potency and permeability of these site 2 inhibitors. Moreover, the third groove pocket noted above can be utilized in the future.

Recently, Novartis scientists combined the pharmacophore in the structure of SHP099 with the compounds obtained through high-throughput screening and successfully generated pyrazolopyrimidinone 19 (the structure-optimized examples 19–23, Fig. 4B) which can effectively inhibit the biochemical activity of SHP2 (IC₅₀ = 0.067 μM).⁸⁷ Analysis of the cocrystal structure of 19 in the SHP2 allosteric binding pocket (PDB: 6MDB) confirmed that it bound to the same allosteric tunnel site as SHP099. Then the SAR study was performed. Extension and conformational restriction of the amine substituent led to the discovery of 20, this maneuver improved both SHP2-mediated phosphorylation of ERK kinase (IC₅₀ = 0.012 μM) and enhanced by 10-fold the antiproliferative activity against KYSE520 cells (IC₅₀ = 0.167 μM), but it had poor ether-a-go-go related gene (hERG) selectivity. After that, decoration of the chloroarene with nitrogen heteroatoms and introduction of the cyclopropane group delivered compound 21, which successfully balanced high biochemical and cellular potency with impressive hERG selectivity (Q-Patch IC₅₀ > 30 μM). Unfortunately, the compound 21 had poor permeability and oral bioavailability (∼2% F), that made it impossible to support further tumor efficacy studies. In order to obtain a more desirable SHP2 allosteric inhibitor, morphing the fused, bicyclic system into a novel monocyclic pyrimidinone scaffold through the SAR study and use of structure-based design, which led to the birth of SHP394 (22, IC₅₀ = 0.023 μM) with high selectivity over hERG (IC₅₀ > 30 μM), and no off-target activity was observed in a set of ion channels and kinases, and G-protein coupled receptor (GPCR) analysis.⁸⁸ In the Detroit-562 mouse model, SHP394 was orally administered, leading to reduction in tumor volume and expression of pharmacodynamic marker DUSP6 mRNA. Overall, aminopyrimidinone SHP394 and additional analogues provide compelling potent, selective, and orally bioavailable SHP2 inhibitors for future investigation of the multifaceted roles of SHP2 in signal transduction and human diseases.

Revolution medicines has been committed to the development of allosteric inhibitors targeting SHP2. Among them, RMC-4630 is an oral effective SHP2 small molecule allosteric inhibitor, which has entered the clinical trial stage. The structure of RMC-4630 has not been disclosed, but the pharmacodynamics and mechanism of its homologue RMC-4550 (23, IC₅₀ = 0.583 nM) have been fully studied and reported. RMC-4550 is an allosteric inhibitor of SHP2 with a mode of inhibition similar to that described previously for SHP099.⁸⁹ In three cell lines carrying class 3 BRAF mutations, NCI-H1666 (BRAF^G466V/+), NCI-H508 (BRAF^G596R/+) and CAL-12T (BRAF^G466V/+), RMC-4550 can cause suppression of both RAS-GTP and p-ERK levels, the same result was also observed in the sensitive NF1^LOF cell lines NCI-H1838 (lung, NF1^N184fs) and MeWo (melanoma, NF1^Q1336*) and KRAS^G12 mutations cell lines NCI-H358 and MIA PaCa-2. Furthermore, RMC-4550 treatment decreases oncogenic RAS/RAF/MEK/ERK signaling and cancer growth by disrupting SOS1-mediated RAS-GTP loading. This study illuminates the key functions of SHP2 in promoting RAS/MAPK pathway activation in RAS-GTP-dependent oncogenic BRAF, NF1 loss, and nucleotide cycling oncogenic KRAS. For cancer patients carrying these oncogenic drivers, inhibition of SHP2 is a promising effective molecular therapeutic strategy.

