Liu, MinPoulose, ShibuSchuman, EliZaitsev, Alexandra D.Dobson, Brittany C.Auerbach, KenSeyb, Kathleen I.Cuny, Gregory D.Glicksman, Marcie A.Stein, Ross L.Yue, Zhenyu2020-03-102020-03-102011-19-15Copyright 2010 Analytical Biochemistry. This is a post-print version of a published paper that is available at: https://www.sciencedirect.com/science/article/pii/S0003269710003520. Recommended citation: Liu, Min, Shibu Poulose, Eli Schuman, Alexandra D. Zaitsev, Brittany Dobson, Ken Auerbach, Kathleen Seyb et al. "Development of a mechanism-based high-throughput screen assay for leucine-rich repeat kinase 2—discovery of LRRK2 inhibitors." Analytical biochemistry 404, no. 2 (2010): 186-192.. doi: 10.1016/j.ab.2010.05.033. This item has been deposited in accordance with publisher copyright and licensing terms and with the author's permission.https://hdl.handle.net/10657/5963LRRK2 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.en-USLRRK2 kinaseAssay and anlaysisArticle