Protein tyrosine phosphatases (PTPs) are a diverse superfamily of enzymes that catalyze the hydrolysis of regulatory phosphate groups on a tyrosine residue of target proteins. The reaction produces an inorganic phosphoric acid salt, and controls the activity of the target protein. These enzymes dominate fundamental cellular processes in mammalian cells, such as cell growth, cellular differentiation, mitotic cycles, oncogenic transformation, receptor endocytosis, and immune cell activation. Consequently, dysregulation of PTPs is associated with various cancers, cardiovascular diseases, autoimmune responses, and diabetes. Nonetheless, our understanding of cellular PTP chemical biology remains limited; the cellular functions of the majority of PTPs are still a mystery, and basic questions concerning the compartmentalization, temporal dynamics, and the role of isoforms in signaling remain unanswered. This lack of information is a historical consequence of the view that PTPs were mere house-keeping enzymes that counteract protein tyrosine kinases (PTKs). This misconception contributed to the imbalance of tools to study PTKs versus PTPs. For example, no cell-based assays capable of directly monitoring PTP enzymatic activity exist. To address this critical need, we envisioned a hybrid protein/small molecule-based assay system in which engineered, high-affinity protein-protein interaction (PPI) domains are used to drive the selectivity and affinity of a small molecule PTP activity probe towards a particular PTP of interest. We hypothesized that the relatively tight binding affinity of the appended PPI domains would increase the local concentration of a generic small molecule activity probe at the target PTP, resulting in a tighter apparent Michaelis-Menten constant (K<sub>m</sub>). Thus, we anticipate the ability to selectively monitor the activity of a target PTP in the presence of excess off-target PTPs. Current progress towards this goal will be presented.

Toward cell-based protein tyrosine phosphatase activity assays

ACS Spring 2022

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