title: Development of a high-throughput AlphaScreen assay measuring full-length LRRK2(G2019S) kinase activity using moesin protein substrate.
abstract: Mutations within the LRRK2 (leucine-rich repeat kinase 2) gene predispose humans to develop late-onset Parkinson's disease (PD). The most prevalent of these mutations, G2019S, has been shown to increase LRRK2 kinase activity. Therefore, the discovery of small molecule inhibitors of LRRK2(G2019S) through high-throughput screening (HTS) may provide a novel therapeutic strategy for treating PD. Current biochemical assays monitoring the activity of LRRK2(G2019S) either are radioactive or use short peptidic substrates. Here we describe the development and optimization of a novel HTS AlphaScreen assay for measuring the catalytic activity of full-length LRRK2(G2019S) using its putative physiological protein substrate moesin. The high sensitivity of this optimized 384-well assay allowed the use of enzyme concentrations as low as 0.75nM. The estimated apparent K(m) value for adenosine triphosphate (6 microM) using the glutathione S-transferase-moesin substrate was much lower than the one previously reported using LRRKtide, a synthetic peptide derived from moesin. Testing of nonselective kinase inhibitors (staurosporine, H-1152, and Y-27632) generated potencies consistent with published data. Finally, robotic validation of the assay yielded an average Z' factor of 0.80. Overall, these results indicate that the present HTS AlphaScreen assay might provide a more relevant biochemical approach for the discovery of novel LRRK2(G2019S) inhibitors.
authors.affiliation: PerkinElmer, Montreal, QC, Canada.
authors.forename: Liliana
authors.initials: L
authors.lastname: Pedro
authors.forename: Jaime
authors.initials: J
authors.lastname: Padrós
authors.forename: Lucille
authors.initials: L
authors.lastname: Beaudet
authors.forename: Hans-Dieter
authors.initials: HD
authors.lastname: Schubert
authors.forename: Frank
authors.initials: F
authors.lastname: Gillardon
authors.forename: Sophie
authors.initials: S
authors.lastname: Dahan
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D057166:High-Throughput Screening Assays; D006801:Humans; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D008840:Microfilament Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
chemical_list: D008840:Microfilament Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; C071058:moesin; D000255:Adenosine Triphosphate; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases