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LRRK2-IN-1 Catalog No.GC10809

LRRK2 inhibitor,cell-permeable and ATP competitive

Size Price Stock Qty
10mM (in 1mL DMSO)
$91.00
In stock
10mg
$65.00
In stock
50mg
$236.00
In stock
100mg
$331.00
In stock

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Sample solution is provided at 25 µL, 10mM.

Quality Control

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Protocol

Kinase experiment:

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.

Cell experiment:

Cells (104 cells 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 20 μM. 48 h post treatment, 10 μL 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. 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.

Animal experiment:

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.

References:

[1]. Hermanson SB, et al. Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation.PLoS One. 2012;7(8):e43580. Epub 2012 Aug 28.
[2]. Deng, Xianming., et al. Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nature Chemical Biology (2011), 7(4), 203-205.
[3]. Koshibu K, et al. Alternative to LRRK2-IN-1 for Pharmacological Studies of Parkinson's Disease. Pharmacology. 2015;96(5-6):240-7.
[4]. Weygant N, et al. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1. Mol Cancer. 2014 May 6;13:103.

Chemical Properties

Cas No. 1234480-84-2 SDF
Synonyms LRRK 2-IN-1
Chemical Name 2-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidine-1-carbonyl]anilino]-5,11-dimethylpyrimido[4,5-b][1,4]benzodiazepin-6-one
Canonical SMILES CN1CCN(CC1)C2CCN(CC2)C(=O)C3=CC(=C(C=C3)NC4=NC=C5C(=N4)N(C6=CC=CC=C6C(=O)N5C)C)OC
Formula C31H38N8O3 M.Wt 570.7
Solubility ≥28.55mg/mL in DMSO, ≥57.2mg/mL in EtOH, <2.87mg/mL in H2O Storage Store at -20°C
General tips For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months.
Shipping Condition Evaluation sample solution : ship with blue ice
All other available size: ship with RT , or blue ice upon request
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Background

LRRK2-IN-1 is a potent and selective inhibitor of LRRK2 with IC50 value of 13 nM. [1]
LRRK2 (Leucine-rich repeat kinase 2) is also known dardarin. LRRK2 belongs to the leucine-rich repeat kinase family. LRRK2 mostly located in the cytoplasm, however, it also associates with outer membrane of the mitochondrial. LRRK2 interacts with parkin at the C-terminal R2 RING finger domain. Parkin interacted with LRRK2 at the COR domain. LRRK2 mutation will induce apoptotic cell death of neuroblastoma cells. Expression of LRRK2 mutants has a close relationship with autosomal dominant Parkinson's disease. The LRRK2 Gly2019Ser mutation is a common cause of familial Parkinson's disease. The Gly2019Ser mutation has been proved to cause Parkinson's disease, even though it is a small number of LRRK2 mutations. LRRK2 also has relationship with Crohn's disease by genomewide association studies.
LRRK2-IN-1 dose-dependent inhibit the activity of wild-type and G2019S LRRK2 with IC50 of 0.17 and 0.04μM respectively in HEK293 cells stably expressing GFP tagged wild-type or mutated LRRK2.[2] LRRK2-IN-1 inhibits the activity of both wild-type and G2019S mutant LRRK2 with IC 50 values of 13 nM and 6 nM respectively in vitro enzyme assay with 0.1 mM ATP. LRRK2-IN-1 competed with ATP. LRRK2-IN-1 has a selective profile compared with other 442 diverse kinases and it has no inhibition effect with LRRK1. LRRK2-IN-1 induced endogenous LRRK2 phosphorylation in lymphoblastoid cells. LRRK2-IN-1 also induced Ser910/Ser935 dephosphorylation of LRRK2 in mice kidney at 100 mg/kg.[1]
References:
[1].    Deng X, Dzamko N, Prescott A, Davies P, Liu Q, Yang Q, Lee JD, Patricelli MP, Nomanbhoy TK, Alessi DR et al: Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2. Nat Chem Biol, 7(4):203-205.
[2].    Hermanson SB, Carlson CB, Riddle SM, Zhao J, Vogel KW, Nichols RJ, Bi K: Screening for novel LRRK2 inhibitors using a high-throughput TR-FRET cellular assay for LRRK2 Ser935 phosphorylation. PLoS One, 7(8):e43580.