LRRK2-IN-1 CAS 1234480-84-2 Kev Ua Yeeb Yam

Hauv Vitro

Tsiaj qus-hom thiab G2019S kev sib hloov ua rau muaj 2.5 npaug siab dua TR-FRET teeb liab uas tuaj yeem raug thaiv los ntawmLRRK2-IN-1kev txhaj tshuaj-siv nrog yam nrog IC50 qhov tseem ceeb ntawm 0.08 µM thiab 0.03 µM, feem [1]. LRRK2-IN-1 muaj IC50 ntawm 45 nM rau kev tiv thaiv ntawm DCLK2 thiab ua kom pom qhov IC50 ntau dua 1 µM thaum ntsuas hauv biochemical assays rau AURKB, CHEK2, MKNK2, MYLK, NUAK1, thiab PLK1. LRRK2-IN-1 tau lees tias yuav inhibit MAPK7 nrog EC50 ntawm 160 nM. LRRK2-IN-1 tsim ib koob tshuaj tiv thaiv kev cia siab ntawm Ser910 thiab Ser935 phosphorylation nrog poob ntawm 14-3-3 khi rau ob hom tsiaj qus LRRK2 thiab LRRK2 [G2019S] stably kis mus rau hauv HEK293 hlwb [2]. LRRK2-IN-1 yog cytotoxic me me nrog IC50 ntawm 49.3 49M hauv HepG2 hlwb. LRRK2-IN-1 nthuav qhia genotoxicity nyob rau hauv muaj thiab tsis muaj S9 ntawm 15.6 thiab 3.9 µM, ntsig txog [3]. LRRK2-IN-1 inhibits kev loj hlob, tsiv teb tsaws, thiab ntxias cov cell tuag nrog kev pom ntawm apoptosis ntawm HCT116 thiab AsPC-1 hlwb [4] ..

Hauv Vivo

LRRK2-IN-1 (100 mg / kg, ip) ua rau dephosphorylation ntawm LRRK2 nyob rau hauv lub raum ntawm cov nas [2]. Peritumoral txhaj tshuaj ntawm LRRK2-IN-1 (100 mg / kg) ua rau muaj qhov txo qis ntawm cov qog loj thiab cov nyhav ntawm AsPC-1 lub qog ua haujlwm ntawm cov leeg [4].

Kinase Assay

Nquag GST-LRRK2 (1326-2527), GST-LRRK2 [G2019S] (1326-2527), GST-LRRK2 [A2016T] (1326-2527) thiab GST-LRRK2 [A2016T + G2019S] (1326-2527) ) enzyme yog huv nrog glutathione sepharose los ntawm HEK293 cell lysate 36 h tom qab hloov cov kev hloov pauv ntawm cDNA tsim nyog. Peptide kinase tshaj tawm, ua nyob rau hauv theej tawm, tau teeb tsa hauv lub ntim tag nrho ntawm 40 µL uas muaj 0.5 Lg LRRK2 kinase (uas kwv yees li 10% purity muab qhov kawg ntawm 8 nM) hauv 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) thiab qhov qhia tau ntau ntawm inhibitor yaj hauv DMSO. Tom qab tsim kom sov rau 15 min ntawm 30 ° C, kev tawm tsam yog kaw los ntawm qhov chaw 35 µL ntawm cov tshuaj tiv thaiv sib xyaw rau P81 phosphocellulose daim ntawv thiab muab tso rau hauv 50 mM phosphoric acid. Cov piv txwv raug ntxuav ntau heev thiab kev koom ua ke ntawm [γ-32P] ATP rau hauv Nictide yog suav los ntawm Cerenkov suav. IC50 qhov tseem ceeb yog muab xam nrog GraphPad Prism siv txoj kev tsom xam cov kab tsis muaj qhov ntsuas.

Cell Ntsuas

Cov hlwb (104 hlwb ib qhov dej) tau ua noob rau hauv 96-lub cev nqaij zoo hauv lub phaj hauv triplicate. Cov qog hlwb tau tawm mus kuaj nrog LRRK2-IN-1 nrog DMSO ua lub tsheb ntawm 0, 0.31, 0.63, 1, 2, thiab 5, 10, thiab 20 μM. 48 h post kev kho mob, 10 μL ntawm TACS MTT Reagent ntxiv rau txhua qhov dej thiab lub hlwb tsim tawm ntawm 37 ° C kom txog thaum tsaus ntuj crystalline nag lossis daus ua kom pom nyob hauv cov hlwb. 100 μL ntawm 266 mM NH4OH hauv DMSO tom qab ntawd ntxiv rau qhov dej thiab muab tso rau ntawm lub plhaws phaj kub ntawm qhov nrawm li 1 feeb. Tom qab tshee, lub phaj tau tso cai rau 10 feeb tiv thaiv los ntawm lub teeb thiab OD550 rau txhua qhov dej yog nyeem siv microplate nyeem ntawv. Cov txiaj ntsig yog qhov nruab nrab thiab suav raws li qhov feem pua ​​ntawm DMSO (tsheb) kev tswj hwm + / - tus qauv yuam kev ntawm qhov phem.

Tsoom thawj coj tsiaj

LRRK2-IN-1yog yaj nyob rau hauv Captisol thiab tswj los ntawm kev txhaj tshuaj rau hauv cov tsiaj qus hom txiv neej C57BL / 6 nas ntawm ib koob ntawm 100 mg / kg. Kev tswj cov nas yog txhaj nrog lub suab sib luag ntawm Captisol. Ntawm 1 thiab 2 teev lub sijhawm ua haujlwm, cov nas tau tua los ntawm lub ncauj tsev menyuam thiab lub raum thiab lub hlwb cov qog ua kom sai thiab tawg-khov hauv cov kua nitrogen.

Ua tim khawv

[1]. Hermanson SB, li al. Kev Kuaj Rau Cov Khoom Tshiab LRRK2 Cov Tshuaj Tso Cai Siv Cov Khoom Siv Muaj Tshaj-TR-FRET Cov Khoom Siv rau LRRK2 Ser935 Phosphorylation.PLoS Ib. 2012; 7 (8): e43580. Epub 2012 Aug 28.

[2]. Deng, Xianming., Li al. Cov yam ntxwv ntawm tus yam tsis haum tshuaj tiv thaiv tus kab mob Parkinson' s kinase LRRK2. Natural Chemology (2011), 7 (4), 203-205.

[3]. Koshibu K, li al. Hloov mus rau LRRK2-IN-1 rau Pharmacological Studies ntawm Parkinson' s Kab Mob. Kws Tshuaj. Xyoo 2015; 96 (5-6): 240-7.

[4]. Weygant N, li al. Cov roj me me molecule kinase inhibitor LRRK2-IN-1 qhia txog kev ua haujlwm muaj zog tiv thaiv mob hnyuv thiab lub qog nqaij hlav ntawm txoj kev ua kom tsis taus cov ntshav zoo li ob lub kinase 1. Mol Cancer. 2014 Lub Tsib Hlis 6; 13: 103