At present, allosteric inhibitors targeting wild-type SHP2 protein have made great progress, but for some carcinogenic SHP2 mutant proteins (such as SHP2^E76K, SHP2^G60V, SHP2^S502P, etc.), the effect of allosteric inhibitors is not ideal.⁹⁰ A biochemical screen against SHP2^E76A was conducted with a library of about 20 000 compounds at 20 μM.⁹¹ And then filtered out the catalytic inhibitors using the SHP2 PTP domain. One compound, 24, was identified to show inhibition against SHP2^E76A with an IC₅₀ of 19.1 μM, but no effect on SHP2^PTP (the SHP2 mutant allosteric inhibitors 24–29, Fig. 4C). A brief replacement of phenyl with biphenyl (25 and 26) or naphthyl (27) led to improved potency with an IC₅₀ of 3.27, 5.32, and 2.55 μM, respectively. The crystal structure of SHP2^E76A in complex with 25 (PDB: 5XZR) shows that compound 25 is accommodated in the same pocket, as previously described for SHP099 but with distinct binding features, so the SAR study was performed. Substitution of various substituted aryl and piperidinyl moieties yields compound 28 directed to the SHP2^E76A mutant with an IC₅₀ of 0.7 μM but no inhibition on SHP2^PTP. Selectivity profiling revealed that 28 exhibited more than 30-fold selectivity over other PTPs. In NCI-H661 cells bearing SHP2^N58S GOF mutation and H1975 cells, 28 strongly inhibited ERK activation. In SF268 and A549 cells, 28 inhibited the yes-associated protein (YAP)-dependent reporter and the expression of YAP target genes, suggesting that YAP activity is modulated by the SHP2 PTP catalytic function. Compound 28 inhibited the proliferation of a variety of cancer cell lines in vitro. And in vivo, it exhibited good PK profiling and antitumor activity in an MV4;11 xenograft model. The results suggest that allosteric inhibition of SHP2 may be a feasible approach for cancer treatment.

To identify small molecular compounds that bind to and stabilize the autoinhibited conformation to functionally inhibit SHP2, SiteMap was used to explore a potential drug-binding site in SHP2 for inhibitor discovery.⁹² Five possible binding sites in SHP2 were identified in this study, among them, site 1 of SHP2 showed specific features, as compared to the corresponding region of the homology SHP1, and it was located at the interface between the C-SH2 and PTP domains of SHP2. So site 1 in SHP2 was chosen for subsequent CADD in silico database screening. Finally, compound 29 was identified as the most active compound with IC₅₀ values of 9.8 μM and 72.7 μM against SHP2 and SHP1, respectively. Docking simulation analyses demonstrated that 29 created three hydrogen bonds with Arg111, Lys129, and Arg229 and occupied site 1 with high binding energy. In the SHP2^E76K mutant, compound 29 inhibited it in a dose-dependent manner, with an IC₅₀ value of 7.67 μM. In BaF3 cells, this compound significantly suppressed cell growth and reduced the activation of SHP2 mediated signaling including ERK, AKT, JAK2 and STAT5 induced by IL3. Moreover, human lung cancer cells H661 carrying the activating mutation (N58S) in PTPN11 are more sensitive to 29 than human lung cancer cells H596 with wild type (WT) PTPN11. And colony formation of myeloid blasts from JMML patients with the PTPN11^E76K/+ mutation was exquisitely sensitive to 29. Therefore, SHP2 is a useful therapeutic target for PTPN11 mutation-associated hematological malignancies. In addition, 29/SHP2 binding affinity is low, further structure–activity relationship analysis is required for this compound, and cocrystallization of 29 and SHP2 is required to reveal the structural bases of their binding interaction.

