A highly sensitive high-throughput luminescence assay for malonyl-CoA decarboxylase.
Malonyl-CoA decarboxylase (MCD) catalyzes the conversion of malonyl-CoA to acetyl-CoA and thereby regulates malonyl-CoA levels in cells. Malonyl-CoA is a potent inhibitor of mitochondrial carnitine palmitoyltransferase-1, a key enzyme involved in the mitochondrial uptake of fatty acids for oxidation. Abnormally high rates of fatty acid oxidation contribute to ischemic damage. Inhibition of MCD leads to increased malonyl-CoA and therefore decreases fatty acid oxidation, representing a novel approach for the treatment of ischemic heart injury. The commonly used MCD assay monitors the production of NADH fluorometrically, which is not ideal for library screening due to potential fluorescent interference by certain compounds. Here we report a luminescence assay for MCD activity. This assay is less susceptible to fluorescent interference by compounds. Furthermore, it is 150-fold more sensitive, with a detection limit of 20 nM acetyl-CoA, compared to 3 muM in the fluorescence assay. This assay is also amenable to automation for high-throughput screening and yields excellent assay statistics (Z' > 0.8). In addition, it can be applied to the screening for inhibitors of any other enzymes that generate acetyl-CoA.
Lo
Mei-Chu
MC
Chemistry Research and Discovery, Amgen, South San Francisco, CA 94080, USA. meil@amgen.com
Wang
Minghan
M
Kim
Ki Won
KW
Busby
James
J
Yamane
Harvey
H
Zondlo
James
J
Yuan
Chester
C
Young
Stephen W
SW
Xiao
Shou-Hua
SH
D002262:Carboxy-Lyases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D049449:Luminescence; D008163:Luminescent Measurements / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase