title: Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.
abstract: To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes.
authors.affiliation: Departments of Bioanalytical and Discovery Analytical Science, Bristol-Myers Squibb Company, Princeton, NJ.
authors.forename: Joelle M
authors.initials: JM
authors.lastname: Onorato
authors.affiliation: Fibrosis Discovery, Bristol-Myers Squibb Company, Princeton, NJ.
authors.forename: Ching-Hsuen
authors.initials: CH
authors.lastname: Chu
authors.affiliation: Fibrosis Discovery, Bristol-Myers Squibb Company, Princeton, NJ.
authors.forename: Zhengping
authors.initials: Z
authors.lastname: Ma
authors.affiliation: Mechanistic Biochemistry, Bristol-Myers Squibb Company, Princeton, NJ.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Kopcho
authors.affiliation: Discovery Chemistry, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ.
authors.forename: Hannguang J
authors.initials: HJ
authors.lastname: Chao
authors.affiliation: Discovery Chemistry, Research and Development, Bristol-Myers Squibb Company, Princeton, NJ.
authors.forename: R Michael
authors.initials: RM
authors.lastname: Lawrence
authors.affiliation: Fibrosis Discovery, Bristol-Myers Squibb Company, Princeton, NJ. Electronic address: dong.cheng@bms.com.
authors.forename: Dong
authors.initials: D
authors.lastname: Cheng
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D004075:Diglycerides / Q000096:biosynthesis*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D006801:Humans; D051379:Mice; D017351:N-Acetylglucosaminyltransferases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009765:Obesity / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D019308:Palmitic Acid / Q000378:metabolism
chemical_list: D004075:Diglycerides; D004791:Enzyme Inhibitors; D019308:Palmitic Acid; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase
keywords: cell-based assay; diacylglycerol acyltransferase; liquid chromatography/mass spectrometry; monoacylglycerol acyltransferase; triglycerides