Identification of Polysulfonated Inhibitors that Target Dual Specificity Phosphatase 5 and Provide New Insights into the Binding Requirements for Dual-Phosphate Substrate Pockets

Dual specific phosphatase‐5 (DUSP5) plays a central role in vascular development and disease. We present a p ‐nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5. pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds with a K m of 7.6 + 0.5 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual‐phosphorylated ERK2 substrate (pThr‐Glu‐pTyr). A first lead molecule has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified an analog that is FDA approved (making it a candidate for repurposing), and is a potent and competitive inhibitor (25 + 5 µM). Further screening and ligand optimization identified additional analogs, including two new scaffolds – one based on a diphenyl‐ether core, and another based on a fused tricyclic core; the latter formerly FDA approved as a drug for topical use. Modeling suggests these inhibitors may have broader relevance for mimicking the dual‐phosphate loops recognized by DUSPs, as they position sulfonates or other negatively charged groups where the two phosphates on the substrate peptide (pThr‐Glu‐pTyr) bind.