A simple homogeneous scintillation proximity assay for acyl-coenzyme A:diacylglycerol acyltransferase.
Acyl-coenzyme A:diacylglycerol acyltransferase (DGAT) is a key enzyme in triacylglycerol synthesis, and inhibiting this enzyme is a promising approach for treating obesity, type II diabetes, and dyslipidemia. There are two distinct DGAT enzymes: DGAT1 and DGAT2. The conventional assay for measuring DGAT activity is a thin layer chromatography (TLC) method, which is not amenable to screening a large number of compounds. To increase the throughput, we have developed a novel, homogeneous scintillation proximity assay (SPA) for DGAT. In this assay, when (3)H-labeled acyl-CoA is used as the acyl donor and diacylglycerol is used as the acyl acceptor, the (3)H-labeled triacylglycerol product formed in the reaction binds to polylysine SPA beads, producing a signal that is measured in a TopCount or LEADseeker. The apparent Michaelis-Menten kinetic parameters determined by this DGAT SPA method agreed well with the values determined with the conventional TLC assay. The statistical values also indicate that the DGAT SPA is a robust assay, with a Z' of more than 0.60 and a signal/background ratio of approximately 9. These results suggest that the current assay provides high-throughput capacity for the identification of DGAT inhibitors.
Seethala
Ramakrishna
R
Division of Drug Discovery, Research and Development, Bristol-Myers Squibb Company, Mail Stop 21-2.16, PO Box 5400, Princeton, NJ 08543-5400, USA. ramakrishna.seethala@bms.com
Peterson
Tara
T
Dong
Jessica
J
Chu
Ching-Hsuen
CH
Chen
Luping
L
Golla
Rajasree
R
Ma
Zhengping
Z
Panemangalore
Reshma
R
Lawrence
R Michael
RM
Cheng
Dong
D
D000818:Animals; D002417:Cattle; D051048:Diacylglycerol O-Acyltransferase / Q000032:analysis* / Q000378:metabolism; D004121:Dimethyl Sulfoxide / Q000378:metabolism; D004800:Enzymes, Immobilized / Q000032:analysis / Q000378:metabolism; D007700:Kinetics; D008863:Microspheres; D051381:Rats; D015203:Reproducibility of Results; D012588:Scintillation Counting; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000378:metabolism; D014280:Triglycerides / Q000096:biosynthesis
D004800:Enzymes, Immobilized; D014280:Triglycerides; D012710:Serum Albumin, Bovine; D051048:Diacylglycerol O-Acyltransferase; D004121:Dimethyl Sulfoxide