Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets.
BACKGROUND
Dual-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.


METHODS
pNPP 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.


RESULTS
The 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 (CSD (3) _2320) with IC₅₀ of 33 μM that is similar to NTS and does not aggregate.


CONCLUSIONS
The 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.
Department of Chemistry and Biochemistry, Texas Wesleyan University, 1201 Wesleyan Ave., Fort Worth, TX, 76105, USA. tneumann@txwes.edu.|Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA. tneumann@txwes.edu.
Terrence S
TS
Neumann
Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA. elise.span@gmail.com.
Elise A
EA
Span
Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA. kekalous@mcw.edu.
Kelsey S
KS
Kalous
Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA. Robert.bongard@cuw.edu.
Robert
R
Bongard
Department of Pediatrics, Department of Obstetrics and Gynecology, Medical College of Wisconsin, Children's Research Institute (CRI) Developmental Vascular Biology Program, Translational and Biomedical Research Center, Milwaukee, WI 53226, USA. agastong@gmail.com.
Adam
A
Gastonguay
Department of Pediatrics, Department of Obstetrics and Gynecology, Medical College of Wisconsin, Children's Research Institute (CRI) Developmental Vascular Biology Program, Translational and Biomedical Research Center, Milwaukee, WI 53226, USA. mlepley@mcw.edu.
Michael A
MA
Lepley
Department of Pediatrics, Department of Obstetrics and Gynecology, Medical College of Wisconsin, Children's Research Institute (CRI) Developmental Vascular Biology Program, Translational and Biomedical Research Center, Milwaukee, WI 53226, USA. rkutty@mcw.edu.
Raman G
RG
Kutty
Department of Pediatrics, Department of Obstetrics and Gynecology, Medical College of Wisconsin, Children's Research Institute (CRI) Developmental Vascular Biology Program, Translational and Biomedical Research Center, Milwaukee, WI 53226, USA. jnayak@mcw.edu.
Jaladhi
J
Nayak
Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA.
Chris
C
Bohl
Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA.
Rachel G
RG
Lange
Department of Chemistry, Wehr Chemistry Building, P.O. Box 1881, 535 N. 14th Street, Milwaukee, WI 53201, USA. majher.sarker@ars.usda.gov.
Majher I
MI
Sarker
Department of Chemistry, Wehr Chemistry Building, P.O. Box 1881, 535 N. 14th Street, Milwaukee, WI 53201, USA. marat.talipov@marquette.edu.
Marat R
MR
Talipov
Department of Chemistry, Wehr Chemistry Building, P.O. Box 1881, 535 N. 14th Street, Milwaukee, WI 53201, USA. rajendra.rathore@marquette.edu.
Rajendra
R
Rathore
Department of Pediatrics, Department of Obstetrics and Gynecology, Medical College of Wisconsin, Children's Research Institute (CRI) Developmental Vascular Biology Program, Translational and Biomedical Research Center, Milwaukee, WI 53226, USA. rramchan@mcw.edu.
Ramani
R
Ramchandran
Center for Structure-based Drug Design and Development, Department of Pharmaceutical Sciences, and School of Pharmacy, Concordia University of Wisconsin, 12800 N. Lake Shore Drive, Mequon, WI 53097, USA. daniel.sem@cuw.edu.
Daniel S
DS
Sem
D020134:Catalytic Domain; D003198:Computer Simulation; D004353:Drug Evaluation, Preclinical; D054637:Dual-Specificity Phosphatases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000378:metabolism; D062105:Molecular Docking Simulation; D010710:Phosphates / Q000378:metabolism*; D011485:Protein Binding; D013498:Suramin / Q000378:metabolism / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008024:Ligands; D010710:Phosphates; D013498:Suramin; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; C442320:DUSP5 protein, human; D054637:Dual-Specificity Phosphatases