Assay development and screening of human DGAT1 inhibitors with an LC/MS-based assay: application of mass spectrometry for large-scale primary screening.
Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.
Zhang
Ji-Hu
JH
Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA. ji-hu.zhang@novartis.com
Roddy
Thomas P
TP
Ho
Pei-I
PI
Horvath
Christopher R
CR
Vickers
Chad
C
Stout
Steven
S
Hubbard
Brian
B
Wang
Y Karen
YK
Hill
W Adam
WA
Bojanic
Dejan
D
D002853:Chromatography, Liquid; D051048:Diacylglycerol O-Acyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D057166:High-Throughput Screening Assays; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D012997:Solvents / Q000737:chemistry; D013997:Time Factors; D017186:Titrimetry
D004791:Enzyme Inhibitors; D012997:Solvents; D051048:Diacylglycerol O-Acyltransferase