title: Development of a high-throughput cell-based assay for 11beta-hydroxysteroid dehydrogenase type 1 using BacMam technology.
abstract: Cortisol is an important glucocorticoid in humans that regulates many physiological processes. Human 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) converts cortisone to cortisol in vivo and has emerged as an appealing therapeutic target for treating metabolic diseases. Here, we report a sensitive and robust high-throughput (HT) cell-based assay for screening 11beta-HSD1 inhibitors. This assay utilizes a HEK293 cell line transduced by a BacMam virus expressing human 11beta-HSD1. The enzyme activity in the cells was measured by quantifying cortisol levels released into the cell culture supernatant via a competitive homogenous time-resolved fluorescence (HTRF) method. We show that 11beta-HSD1 activity in supernatant of BacMam-transduced HEK293 cells increases with 11beta-HSD1 BacMam virus load in a dose-dependent manner, and is comparable to the enzyme activity detected in differentiated mouse adipocytes. In addition, we show that co-expression of hexose-6-phosphate dehydrogenase (H6PDH) is not required for the enzyme to function effectively as an oxo-reductase. This assay has been developed in low-volume 384-well format and it is sensitive, robust, and amenable to HT screening.
authors.affiliation: Biological Reagents and Assay Development, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.
authors.forename: Da-Yuan
authors.initials: DY
authors.lastname: Wang
authors.forename: Quinn
authors.initials: Q
authors.lastname: Lu
authors.forename: Stacey L
authors.initials: SL
authors.lastname: Walsh
authors.forename: Lisa
authors.initials: L
authors.lastname: Payne
authors.forename: Sundip S
authors.initials: SS
authors.lastname: Modha
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Scott
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sweitzer
authors.forename: Robert S
authors.initials: RS
authors.lastname: Ames
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Krosky
authors.forename: Hu
authors.initials: H
authors.lastname: Li
mesh_terms: D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D041721:3T3-L1 Cells; D017667:Adipocytes / Q000201:enzymology; D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000378:metabolism; D002470:Cell Survival; D003348:Cortisone / Q000378:metabolism; D003470:Culture Media / Q000032:analysis; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D006854:Hydrocortisone / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D003470:Culture Media; D002237:Carbohydrate Dehydrogenases; C020552:galactose-6-phosphate dehydrogenase; D043205:11-beta-Hydroxysteroid Dehydrogenase Type 1; C468386:HSD11B1 protein, human; D003348:Cortisone; D006854:Hydrocortisone