An automated multistep high-throughput screening assay for the identification of lead inhibitors of the inducible enzyme mPGES-1.

Prostaglandin E2 synthase (mPGES-1), the enzyme which catalyzes the synthesis of PGE2, is induced during the inflammatory response. For this reason, mPGES-1 could be a potential therapeutic target. A high-throughput screening assay was developed to identify potential inhibitors of mPGES-1. The assay consisted of a 30-s mPGES-1 enzymatic reaction followed by the detection of PGE2 by enzyme immunoassay (EIA). The enzymatic reaction was performed in a batch mode because the instability of the substrate (10 min) limited the number of plates assayed within a working day. The detection of the product by EIA was performed on 3 instruments requiring 14 different steps for complete automation. The authors describe here the optimization and implementation of a 2-part assay on a Thermo CRS robotic system. More than 315,000 compounds were tested, and a hit rate of 0.84% was obtained for this assay. Although the entire assay required multiple steps, the assay was successfully miniaturized and automated for a high-throughput screening campaign.

Resorufin butyrate as a soluble and monomeric high-throughput substrate for a triglyceride lipase.

Triglyceride lipases such as lipoprotein lipase, endothelial lipase, and hepatic lipase play key roles in controlling the levels of plasma lipoprotein. Accordingly, small-molecule modulation of these species could alter patient lipid profiles with corresponding health effects. Screening of these enzymes for small-molecule therapeutics has historically involved the use of lipid-based particles to mimic native substrates. However, particle-based artifacts can complicate the discovery of therapeutic molecules. As a simplifying solution, the authors sought to develop an approach involving a soluble and monomeric lipase substrate. Using purified bovine lipoprotein lipase as a model system, they show that the hydrolysis of resorufin butyrate can be fluorescently monitored to give a robust assay (Z' > 0.8). Critically, using parallel approaches, they show that resorufin butyrate is soluble and monomeric under assay conditions. The presented assay should be useful as a simple and inexpensive primary or secondary screen for the discovery of therapeutic lipase modulators.

Effect of fish oil on LDL oxidation and plasma homocysteine concentrations in health.

Oxidation of low-density lipoprotein (LDL) and hyperhomocysteinemia are believed to play a role in therogenesis. Whether n-3 polyunsaturated fatty acids increase LDL susceptibility to oxidation or influence homocysteine (Hcy) metabolism has long been a subject of controversy. In this study, we evaluated the effect of 8 weeks of dietary supplementation with 6 g/day of fish oil (FO; 3 g of n-3 fatty acids) on plasma lipoproteins, in vitro LDL peroxidation, antioxidant status, and plasma Hcy concentrations in 16 normolipidemic subjects. FO rapidly and significantly (P < .01) decreased plasma total and very low density lipoprotein triglyceride concentrations and had no effect on LDL or high-density-lipoprotein cholesterol. The mean lag time before onset of Cu(2+)-induced LDL oxidation, as well as plasma and LDL alpha-tocopherol and beta-carotene concentrations, was unchanged. However, changes in plasma aminothiol concentrations occurred during the study. Specifically, a progressive and significant increase in total Hcy plasma concentrations was observed (13.4% and 20% after 4 and 8 weeks, respectively; P < .01). Total glutathione concentrations were significantly higher after 8 weeks (P < .05). The tHcy increase was not associated with changes in plasma folate or vitamin B(12) concentrations. However, concentrations of plasma nitric oxide metabolites (NO(x) = NO(2) + NO(3)) were significantly higher than at baseline after 8 weeks of FO intake (74%; P < .01). Further, the changes in total Hcy and NO(x) plasma concentrations observed after 8 weeks of FO were found to be significantly correlated (r = .78, P < .001). With this study, we report for the first time the apparent interaction of n-3 fatty acids and nitric oxide on Hcy metabolism.