LRRK2-IN-1 is an effective and selective LRRK2 inhibitor.

LRRK2 (G2019S):6 nM, DCLK2:45 nM, LRRK2 (WT):13 nM

在稳定表达GFP-LRRK2[G2019S]的HEK 293细胞中，LRRK2-IN-1改变了LRRK2的细胞质定位。在人源神经母细胞瘤SHSY5Y细胞和小鼠Swiss 3T3细胞中，LRRK2-IN-1引起剂量依赖性的Ser910和Ser935去磷酸化以及14-3-3蛋白与内源性LRRK2的结合丧失。[1] 在表达人类R1441C-和G2019S-LRRK2的转基因C. elegans中，LRRK2-IN1挽救了多巴胺能受损特有的行为缺陷。[2] 在小鼠成纤维细胞中，LRRK2-IN1降低了细胞移动性。[3] 在AsPC-1和HCT116细胞系中，LRRK2-IN-1显示出抗增殖和促凋亡属性，诱导G1和G2/M细胞周期阻滞，并抑制DCLK1 mRNA和蛋白表达。[4]

在野生型雄性C57BL/6小鼠中，LRRK2-IN-1（100 mg/kg，i.p.）抑制肾脏中LRRK2的Ser910和Ser935去磷酸化，而在大脑中无效果。[1]

IC50 determination: Active GST-LRRK2 (1326-2527), GST-LRRK2 [G2019S] (1326-2527), GST-LRRK2 [A2016T] (1326-2527) and GST-LRRK2 [A2016T+G2019S] (1326-2527) enzyme is purified with glutathione sepharose from HEK293 cell lysate 36 h following transient transfection of the appropriate cDNA constructs. Peptide kinase assays, performed in duplicate, are set up in a total volume of 40 μL containing 0.5 μg LRRK2 kinase (which at approximately 10% purity gives a final concentration of 8 nM) in 50 mM Tris/HCl, pH 7.5, 0.1 mM EGTA, 10 mM MgCl2, 20 μM Nictide, 0.1 μM [γ-32P]ATP (~500 cpm/pmol) and the indicated concentrations of inhibitor dissolved in DMSO. After incubation for 15 min at 30 °C, reactions are terminated by spotting 35 μL of the reaction mix onto P81 phosphocellulose paper and immersion in 50 mM phosphoric acid. Samples are washed extensively and the incorporation of [γ-32P]ATP into Nictide is quantified by Cerenkov counting. IC50 values are calculated with GraphPad Prism using non-linear regression analysis.

Cells are seeded into a 96-well tissue culture plate in triplicate. The cells are cultured in the presence of LRRK2-IN-1 with DMSO as a vehicle at 0, 0.31, 0.63, 1, 2, and 5, 10, and 20 μM. 48 h post treatment, 10 μL of TACS MTT Reagent (RND Systems) is added to each well and the cells are incubated at 37°C until dark crystalline precipitate became visible in the cells. 100 μL of 266 mM NH4OH in DMSO is then added to the wells and placed on a plate shaker at low speed for 1 minute. After shaking, the plate is allowed to incubate for 10 minutes protected from light and the OD550 for each well is read using a microplate reader. The results are averaged and calculated as a percentage of the DMSO (vehicle) control +/- the standard error of the mean.(Only for Reference)