title: A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson's Disease Using RapidFire Mass Spectrometry.
abstract: LRRK2 is a large multidomain protein containing two functional enzymatic domains: a GTPase domain and a protein kinase domain. Dominant coding mutations in the LRRK2 protein are associated with Parkinson's disease (PD). Among such pathogenic mutations, Gly2019Ser mutation in the LRRK2 kinase domain is the most frequent cause of familial PD in Caucasians and is also found in some apparently sporadic PD cases. This mutation results in 2- to 3-fold elevated LRRK2 kinase activity compared with wild type, providing a clear clinical hypothesis for the application of kinase inhibitors in the treatment of this disease. To date, reported screening assays for LRRK2 have been based on detection of labeled adenosine triphosphate and adenosine diphosphate or on antibody-based detection of phosphorylation events. While these assays do offer a high-throughput method of monitoring LRRK2 kinase activity, they are prone to interference from autofluorescent compounds and nonspecific events. Here we describe a label-free assay for LRRK2 kinase activity using the RapidFire mass spectrometry system. This assay format was found to be highly robust and enabled a screen of 100,000 lead-like small molecules. The assay successfully identified a number of known LRRK2 chemotypes that met stringent physicochemical criteria.
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK melanie.v.leveridge@gsk.com.
authors.forename: Melanie
authors.initials: M
authors.lastname: Leveridge
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK Cancer Research Technology, Babraham Research Campus, Cambridge, UK.
authors.forename: Lee
authors.initials: L
authors.lastname: Collier
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK.
authors.forename: Colin
authors.initials: C
authors.lastname: Edge
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK.
authors.forename: Phil
authors.initials: P
authors.lastname: Hardwicke
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK.
authors.forename: Bill
authors.initials: B
authors.lastname: Leavens
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK.
authors.forename: Steve
authors.initials: S
authors.lastname: Ratcliffe
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK.
authors.forename: Mike
authors.initials: M
authors.lastname: Rees
authors.affiliation: Neurodegeneration DPU, Neurosciences Therapy Area Unit, GlaxoSmithKline, Pharmaceuticals R&D, Hertfordshire, UK, and Pudong, China Nuevolution A/S, Rønnegade 8, DK-2100 Copenhagen, Denmark.
authors.forename: Luigi Piero
authors.initials: LP
authors.lastname: Stasi
authors.affiliation: Department of Platform Technology and Science, GlaxoSmithKline Pharmaceuticals R&D, Hertfordshire, UK.
authors.forename: Alan
authors.initials: A
authors.lastname: Nadin
authors.affiliation: Neurodegeneration DPU, Neurosciences Therapy Area Unit, GlaxoSmithKline, Pharmaceuticals R&D, Hertfordshire, UK, and Pudong, China.
authors.forename: Alastair D
authors.initials: AD
authors.lastname: Reith
mesh_terms: D000818:Animals; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D013058:Mass Spectrometry / Q000379:methods; D009154:Mutation / Q000235:genetics; D010300:Parkinson Disease / Q000188:drug therapy*; D010766:Phosphorylation / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D017434:Protein Structure, Tertiary / Q000235:genetics; D061987:Sf9 Cells
chemical_list: D018076:DNA, Complementary; D047428:Protein Kinase Inhibitors; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases; D020558:GTP Phosphohydrolases
keywords: LRRK2; LRRKtide; Parkinson’s disease; RapidFire; mass spectrometry