Although small molecule inhibitors that stabilize SHP2 autoinhibition (e.g., SHP099) have great potential for targeting cancers, the potency of such compounds can be strongly attenuated by cancer-associated GOF mutations that disrupt the integrity of the autoinhibitory interaction.⁹³ Therefore, another allosteric pocket with a nonconserved Cys333 on the PTP domain was identified. To identify compounds that can allosterically inhibit SHP2 through the covalent reaction with C333 and avoid reaction with conserved cysteines within the PTP domain, a library was screened through the SHP2^WT and SHP2^C333P mutant assay. A compound that selectively inhibited SHP2^WT over SHP2^C333P was likely to act at SHP2's allosteric site, as the 333 position contained the only molecular difference between the two enzymes. After screening, they found that compound 30 (Fig. 4C) had significantly higher inhibitory activity against SHP2^WT than SHP2^C333P. A simple SAR study indicated that the quinoline ring of compound 30 was necessary. Substitution of cyanoacrylamide with acrylamide produced compound 31 (Fig. 4C), which had an IC₅₀ of 35 μM for the SHP2^WT PTP domain. Meanwhile, compound 31 retained its specificity for Cys333 on the PTP domain, even at the highest compound concentration (150 μM), it did not significantly inhibit the SHP2^C333P mutant. The liquid-chromatographic tandem-mass-spectrometric analysis (LC–MS/MS) showed that about 20% of compound 31 covalently and irreversibly acted on Cys333 and did not bind to conserved cysteine residues (Cys459, Cys367) in SHP2. In addition to Cys333, compound 31 also acted on other non-conserved cysteine residues (Cys259, Cys318, and Cys486) on the surface and/or loop of the protein, but these effects may not have a significant impact on SHP2 activity. In a word, the interaction of compound 31 with the non-conserved cysteine residue Cys333 in SHP2 demonstrates the possibility of targeting SHP2 allosteric sites with covalent inhibitors. This lays the foundation for the development of highly effective and specific SHP2 allosteric site covalent inhibitors.

6. Conclusions

SHP2 has attracted widespread attention as a carcinogenic tyrosine phosphatase. Initially, researchers focused on inhibitors targeting the SHP2 PTP catalytic domain. However, due to the highly conserved PTP domain, as well as its polar and positively charged environment, catalytic site inhibitors often lack selectivity, cell permeability and oral bioavailability, which has led to the failure of SHP2 catalytic site inhibitors to advance to the clinic. Since Novartis reported the first allosteric site inhibitor in 2016, several small molecule SHP2 allosteric inhibitors have entered clinical trials, including TNO155, RMC4630, JAB3068 and JAB3312 which are used to evaluate the therapeutic effects on solid tumors.

Although SHP2 is a reasonable target for tumors due to hyperactivation involved in the development and promotion of certain cancers, mutations in PTPN11 have been detected in human developmental diseases and several cancers. Some molecules against these mutants have been reported, for example molecule 28 against SHP2^E76A. More effective inhibitors of SHP2 activating mutants need further development. The combination of inhibitors that target different allosteric sites may be a strategy for inhibiting SHP2 oncogenic mutants. In addition, SHP2 inhibitors can be combined with a protein kinase inhibitor such as a MEK inhibitor to doubly inhibit interconnected signaling pathways, which may be more effective than monotherapy and less drug resistant.

Recent reports indicate that SHP2 inhibitors have clinical utility in the management of mutant KRAS-driven cancers,^94–96 and with the successful development of KRAS inhibitors,^97–99 the combined application of SHP2 and KRAS inhibitors may achieve better therapeutic effects. Moreover, SHP2 is a key downstream effector of the immune checkpoint receptor PD-1 in T cells, therefore the design of small molecules targeting the SHP2-PD-1 interaction during immune response holds promise for the development of a new class of immune modulators.

Conflicts of interest

There are no conflicts to declare.

Biographies

Biography

Jiao Kong.

Jiao Kong received her B. S. in Pharmaceutical Engineering in 2017 from Shandong University of Traditional Chinese Medicine. In 2021, she completed her M.S. in medicinal chemistry at Soochow University, Suzhou, China, under the supervision of Prof. Ya-Qiu Long, working on the design, synthesis and SAR study of druggable Protacs against SHP2 protein. Since July of 2021 she has been a Research Associate at CellCarta biopharmaceutical company, focusing on delivering customized biomarker testing solutions for precision medicine.

Biography

Ya-Qiu Long.

Ya-Qiu Long obtained a B.S. from Sichuan University, a M.S. and a Ph.D. in organic chemistry from Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China, and completed postdoctoral studies in medicinal chemistry at National Institutes of Health, the U.S.A. She is currently Distinguished Professor and Vice Dean at Suzhou Medical College of Soochow University, China. She is interested in the design and discovery of molecular targeted antitumor agents and antivirals with new structures and new mechanisms, and has established a featured and generally applied system to design non-enzymatic site binding inhibitors and multiple targeting ligands, for traditionally “undruggable” targets.