NQDI 1

Kinase experiment:

Enzyme activity of human protein kinases ASK1, Aurora A, ROCK1, HGFR, FGFR1, Tie2, JNK3, and CK2 is determined using in vitro kinase assay (γ-32P-ATP method). Each reaction mixture contains 6 μL of buffer solution (25 mM MOPS, pH 7.2, 2.5 mM EGTA, 2.5 mM EDTA, 0.5 mM DTT, 0.25 mg/mL BSA, 20 mM β-glycerophosphate), 3 μL of substrate solution (MBP, Long S6 kinase substrate peptide, KKKSPGEYVNIEFG, IGF-IRtide (12-527), TK substrate 2, JNK3tide, or RRRDDDSDDD for each kinase, respectively) (5.0 μg/μL), 0.3 μL of enzyme (protein kinase catalytic subunit, 0.1 μg/μL≈32 mU/μL), and 10.25 μL of H2O. The reaction mixture (total volume of 19 μL) is quickly added to 1.5 mL tubes at room temperature. The stock solutions of inhibitors (e.g., NQDI-1) are prepared in DMSO, and the concentration of inhibitor is 1 mM. The concentration of DMSO in the reaction does not exceed 3%. Then 1 μL of inhibitor solution in DMSO is added to each tube and mixed by pipetting. ATP solution is prepared separately. For each sample 0.05 mCi γ-[P32]ATP is taken (specific activity of 100 μCi/μM). The total concentration of labeled and unlabeled ATP is 100 μM. The reaction is started with addition of ATP solution (150 μM ATP, 30 mM MgCl2, 15 mM MOPS, pH 7.2). The time of reaction is 20 min at 30°C. The reaction is stopped by adding 20 μL of 0.5 M orthophosphoric acid. Then the reaction mixture is loaded on the 20 mm filter disks of the cellulose phosphate paper. Filters are washed three times with 0.075 M orthophosphoric acid at room temperature and dried. For detection of products, dried filters are counted by Tri-Carb 2800-TR liquid scintillation analyzer. Then 1 μL of DMSO is added to the reaction volume instead of the inhibitor stock solution for a positive control[1].

Animal experiment:

Rats[2]A total of 12 female Sprague-Dawley rats with litters of mixed gender pups are used. The mothers are housed at 25°C under a 12-h light/dark cycle, with ad libitum access to food and water, until the pups are 7-days-old. The HI model is established. The pups are anesthetized with 2.5% halothane and are intracerebroventricularly infused with DMSO or 250 nmol NQDI-1, dissolved in DMSO into the right cerebral hemisphere 30 min prior to HI using a 30-gauge needle with a 5 μL Hamilton syringe (infusion rate, 1 μL/min).

References:

[1]. Volynets GP, et al. Identification of 3H-naphtho[1,2,3-de]quinoline-2,7-diones as inhibitors of apoptosis signal-regulating kinase 1 (ASK1). J Med Chem. 2011 Apr 28;54(8):2680-6.
[2]. Hao H, et al. NQDI-1, an inhibitor of ASK1 attenuates acute perinatal hypoxic-ischemic cerebral injury by modulating cell death. Mol Med Rep. 2016 Jun;13(6):4585-92.