Seethala Ramakrishna, Peterson Tara, Dong Jessica, Chu Ching-Hsuen, Chen Luping, Golla Rajasree, Ma Zhengping, Panemangalore Reshma, Lawrence R Michael, Cheng Dong
Division of Drug Discovery, Research and Development, Bristol-Myers Squibb Company, Mail Stop 21-2.16, PO Box 5400, Princeton, NJ 08543-5400, USA.
Anal Biochem. 2008 Dec 15;383(2):144-50. doi: 10.1016/j.ab.2008.09.013. Epub 2008 Sep 15.
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.
酰基辅酶A：二酰基甘油酰基转移酶（DGAT）是三酰甘油合成中的关键酶，抑制该酶是治疗肥胖症、II型糖尿病和血脂异常的一种有前景的方法。有两种不同的DGAT酶：DGAT1和DGAT2。用于测量DGAT活性的传统方法是薄层色谱（TLC）法，该方法不适用于大量化合物的筛选。为了提高通量，我们开发了一种用于DGAT的新型均相闪烁邻近分析（SPA）。在该分析中，当使用（3）H标记的酰基辅酶A作为酰基供体且二酰基甘油作为酰基受体时，反应中形成的（3）H标记的三酰甘油产物与聚赖氨酸SPA珠结合，产生可在TopCount或LEADseeker中测量的信号。通过这种DGAT SPA方法确定的表观米氏动力学参数与用传统TLC分析确定的值非常吻合。统计值还表明DGAT SPA是一种稳健的分析方法，Z'大于0.60，信号/背景比约为9。这些结果表明当前的分析方法为鉴定DGAT抑制剂提供了高通量能力。
