K-604 dihydrochloride

Kinase experiment:

ACAT activity is determined by measuring the production of cholesteryl[14C]oleate. Microsomal fractions derived from CHO-ACAT-1 or CHO-ACAT-2 cells are diluted with CHAPS in 0.5 M KCl, 0.5 mM EDTA, and 25 mM Tris-HCl (pH 7.8) (buffer A) to a final protein concentration of 1.0 mg/mL. The microsomal fractions containing 9.2 μg of protein for ACAT-1 or 4.9 μg for ACAT-2 in 10 μL of buffer A are mixed with various concentrations of K-604 (0.4, 0.6 and 0.8 μM) or CI-1011 in 5 μL of DMSO, and reconstituted with 152.5 μL of mixed micelles containing 1.6 mM Cholesterol, 11.2 mM Phosphatidylcholine, and 9.3 mM Taurocholate in 25, 0.5 mM EDTA, and Tris-HCl (pH 7.8). The reaction is started by adding 10 μL of 0.45 μM [14C]oleoyl-CoA, 10 nM fatty acid-free bovine serum albumin (fatty acid-free BSA) and 25 mM Tris-HCl (pH 7.8). Reaction mixtures are incubated for 25 min at 37°C and terminated by adding 150 μL of Chloroform/Methanol (2:1). The organic phase is dried under a stream of nitrogen. The lipids are resuspended with 150 μL of Chloroform/Methanol (2:1) and developed by thin layer chromatography (TLC) using hexane/diethyl ether/acetic acid (75:25:1). Radioactivities of cholesterol [14C]oleate are determined using a BAS 2500 image analyzer[1].

Cell experiment:

The mouse neuroblastoma cell line N2a is cultured in DMEM/Opti-MEM (50:50) with 10% fetal bovine serum (FBS) at 37°C with 5% CO2 in a humidified incubator. Cells are incubated for 24 h with the ACAT1-specific inhibitor K604 (0.1, 0.5 and 1 µM) or isotype-nonspecific ACAT inhibitor CI-1011. Primary cortical neurons are isolated from mouse brains at postnatal day 0-3. Cortical neurons are plated in 6 well plates at 350,000 cells/well and grown in 2 mL/well Neurobasal A with 1×B27, 0.5 mM L-glutamine, and 5 ng/mL fibroblast growth factor. Half of the media is replaced with fresh media once every 7 days. After 14-21 days in culture, cells are used for individual experiments[2].

Animal experiment:

Hamsters[1]Male F1B hamsters (n=30) at 8 weeks old are used for animal experiments. The animal room is controlled at 23±3°C and relative humidity of 50±20%. Animals are fed a CE-2 chow diet, followed by supplementation with CE-2 containing 0.3% cholesterol and 10% coconut oil for 10 weeks. During fat loading, K-604 is administered orally at 1, 3, 10, or 30 mg/kg/day (n=6 for each dose group). The control group (n=6) receive an aqueous solution of 0.5% methylcellulose instead of K-604. Tap water is given ad libitum. Body weight is measured at the time of drug administration. Blood is sampled for determination of plasma cholesterol levels using a commercially available kit (Cholesterol E-Test).

References:

[1]. Ikenoya M, et al. A selective ACAT-1 inhibitor, K-604, suppresses fatty streak lesions in fat-fed hamsters without affecting plasma cholesterol levels. Atherosclerosis. 2007 Apr;191(2):290-7.
[2]. Shibuya Y, et al. Acyl-coenzyme A:cholesterol acyltransferase 1 blockage enhances autophagy in the neurons of triple transgenic Alzheimer's disease mouse and reduces human P301L-tau content at the presymptomatic stage. Neurobiol Aging. 2015 Jul;36(7):2248-2259.