title: High-throughput scintillation proximity assay for stearoyl-CoA desaturase-1.
abstract: Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of monounsaturated fatty acids and has been implicated in a number of disease states, including obesity and diabetes. To find small-molecule inhibitor leads, a high-throughput scintillation proximity assay (SPA) was developed using the hydrophobic binding characteristics of a glass microsphere scintillant bead to capture SCD1 from a crude lysate of recombinant SCD1 in Sf9 lysate coupled with the strong binding characteristics of an azetidine compound ([(3)H]AZE). The SPA assay was stable over 24 h and could detect compounds with micromolar to nanomolar potencies. A robust 1536-well high-throughput screening assay was developed with good signal-to-noise ratio (10:1) and excellent Z' factor (0.8). A screening collection of 1.6 million compounds was screened at 11 µM, and approximately 7700 compounds were identified as initial hits, exhibiting at least 35% inhibition of [(3)H]AZE binding. Further screening and confirmation with an SCD enzyme activity assay led to a number of new structural leads for inhibition of the enzyme. The SPA assay complements the enzyme activity assay for SCD1 as a tool for the discovery of novel leads in drug discovery.
authors.affiliation: Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Québec, Canada.
authors.forename: Paul
authors.initials: P
authors.lastname: Tawa
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Falgueyret
authors.forename: Sebastien
authors.initials: S
authors.lastname: Guiral
authors.forename: Elise
authors.initials: E
authors.lastname: Isabel
authors.forename: David A
authors.initials: DA
authors.lastname: Powell
authors.forename: Paul
authors.initials: P
authors.lastname: Zuck
authors.forename: Kathryn
authors.initials: K
authors.lastname: Skorey
mesh_terms: D000818:Animals; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006801:Humans; D008024:Ligands; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D011485:Protein Binding; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012588:Scintillation Counting; D013230:Stearoyl-CoA Desaturase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D014316:Tritium / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D008024:Ligands; D011994:Recombinant Proteins; D014316:Tritium; D013230:Stearoyl-CoA Desaturase