No abstract

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.

The impact of 1-methylcyclopropene (1-MCP) on the synthesis and retention of flavonoid compounds during storage and ripening of red Delicious (Malus x domestica Borkh.) apples was investigated. Numerous anthocyanins, flavonols, flavan-3-ols, and a hydroxycinnamic acid from three different fruit harvest maturities were monitored after a 120 day storage and 1 week shelf life period using high-performance liquid chromatography/diode array detector analysis. The total flavonoid concentration was 5% greater in fruit treated with 1-MCP, whereas chlorogenic acid levels were 24% lower. All compounds analyzed increased in concentration during fruit harvest; however, the anthocyanins generally declined after storage, while chlorogenic acid levels increased. 1-MCP treatment resulted in the retention of anthocyanins in the latter stages of storage but did not affect the flavonols and flavan-3-ols. Chlorogenic acid biosynthesis from early and optimal fruit harvest maturities was greatly inhibited by 1-MCP during storage and the 1 week shelf life period. However, 1-MCP did not affect chlorogenic acid concentrations in late-harvested fruit. Results suggest that 1-MCP may inhibit the activity of phenylalanine ammonia-lyase and subsequent biosynthesis of flavonoid compounds. However, because very little postharvest biosynthesis of flavonoids occurs in apples, 1-MCP treatment may be useful for maintaining some of the intrinsic flavonoid levels of red Delicious apples, if applied at the proper harvest maturity.

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.