Targeting Nonconserved and Pathogenic Cysteines of Protein Tyrosine Phosphatases with Small Molecules.

Protein tyrosine phosphatases (PTPs) are important therapeutic targets for a range of human pathologies. However, the common architecture of PTP active sites impedes the discovery of selective PTP inhibitors. Our laboratory has recently developed methods to inhibit PTPs allosterically by targeting cysteine residues that either (i) are not conserved in the PTP family or (ii) result from pathogenic mutations. Here, we describe screening protocols for the identification of selective inhibitors that covalently engage such "rare" cysteines in target PTPs. Moreover, to elucidate the breadth of possible applications of our cysteine-directed screening protocols, we provide a brief overview of the nonconserved cysteines present in all human classical PTP domains.

Quantitation of autoinhibitory defects in pathogenic SHP2 mutants by differential scanning fluorimetry.

Src-homology-2-domain-containing protein tyrosine phosphatase-2 (SHP2) is a signaling enzyme whose activity is governed by an equilibrium between autoinhibited and activated states. Regulation of SHP2 activity is critical for cellular homeostasis, and mutations that alter its autoregulatory equilibrium cause cancers and developmental disorders. Several methods for assessing the strength of autoinhibitory interactions in SHP2 mutants have been previously reported, but each has limitations. We show that differential scanning fluorimetry provides a rapid, quantitative measure of SHP2 autoinhibition that is independent of the intrinsic activity of the SHP2 mutant being analyzed, does not involve protein labeling, and does not require specialized instrumentation.