A high-throughput screen for endothelial lipase using HDL as substrate.
Endothelial lipase (EL) is a 482-amino-acid protein from the triglyceride lipase gene family that uses a Ser-His-Asp triad for catalysis. Its expression in endothelial cells and preference for phospholipids rather than triglycerides are unique. Animal models in which it is overexpressed or knocked out indicate EL levels are inversely correlated with high-density lipoprotein cholesterol (HDL-C). HDL-C is commonly referred to as the good form of cholesterol because it is involved in the reverse cholesterol transport pathway, in which excess cholesterol is effluxed from peripheral tissues for excretion or reabsorption. Thus, EL inhibition in humans is expected to lead to increases in HDL levels and possibly a decrease in cardiovascular disease. To discover inhibitors of EL, a coupled assay for EL has been developed, using its native substrate, HDL. Hydrolysis of HDL by EL yields free fatty acids, which are coupled through acyl-CoA synthetase, acyl-CoA oxidase, and horseradish peroxidase to produce the fluorescent species resorufin. This assay was developed into a 5-microL, 1536-well assay format, and a high-throughput screen was executed against the GSK collection. In addition to describing the screening results, novel post-HTS mechanism-of-action studies were developed for EL and applied to 1 of the screening hits as an example.
GSK Screening & Compound Profiling, Collegeville, Pennsylvania, USA.
Paul M
PM
Keller
Timothy
T
Rust
Dennis J
DJ
Murphy
Rosalie
R
Matico
John J
JJ
Trill
John A
JA
Krawiec
Anthony
A
Jurewicz
Michael
M
Jaye
Mark
M
Harpel
Sara
S
Thrall
Benjamin
B
Schwartz
D042962:Acyl-CoA Oxidase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D003066:Coenzyme A Ligases / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005230:Fatty Acids, Nonesterified / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008075:Lipoproteins, HDL / Q000378:metabolism*; D008954:Models, Biological; D010078:Oxazines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D005230:Fatty Acids, Nonesterified; D008075:Lipoproteins, HDL; D010078:Oxazines; D011994:Recombinant Proteins; C014180:resorufin; D000255:Adenosine Triphosphate; D006735:Horseradish Peroxidase; D042962:Acyl-CoA Oxidase; D008049:Lipase; D003066:Coenzyme A Ligases