High capacity homogeneous non-radioactive cortisol detection assays for human 11beta-hydroxysteroid dehydrogenase type 1.
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.
Yu
Violeta
V
Amgen Inc., Thousand Oaks, CA 91320-1799, USA. vyu@amgen.com
Tudor
Yanyan
Y
Hale
Clarence
C
Plant
Matthew
M
Kim
Ki Won
KW
Wang
Minghan
M
Nguyen
Yen
Y
Miguel
Tisha San
TS
Chen
Michelle
M
Nybo
Rebecca
R
Baumgartner
Jamie
J
Kurzeja
Robert J M
RJ
Powers
David
D
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
D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; D006854:Hydrocortisone