title: Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.
abstract: Pre-receptor metabolism of glucocorticoids by the 11beta-hydroxysteroid dehydrogenase (11betaHSD) enzymes has been implicated in the etiology of the metabolic syndrome. Recent studies have shown that alterations in the activity of the type 1 isozyme can affect many aspects of the disease. This paper describes the optimization and application of a high-throughput scintillation proximity assay (SPA) developed to identify selective specific inhibitors of 11betaHSD1. Microsomes containing 11betaHSD1 were incubated in the presence of NADPH and [3H]cortisone, and the product, [3H]cortisol, was specifically detected in the mixture by a monoclonal antibody coupled to protein A-coated SPA beads with greater than 2 log higher affinity for cortisol than cortisone. Dimethyl sulfoxide and NADPH co-substrate additions were optimized for 11betaHSD1 reductase activity. Titrated test compound, when introduced into the optimized assay, reproducibly inhibited the enzyme and yielded consistent IC50 data in either 96- or 384-well format. An 11betaHSD2 counterscreen was performed by incubating 11betaHSD2 microsomes with [3H]cortisol and NAD+ and monitoring substrate disappearance.
authors.affiliation: Department of Cardiovascular Diseases, Merck & Co., Rahway, NJ 07065, USA. steven_mundt@merck.com
authors.forename: S
authors.initials: S
authors.lastname: Mundt
authors.forename: K
authors.initials: K
authors.lastname: Solly
authors.forename: R
authors.initials: R
authors.lastname: Thieringer
authors.forename: A
authors.initials: A
authors.lastname: Hermanowski-Vosatka
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000032:analysis / Q000494:pharmacology* / Q000627:therapeutic use; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D024821:Metabolic Syndrome / Q000188:drug therapy / Q000209:etiology / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D009249:NADP / Q000378:metabolism; D012588:Scintillation Counting / Q000379:methods*; D014162:Transfection; D014316:Tritium
chemical_list: D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D014316:Tritium; D009249:NADP; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D043209:11-beta-Hydroxysteroid Dehydrogenase Type 2; D006854:Hydrocortisone; D004121:Dimethyl Sulfoxide