title: Development of a high-throughput screening method for LIM kinase 1 using a luciferase-based assay of ATP consumption.
abstract: Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)-binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases.
authors.affiliation: The Beatson Institute for Cancer Research, Glasgow Scotland, UK.
authors.forename: Mokdad
authors.initials: M
authors.lastname: Mezna
authors.forename: Ai Ching
authors.initials: AC
authors.lastname: Wong
authors.forename: Margaret
authors.initials: M
authors.lastname: Ainger
authors.forename: Rebecca W
authors.initials: RW
authors.lastname: Scott
authors.forename: Tim
authors.initials: T
authors.lastname: Hammonds
authors.forename: Michael F
authors.initials: MF
authors.lastname: Olson
mesh_terms: D051339:Actin Depolymerizing Factors / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D004789:Enzyme Activation / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D054461:Lim Kinases / Q000037:antagonists & inhibitors*; D008156:Luciferases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D054852:Small Molecule Libraries
chemical_list: D051339:Actin Depolymerizing Factors; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008156:Luciferases; D054461:Lim Kinases