MK-4074

Kinase experiment:

Recombinant ACC protein is purified from FM3A or Sf9 cells expressing recombinant ACC by chelating chromatography or from liver by Softlink avidin resin chromatography. Purified ACC protein is incubated with MK-4074 in assay buffer containing 5 mM ATP, 250 mM acetyl-CoA, 4.1 mM NaHCO3, 0.086 mM NaH14CO3, 20 mM potassium citrate, 20 mM MgCl2, 2 mM DTT, 0.5 mg/mL BSA and 50 mM HEPES-Na (pH 7.5) for 40 min at 37°C[1].

Cell experiment:

For cellular assays of DNL and FAO, cells are pre-incubated with MK-4074 for 1 hr. Then the cells are incubated for additional 1-3 hr with either 65-260 mM 14C-labeled acetate or 0.018 mM 3H-labeled palmitate for DNL or FAO assay, respectively. After incubation, intracellular 14C-labeled lipids and released 3H-labeled fatty acids are extracted and measured for DNL and FAO, respectively[1].

Animal experiment:

Mice[1]Studies are performed in male KKAy mice or C57BL/6J mice. KKAy mice are fed a chow diet while C57BL/6J mice are fed a high-fat diet (45% fat) for 3 weeks prior to study. Mice are treated for 7 days with vehicle (distilled water, 0.2 mL/mouse) before MK-4074 administration to acclimate mice to oral dosing. Animals are drug naive at the time of study. Mice are housed individually. Male KKAy mice (n=10-11/group) are administered a single oral dose of MK-4074 (0.3 to 3 mg/kg) prior to liver slice studies. Male KKAy mice (n=5/group) are administered a single oral dose of MK-4074 (3 to 30 mg/kg) prior to measurement of liver DNL rates. Male KKAy mice (n=8/group) are administered a single oral dose of MK-4074 (10 to 100 mg/kg) and plasma ketone bodies are measured at the indicated times. Male C57BL/6J mice (n=5, veh; n=10, MK-4074) are fed chow or a high-fat/high-sucrose (HF/HS) diet for 7 weeks and vehicle or MK-4074 is administered orally (10 or 30 mg/kg/day) for 4 weeks prior to study[1].

References:

[1]. Kim CW, et al. Acetyl CoA Carboxylase Inhibition Reduces Hepatic Steatosis but Elevates Plasma Triglycerides in Mice and Humans: A Bedside to Bench Investigation. Cell Metab. 2017 Aug 1;26(2):394-406.e6.