LRRK2-IN-1

LRRK2-IN-1 is a potent and selectiveLRRK2inhibitor withIC50of 6 nM and 13 nM for LRRK2 (G2019S) and LRRK2 (WT), respectively.

IC50& Target

IC50: 13 nM (WT), 6 nM (G2019S)

体外

Wild-type and G2019S transduction results in 2.5 fold higher TR-FRET signal which can be inhibited by LRRK2-IN-1 in a dose-dependent manner with IC50values of 0.08 µM and 0.03 µM, respectively. LRRK2-IN-1 possessed an IC50of 45 nM for inhibition of DCLK2 and exhibits an IC50of greater than 1 µM when evaluated in biochemical assays for AURKB, CHEK2, MKNK2, MYLK, NUAK1, and PLK1. LRRK2-IN-1 is confirmed to inhibit MAPK7 with an EC50of 160 nM. LRRK2-IN-1 induces a dose dependent inhibition of Ser910 and Ser935 phosphorylation accompanied by loss of 14-3-3 binding to both wild type LRRK2 and LRRK2[G2019S] stably transfected into HEK293 cells. LRRK2-IN-1 is moderately cytotoxic with IC50of 49.3 µM in HepG2 cells. LRRK2-IN-1 exhibits genotoxicity in the presence and absence of S9 at 15.6 and 3.9 µM, respectively. LRRK2-IN-1 inhibits proliferation, migration, and induces cell death with hallmarks of apoptosis of HCT116 and AsPC-1 cells.

体内

LRRK2-IN-1 (100 mg/kg, i.p.) induces dephosphorylation of LRRK2 in the kidney of the mice. Peritumoral injection of LRRK2-IN-1 (100 mg/kg) results in a significant decrease in tumor volume and weight of AsPC-1 tumor xenografts.

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. IC50values are calculated with GraphPad Prism using non-linear regression analysis.

细胞实验：

Cells (104cells per well) 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 20μM. 48h post treatment, 10μL of TACS MTT Reagent is added to each well and the cells are incubated at 37°C until dark crystalline precipitate become visible in the cells. 100μL of 266mM NH4OH in DMSO is then added to the wells and placed on a plate shaker at low speed for 1minute. After shaking, the plate is allowed to incubate for 10minutes protected from light and the OD550for 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.

动物实验

LRRK2-IN-1 is dissolved in Captisol and administered by intraperitoneal injection into wild type male C57BL/6 mice at a dose of 100 mg/kg. Control mice are injected with an equal volume of Captisol. At 1 and 2 h time points, mice are extinguwashed by cervical dislocation and kidney and brain tissue rapidly dissected and snap-frozen in liquid nitrogen.

URMC-099

MB1136

Lenalidomide

MB1040

Bortezomib(PS-341)

1 mg

5 mg

10 mg

1 mM

1.7523 mL

8.7613 mL

17.5226 mL

5 mM

0.3505 mL

1.7523 mL

3.5045 mL

10 mM

0.1752 mL

0.8761 mL

1.7523 mL