Identification and characterization of novel tissue-nonspecific alkaline phosphatase inhibitors with diverse modes of action.
Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme expressed at high levels in bone, liver, and kidney. It appears involved in dephosphorylation of numerous phosphate monoesters, but only 2 of them, pyrophosphate and pyridoxal phosphate, have yet been unequivocally documented. Discovery and characterization of other substrates could be considerably facilitated if specific and potent modulators of TNAP activity with various modes of action were available. Here, the authors describe in detail a high-throughput screening campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A novel homogeneous luminescent TNAP assay was developed and optimized with respect to the enzyme and substrate concentrations, enabling identification of a large number of compounds overlooked by a conventional colorimetric assay. Several new chemical series were identified from screening the Molecular Libraries Small Molecule Repository (MLSMR) collection and demonstrated to have diverse selectivity and mode of inhibition profiles. The nanomolar potency of some of these scaffolds surpasses currently known inhibitors. This article provides an example of a success where the Roadmap Initiative collaborative model, sponsored by the National Institutes of Health, brought together a deep knowledge of target biology from a principal investigator's laboratory, a well-designed compound collection from the MLSMR, and an industrial-level screening facility and staff at the MLSCN center to identify pharmacologically active compounds, with outstanding selectivity data from a panel of more than 200 publicly accessible assays, through a high-throughput screen.
Sergienko
Eduard
E
Conrad Prebys Center for Chemical Genomics, Burnham Institute for Medical Research, La Jolla, California 92037, USA. esergien@burnham.org
Su
Ying
Y
Chan
Xochella
X
Brown
Brock
B
Hurder
Andrew
A
Narisawa
Sonoko
S
Millán
José Luis
JL
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors*; D000818:Animals; D055162:Biocatalysis / Q000187:drug effects; D019556:COS Cells; D002522:Chlorocebus aethiops; D003124:Colorimetry; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D008163:Luminescent Measurements; D009928:Organ Specificity / Q000187:drug effects; D015203:Reproducibility of Results; D013379:Substrate Specificity / Q000187:drug effects; D000077145:Sulfanilamide; D013424:Sulfanilamides / Q000737:chemistry
D004791:Enzyme Inhibitors; D013424:Sulfanilamides; D000077145:Sulfanilamide; D000469:Alkaline Phosphatase