Mass spectrometric techniques for label-free high-throughput screening in drug discovery.
High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.
Roddy
Thomas P
TP
Discovery Technologies, Novartis Institute for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA. thomas.roddy@novartis.com
Horvath
Christopher R
CR
Stout
Steven J
SJ
Kenney
Kristin L
KL
Ho
Pei-I
PI
Zhang
Ji-Hu
JH
Vickers
Chad
C
Kaushik
Virendar
V
Hubbard
Brian
B
Wang
Y Karen
YK
D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods; D020650:Combinatorial Chemistry Techniques; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods; D004364:Pharmaceutical Preparations / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000295:instrumentation / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
D004364:Pharmaceutical Preparations