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Abstract
BackgroundDual-specificity 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.MethodspNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 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 tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate.ResultsThe most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 μM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD3_2320) with IC50 of 33 μM that is similar to NTS and does not aggregate.ConclusionsThe new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12858-015-0048-3) contains supplementary material, which is available to authorized users.
Highlights
Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease
Docking and ligand-based in silico searches yield candidate small molecules that target the DUSP5 phosphatase domain (PD) domain In this study, we were interested in identifying inhibitors that could selectively target dual-specificity phosphatase 5 (DUSP5), which we have shown previously to be mutated in patients with vascular anomalies
The X-ray structure of PD of human DUSP5 was previously reported (PDB:2G6Z) [16], while the structure of ERK binding domain (EBD) was constructed using homology modeling based on the solution structure (21 % identity and 35 % homology) of human MKP-3 protein (PDB:1HZM) as a template [35]
Summary
Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. MAPKs can be deactivated by phosphatases that remove these phosphate groups from their activation loop One such class of phosphatases, dual-specificity phosphatases (DUSPs), is unique in that it can dephosphorylate both serine/threonine and tyrosine residues. We identified a clinically relevant serine to proline mutation (S147P) that is associated with vascular defects [2] This mutation has been shown previously by our group to interfere with the dephosphorylating activity of DUSP5 protein [8], and makes the protein hypoactive. In terms of the vasculature, the DUSP5 KO rat displays enhanced myogenic response and autoregulation of cerebral blood flow [15] Taken together, these studies demonstrate that inhibition of DUSP5 will result in biologically relevant changes in vivo
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