Active site variability of type 1 11beta-hydroxysteroid dehydrogenase revealed by selective inhibitors and cross-species comparisons.

The NADPH-dependent enzyme type 1 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) activates in a tissue-specific manner circulating pro-glucocorticoid hormones (cortisone in humans) to the 11beta-OH ligand (cortisol in humans), which is able to bind to its cognate receptor and regulate gene transcription. Modulation of this pre-receptor activation mechanism by selective enzyme inhibitors is a desirable goal in the treatment of insulin resistance and related metabolic disorders. Like most other hydroxysteroid dehydrogenases 11beta-HSD1 belongs to the evolutionarily conserved enzyme superfamily of short-chain dehydrogenases/reductases (SDR). The enzyme is anchored within the endoplasmic reticulum through an N-terminal transmembrane domain. In this study we aimed to characterize the active site of mammalian 11beta-HSD1 by determining primary structures from several mammalian lines (cat, hamster, cynomolgus, chimpanzee, dog) thus increasing substantially available sequence information, and allowing us to determine highly variable and constant parts within the primary structure. These regions were mapped to the recently determined three-dimensional structure and are mostly found around the substrate binding site. Furthermore we performed inhibition studies by using different series of inhibitors, comprising 11beta-HSD1 selective arylsulfonamidothiazoles and the unselective steroid-based compound carbenoxolone. The different arylsulfonamidothiazoles display distinct inhibition profiles versus the mammalian species tested, with several tight binding inhibitors for the human enzyme (Ki approximately 50 nM), intermediate for mouse, and weak or not binding inhibitors for rat and guinea pig (Ki>3 microM). Analysis of the inhibition mode reveals that the tight binding inhibitor BVT.528 is a competitive inhibitor for the human form, whereas the related compound BVT.2733 displays a mixed-type inhibition pattern versus the mouse enzyme. Taken together, this structure-activity study provides increased insight into active site complexity and catalytic mechanism of 11beta-HSD1, useful for further inhibitor design.