Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dual-specificity protein phosphatase inhibitors.
We report here the miniaturization, development, and implementation of a homogeneous 384-well fluorescence intensity high-throughput screening (HTS) assay for identifying mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) dual-specificity phosphatase inhibitors. As part of the National Institutes of Health (NIH) Molecular Libraries Screening Center Network (MLSCN), the MKP-1 assay was utilized to screen an NIH diversity library of 65,239 compounds for inhibitors of MKP-1 activity at 10 microM and was also used to confirm the concentration dependence of active agents identified in the primary screen. We observed 100 (0.15%) compounds that inhibited MKP-1 in vitro by > or =50% at 10 microM in the primary assay, and 46 of the 100 compounds were confirmed as concentration-dependent inhibitors of MKP-1 with 50% inhibitory concentration (IC(50)) values of <50 microM; four exhibited IC(50) values <1.0 microM, six produced IC(50) values in the 1-10 microM range, and 36 produced IC(50) values in the 10-50 microM range. A clustering and classification analysis of the compound structures of the 46 confirmed MKP-1 inhibitors produced 29 singleton structures and seven clusters of related structures. Some MKP-1 inhibitors were members of structural classes or contained substructure pharmacophores that previously were reported to inhibit either MKP-1 or other protein tyrosine phosphatases, validating the HTS assay. Importantly, we have identified several attractive and novel MKP-1 inhibitor structures that warrant further investigation as potential probes to study the biology of MKP-1 and its role in controlling the amplitude and/or duration of MAPK signaling, cell survival, and tumor progression.
Johnston
Paul A
PA
Pittsburgh Molecular Libraries Screening Center, Department of Pharmacology, University of Pittsburgh Drug Discovery Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA. paj18@pitt.edu
Foster
Caleb A
CA
Shun
Tong Ying
TY
Skoko
John J
JJ
Shinde
Sunita
S
Wipf
Peter
P
Lazo
John S
JS
D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors*; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D054638:Dual Specificity Phosphatase 1; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D017874:Immediate-Early Proteins / Q000037:antagonists & inhibitors*; D020935:MAP Kinase Signaling System / Q000502:physiology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*
D018797:Cell Cycle Proteins; D004791:Enzyme Inhibitors; D017874:Immediate-Early Proteins; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases