Development and Application of a Scintillation Proximity Assay (SPA) for Identification of Selective Inhibitors of 11β-Hydroxysteroid Dehydrogenase Type 1

Pre-receptor metabolism of glucocorticoids by the 11β-hydroxysteroid dehydrogenase(11βHSD) 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 11βHSD1. Microsomes containing 11βHSD1 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 11βHSD1 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 11βHSD2 counterscreen was performed by incubating 11βHSD2 microsomes with [³H]cortisol and NAD⁺ and monitoring substrate disappearance.