Whole-cell LRRK2 radioligand binding. (A) Chemical structure of LRRK2 radioligand ([³⁵S]MLi-A). (B) Whole-cell saturation binding with increasing concentrations of [³⁵S]MLi-A in Tet-inducible wild-type LRRK2 expressing SHSY5Y cells. Binding was performed in the absence of induction (noninduced) or where expression was induced for 48 hours in the presence of tetracycline (Tet-induced). The results are presented as specific binding and are averages of triplicate reactions. The K_D value, in nM, is derived from the graph. (C) Displacement of [³⁵S]MLi-A binding with MLi-2. Experiments were performed with 0.7 nM [³⁵S]MLi-A and increasing concentrations of MLi-2. The results are presented as specific binding and are averages of triplicate reactions. The K_i value, in nM, is derived from the graph. CPM, counts per minute.

Effect of oral administration of various doses of MLi-2 on pSer935 LRRK2 in the brains of mice after acute, single administration. (A) Western blot and (B) quantification of LRRK2 pSer935 LRRK2 levels. (C) Unbound brain concentration of MLi-2. Data are mean ± S.E.M.; n = 5 mice per treatment group.*P < 0.05–P < 0.001 compared with the vehicle-treated animals.

Effect of MLi-2 (3–120 mg/kg per day) on pSer935 LRRK2 (ratio of pSer935/total LRRK2) and total LRRK2 (total LRRK2/GAPDH) in the brains of mice after 11 days of in-diet dosing. (A) Western blot showing pSer935 LRRK2 and total LRRK2. (B) and (C) shows the quantification of pSer935 LRRK2 and total LRRK2, respectively. (D) Unbound brain concentrations of MLi-2 on day 11 in mice administered MLi-2 in diet for 11 days. Open circles, 3 mg/kg per day; 10 mg/kg per day, open squares; 30 mg/kg per day, closed triangles; 60 mg/kg per day, open triangles; and 100 mg/kg, closed circles. (E) pSer935 LRRK2 levels (ratio of pSer935/total LRRK2 expressed as a percentage of the vehicle control group) in the brains of mice versus unbound brain concentrations of MLi-2 (3–120 mg/kg per day) on day 11 of in-diet dosing. Mice were euthanized a 4, 8, and 24 hours after the start of the dark cycle on day 11 of dosing. Data are mean ± S.E.M.; n = 5 per treatment group; ***P < 0.001 compared with the vehicle diet–treated animals.

Chronic in-diet dosing with MLi-2 (30 mg/kg per day) is well tolerated and leads to sustained inhibition of CNS LRRK2 kinase activity. (A) Bodyweight, (B) food intake, and (C) dose achieved per week of the study. Effect of MLi-2 at 30 mg/kg per day on pSer935 LRRK2 levels in the cortex of mice after (D) 3 weeks on treatment, (F) 9 weeks on treatment, and (H) 15 weeks on treatment. Panels (E), (G), and (I) show the Western blot data for pSer935 LRRK2, total LRRK2, and GAPDH from each time point. MLi-2 treatment started at 5 weeks of age. N = 10 mice/group per time point. Data are mean ± S.E.M.; n = 10 per treatment group; *P < 0.001 compared with the vehicle diet–treated animals.

Chronic in-diet dosing with MLi-2 (30 mg/kg per day) does not attenuate the behavioral phenotype in in MitoPark mice. Effect of MLi-2 on (A) distance traveled (B) rearing measured for 60 minutes. MLi-2 treatment started at 5 weeks of age. Data are mean ± S.E.M.; n = 10 per treatment group. *P < 0.05; ***P < 0.001 compared with the vehicle diet–treated animals.