Protein phosphatases, while long overlooked, have recently become appreciated as drivers of both normal- and disease-associated signaling events. As a result, the spotlight is now turning torwards this enzyme family and efforts geared towards the development of modern chemical tools for studying these enzymes are well underway. This Minireview focuses on the evolution of chemical activity probes, both optical and covalent, for the study of protein phosphatases. Small-molecule probes, global monitoring of phosphatase activity through the use of covalent modifiers, and targeted fluorescence-based activity probes are discussed. We conclude with an overview of open questions in the field and highlight the potential impact of chemical tools for studying protein phosphatases.

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The ability for density functional theory with the B3LYP functional with the lanl2dz basis set to predict the 1st (Wave 1) and 2nd (Wave 2) reductions of the diazine ring in a series of thirty-seven (37) 1,4-di-N-oxide quinoxaline-2-carboxamide derivatives in dimethylformamide was examined. The B3LYP/lanl2dz method had a strong correlation and low correlation to the experimental potentials for Wave 1 and Wave 2, respectively. There are nine identifiable analogs based on similarities of structure. The predicted reduction potentials for the derivatives of each analog generally fit the modified Hammett equation. The B3LYP/lanl2dz method is shown to be useful in accurately predicting the Wave 1 potentials for quinoxaline-di-N-oxide derivatives. For derivatives with assessable anti-tuberculosis activity, the predicted Wave 1 potentials have a similar correlation with the bioactivity when compared to the experimental wave 1 potentials.

Protein phosphatases act in concert with protein kinases to regulate and maintain the phosphoproteome. However, the catalog of chemical tools to directly monitor the enzymatic activity of phosphatases has lagged behind their kinase counterparts. In this chapter, we provide protocols for repurposing the phosphorylation-sensitive sulfonamido-oxine fluorophore known as Sox to afford direct activity probes for phosphatases. With validated activity probes in-hand, inhibitor screens can be conducted with recombinant enzyme and the role of phosphatases in cell signaling can be investigated in unfractionated cell lysates.

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