title: High capacity homogeneous non-radioactive cortisol detection assays for human 11beta-hydroxysteroid dehydrogenase type 1.
abstract: 11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.
authors.affiliation: Amgen Inc., Thousand Oaks, CA 91320-1799, USA. vyu@amgen.com
authors.forename: Violeta
authors.initials: V
authors.lastname: Yu
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Tudor
authors.forename: Clarence
authors.initials: C
authors.lastname: Hale
authors.forename: Matthew
authors.initials: M
authors.lastname: Plant
authors.forename: Ki Won
authors.initials: KW
authors.lastname: Kim
authors.forename: Minghan
authors.initials: M
authors.lastname: Wang
authors.forename: Yen
authors.initials: Y
authors.lastname: Nguyen
authors.forename: Tisha San
authors.initials: TS
authors.lastname: Miguel
authors.forename: Michelle
authors.initials: M
authors.lastname: Chen
authors.forename: Rebecca
authors.initials: R
authors.lastname: Nybo
authors.forename: Jamie
authors.initials: J
authors.lastname: Baumgartner
authors.forename: Robert J M
authors.initials: RJ
authors.lastname: Kurzeja
authors.forename: David
authors.initials: D
authors.lastname: Powers
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000032:analysis* / Q000737:chemistry*; D004563:Electrochemistry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D006854:Hydrocortisone / Q000032:analysis*; D008163:Luminescent Measurements / Q000379:methods*; D008832:Microchemistry / Q000379:methods*; D011865:Radioisotope Dilution Technique; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D006854:Hydrocortisone