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P2030
Development of a Plasma Based Assay for Endothelial Lipase
Presenter Benjamin Schwartz, GlaxoSmithKline, USA
Additional Authors: Thomas Sweitzer, John Krawiec
Recently, a new member of the triglyceride lipase gene family has been cloned from endothelial cells. This enzyme (called endothelial lipase, or EL) is a 500 amino acid protein, and like other triglyceride lipases utilizes a Ser-His-Asp triad for catalysis. Animal models in which it is over-expressed or knocked out indicate that levels of EL are inversely correlated with HDL. HDL is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, where excess cholesterol is effluxed from peripheral tissues either for excretion or reabsorbtion. Thus, inhibition of EL in humans is expected to lead to increases in HDL levels, and a decrease in cardiovascular disease.
As a prerequisite to in vivo studies, compounds need to demonstrate efficacy against EL in plasma. Assays using generic substrates for measuring EL activity have been found to be incompatible with even low amounts of plasma, likely due to the presence of other (phospho)lipases. In order to overcome this issue, we produced several new fluorescent HDL substrates, and found one (HDL  PED6/TAG) that allowed us to measure EL activity in plasma. Herein we describe our reagent and assay development efforts, and show some exemplary data from compound profiling efforts.