title: Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2--discovery of LRRK2 inhibitors.
abstract: LRRK2 is a large and complex protein that possesses kinase and GTPase activities and has emerged as the most relevant player in PD pathogenesis possibly through a toxic gain-of-function mechanism. Kinase activity is a critical component of LRRK2 function and represents a viable target for drug discovery. We now report the development of a mechanism-based TR-FRET assay for the LRRK2 kinase activity using full-length LRRK2. In this assay, PLK-peptide was chosen as the phosphoryl acceptor. A combination of steady-state kinetic studies and computer simulations was used to calculate the initial concentrations of ATP and PLK-peptide to generate a steady-state situation that favors the identification of ATP noncompetitive inhibitors. The assay was also run in the absence of GTP. Under these conditions, the assay was sensitive to inhibitors that directly interact with the kinase domain and those that modulate the kinase activity by directly interacting with other domains including the GTPase domain. The assay was optimized and used to robustly evaluate our compound library in a 384-well format. An inhibitor identified through the screen was further characterized as a noncompetitive inhibitor with both ATP and PLK-peptide and showed similar inhibition against LRRK2 WT and the mutant G2019S.
authors.affiliation: Laboratory for Drug Discovery in Neurodegeneration, Brigham and Women's Hospital, 65 Landsdowne Street, Fourth Floor, Cambridge, MA 02139, USA. mliu@rics.bwh.harvard.edu
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Shibu
authors.initials: S
authors.lastname: Poulose
authors.forename: Eli
authors.initials: E
authors.lastname: Schuman
authors.forename: Alexandra D
authors.initials: AD
authors.lastname: Zaitsev
authors.forename: Brittany
authors.initials: B
authors.lastname: Dobson
authors.forename: Ken
authors.initials: K
authors.lastname: Auerbach
authors.forename: Kathleen
authors.initials: K
authors.lastname: Seyb
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Cuny
authors.forename: Marcie A
authors.initials: MA
authors.lastname: Glicksman
authors.forename: Ross L
authors.initials: RL
authors.lastname: Stein
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Yue
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D018797:Cell Cycle Proteins / Q000737:chemistry; D055808:Drug Discovery; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007700:Kinetics; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000737:chemistry; D000097869:Polo-Like Kinase 1
chemical_list: D000255:Adenosine Triphosphate; D018797:Cell Cycle Proteins; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D010455:Peptides; D047428:Protein Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D000097869:Polo-Like Kinase 1; C495280:LRRK2 protein, human