Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson's disease. We employed a new, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor, LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910 and Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson's disease.

Figure 1. Enzymatic activity of LRRK2-IN-1 and its selectivity

(a) Chemical structure of the LRRK2 inhibitor LRRK2-IN-1. (b) GST-LRRK2 (1326-2517), GST-LRRK2 [G2019S] (1326-2527), GST-LRRK2 [A2016T] (1326-2517) and GST-LRRK2 [A2016T+G2019S] (1326-2517) were assayed using 20µM Nictide in the presence of 100 µM ATP with the indicated concentrations of LRRK2-IN-1. The results are presented as percentage of kinase activity relative to the DMSO treated control. Results are averages of duplicate reactions with similar results obtained in at least one other experiment. The IC₅₀ values, in µM, were derived from the graphs. (c) The combined kinase profiling hits with biochemical activities. Kinase hits from Ambit profiling with a score of less than 10% of the DMSO control at a concentration of 10 µM, ActivX profiling with higher than 50% inhibition at 1 µM, and Dundee profiling with greater than 50% inhibition at 1 µM are listed. The biochemical IC₅₀ values of hits were measured by Invitrogen Adapta®, Z´-Lyte®, Lantha-Screen® assays (http://www.invitrogen.com/site/us/en/home/Products-and-Services/Services/Screening-and-Profiling-Services/SelectScreen-Profiling-ervice/SelectScreen-Kinase-Profiling-Service.html) and Dundee radioactive-base enzyme assay. *Assessment of PLK1 cellular activity of LRRK2-IN-1 (see Supplementary Results).

Figure 2. LRRK2-IN-1 inhibits LRRK2 in vivo

(a) HEK 293 cells stably expressing wild type GFP-LRRK2 were treated with DMSO or increasing concentrations of LRRK2-IN-1 for 90 min. Cell lysates were subjected to immunoblotting for detection of LRRK2 phosphorylated at S910 and S935 and for total GFP-LRRK2. 14-3-3 binding was measured by overlay assay following immunoprecipitation of LRRK2 with GFP-binder Sepharose. (b) As in (a) except HEK 293 cells stably expressing GFP-LRRK2 [G2019S] were utilized. (c) As in (a) except HEK 293 cells stably expressing drug resistant GFP-LRRK2 [A2016T] were utilized. (d) As in (a) except HEK 293 cells stably expressing drug resistant GFPLRRK2[ A2016T+G2019S] cells were utilized. (e) Endogenous LRRK2 was immunoprecipitated with anti-LRRK2 100-500 from EBV immortalized human lymphoblastoid cells from a control subject and a Parkinson’s disease patient homozygous for the LRRK2 [G2019S] mutation. (f) Following treatment of the cells with DMSO or the indicated concentrations of LRRK2-IN-1 for 90 min. Immunoprecipitates were subjected to immunoblot analysis with the indicated antibody as well as 14-3-3 overlay for western analysis. Immunoprecipitations were performed in duplicate and results are representative of at least two independent experiments. (g) As in (e) except that human SHSY5Y cells were utilized. (h) 100 mg/kg LRRK2-IN-1 was administered by intraperitoneal injection into wild type mice (n=3 per group). Tissues were collected and endogenous LRRK2 was immunoprecipitated and analysed for S910 and S935 phosphorylation as above. Quantitation of immunoblots along with original images are available in Supplementary Fig. 11

Figure 3. LRRK2-IN-1 alters the cytoplasmic localisation of LRRK2

HEK 293 cells stably expressing GFP-LRRK2 [G2019S] were treated with 1 µM LRRK2-IN-1 for 2 h. Cells were then washed and fixed with 4% paraformaldehyde for fluorescence imaging. To visualize LRRK2 localization following removal of LRRK2-IN-1, inhibitor-containing media was removed 2 h after treatment. Cells were then washed twice and incubated with fresh media without inhibitor for 2 h before again being washed and then fixed for fluorescence imaging. Representative micrographs of three independent experiments are presented.