DrugLib.com — Drug Information Portal

Rx drug information, pharmaceutical research, clinical trials, news, and more

Development and application of a scintillation proximity assay (SPA) for identification of selective inhibitors of 11beta-hydroxysteroid dehydrogenase type 1.

Author(s): Mundt S, Solly K, Thieringer R, Hermanowski-Vosatka A

Affiliation(s): Department of Cardiovascular Diseases, Merck & Co., Rahway, NJ 07065, USA. steven_mundt@merck.com

Publication date & source: 2005-08, Assay Drug Dev Technol., 3(4):367-75.

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.

Page last updated: 2007-02-12

-- advertisement -- The American Red Cross
Home | About Us | Contact Us | Site usage policy | Privacy policy

All Rights reserved - Copyright DrugLib.com, 2006-2017