Inhibition of SHP2 and SHP1 Protein Tyrosine Phosphatase Activity by Chemically Induced Dimerization.

Protein tyrosine phosphatases (PTPs), the enzymes that catalyze the dephosphorylation of phosphotyrosine residues, are important regulators of mammalian cell signaling, whose activity is misregulated in numerous human diseases. PTPs are also notoriously difficult to selectively modulate with small molecules, and relatively few small-molecule tools for controlling their activities in the context of complex signaling pathways have been developed. Here, we show that a chemical inducer of dimerization (CID) can be used to selectively and potently inhibit constructs of Src-homology-2-containing PTP 2 (SHP2) that have been engineered to contain dimerization domains. Our strategy was inspired by the naturally occurring mechanism of SHP2 regulation, in which the PTP activity of SHP2's catalytic domain is autoinhibited through an intramolecular interaction with the protein's N-terminal SH2 (N-SH2) domain. We have re-engineered this inhibitory interaction to function intermolecularly by independently fusing the SHP2 catalytic and N-SH2 domains to protein domains that heterodimerize upon the introduction of the CID rapamycin. We show that rapamycin-induced protein dimerization leads to potent inhibition of SHP2's catalytic activity, which is driven by increased proximity of the SHP2 catalytic and N-SH2 domains. We also demonstrate that CID-based inhibition of PTP activity can be applied to an oncogenic gain-of-function SHP2 mutant (E76K SHP2) and to the catalytic domain of the SHP2's closest homologue, SHP1. In sum, CID-driven inhibition of PTP activity provides a broadly applicable tool for inhibiting dimerizable forms of the SHP PTPs and represents a novel paradigm for selective PTP inhibition through inducible protein-protein interactions.

Targeting a Pathogenic Cysteine Mutation: Discovery of a Specific Inhibitor of Y279C SHP2.

An intriguing challenge of drug discovery is targeting pathogenic mutant proteins that differ from their wild-type counterparts by only a single amino acid. In particular, pathogenic cysteine mutations afford promising opportunities for mutant-specific drug discovery, due to the unique reactivity of cysteine's sulfhydryl-containing side chain. Here we describe the first directed discovery effort targeting a pathogenic cysteine mutant of a protein tyrosine phosphatase (PTP), namely Y279C Src-homology-2-containing PTP 2 (SHP2), which has been causatively linked to the developmental disorder Noonan syndrome with multiple lentigines (NSML). Through a screen of commercially available compounds that contain cysteine-reactive functional groups, we have discovered a small-molecule inhibitor of Y279C SHP2 (compound 99; IC50 ≈ 6 µM) that has no appreciable effect on the phosphatase activity of wild-type SHP2 or that of other homologous PTPs (IC50 ≫ 100 µM). Compound 99 exerts its specific inhibitory effect through irreversible engagement of Y279C SHP2's pathogenic cysteine residue in a manner that is time-dependent, is substrate-independent, and persists in the context of a complex proteome. To the best of our knowledge, 99 is the first specific ligand of a disease-causing PTP mutant to be identified. This study therefore provides both a starting point for the development of NSML-directed therapeutic agents and a precedent for the identification of mutant-specific inhibitors of other pathogenic PTP mutants.

Chemical activation of divergent protein tyrosine phosphatase domains with cyanine-based biarsenicals.

Strategies for the direct chemical activation of specific signaling proteins could provide powerful tools for interrogating cellular signal transduction. However, targeted protein activation is chemically challenging, and few broadly applicable activation strategies for signaling enzymes have been developed. Here we report that classical protein tyrosine phosphatase (PTP) domains from multiple subfamilies can be systematically sensitized to target-specific activation by the cyanine-based biarsenical compounds AsCy3 and AsCy5. Engineering of the activatable PTPs (actPTPs) is achieved by the introduction of three cysteine residues within a conserved loop of the PTP domain, and the positions of the sensitizing mutations are readily identifiable from primary sequence alignments. In the current study we have generated and characterized actPTP domains from three different subfamilies of both receptor and non-receptor PTPs. Biarsenical-induced stimulation of the actPTPs is rapid and dose-dependent, and is operative with both purified enzymes and complex proteomic mixtures. Our results suggest that a substantial fraction of the classical PTP family will be compatible with the act-engineering approach, which provides a novel chemical-biological tool for the control of PTP activity and the study of PTP function.

The Allosteric Site on SHP2's Protein Tyrosine Phosphatase Domain is Targetable with Druglike Small Molecules.

Difficulties in developing active-site-directed protein tyrosine phosphatase (PTP) inhibitors have led to the perception that PTPs are "undruggable", highlighting the need for new means to target pharmaceutically important PTPs allosterically. Recently, we characterized an allosteric-inhibition site on the PTP domain of Src-homology-2-domain-containing PTP 2 (SHP2), a key anticancer drug target. The central feature of SHP2's allosteric site is a nonconserved cysteine residue (C333) that can potentially be labeled with electrophilic compounds for selective SHP2 inhibition. Here, we describe the first directed discovery effort for C333-targeted allosteric SHP2 inhibitors. By screening a previously reported library of reversible, covalent inhibitors, we identified a lead compound, which was modified to yield an irreversible inhibitor (12), that inhibits SHP2 allosterically and selectively through interaction with C333. These findings provide a novel paradigm for allosteric-inhibitor discovery on SHP2, one that may help to circumvent the challenges inherent in targeting SHP2's active site.