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.

The selectivity of tyrosine 280 of human 11beta-hydroxysteroid dehydrogenase type 1 in inhibitor binding.

11beta-Hydroxysteroid dehydrogenase type 1 is a homodimer where the carboxyl terminus of one subunit covers the active site of the dimer partner. Based on the crystal structure with CHAPS, the carboxyl terminal tyrosine 280 (Y280) has been postulated to interact with the substrate/inhibitor at the binding pocket of the dimer partner. However, the co-crystal structure with carbenoxolone argues against this role. To clarify and reconcile these findings, here we report our mutagenesis data and demonstrate that Y280 is not involved in substrate binding but rather plays a selective role in inhibitor binding. The involvement of Y280 in inhibitor binding depends on the inhibitor chemical structure. While Y280 is not involved in the binding of carbenoxolone, it is critical for the binding of glycyrrhetinic acid.

The role of tyrosine 177 in human 11beta-hydroxysteroid dehydrogenase type 1 in substrate and inhibitor binding: an unlikely hydrogen bond donor for the substrate.

The catalytic motif (YSASK) at the active site of 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is conserved across different species. The crystal structures of the human, guinea pig and mouse enzymes have been resolved to help identify the non-conserved residues at the active site. A tyrosine residue (Y177) upstream of the catalytic motif in human 11beta-HSD1 represents the largest difference at the active sites between the human and the rodent enzyme where the corresponding residue is glutamine. Although Y177 was postulated as a potential hydrogen bond donor in substrate binding in crystal structure-based modeling, no experimental evidence is available to support this notion. Here, we report that Y177 is not a hydrogen bond donor in substrate binding because removal of the hydroxyl group from its side chain by mutagenesis (Y177F) did not significantly change the Km value for cortisone. However, removal of the hydrophobic side chain by changing tyrosine to alanine (Y177A) or substitution with a hydrophilic side chain by changing tyrosine to glutamine (Y177Q) increased Km values for cortisone. These data suggest that Y177 is involved in substrate binding through its hydrophobic side chain but not by hydrogen bonding. In addition, the three mutations had little effect on the binding of the rodent substrate 11-dehydrocorticosterone, suggesting that Y177 does not confer substrate specificity. However, the same mutations reduced the affinity of the licorice derived 11beta-HSD1 inhibitor glycyrrhetinic acid by about 6- to 10-fold. Interestingly, the affinity of carbenoxolone, the hemisuccinate ester of glycyrrhetinic acid with a similar potency against the wildtype enzyme, was not drastically affected by the same mutations at Y177. These data suggest that Y177 has a unique role in inhibitor binding. Molecular modeling with glycyrrhetinic acid led to findings consistent with the experimental data and provided potential interaction mechanisms. Our data suggest that Y177 plays an important role in both substrate and inhibitor binding but it is unlikely a hydrogen bond donor for the substrate.

Enhanced stability of recombinant keratinocyte growth factor by mutagenesis.

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.

Crystal structure of murine 11 beta-hydroxysteroid dehydrogenase 1: an important therapeutic target for diabetes.

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the conversion of 11-dehydrocorticosterone to its active form corticosterone in rodents (or cortisone to cortisol in humans). The reductive reaction of the 11-keto to 11-hydroxyl is the pivotal switch in the activation of glucocorticoids. An excess of active glucocorticoids has been shown to play a key role in metabolic disorders such as diabetes and obesity. Therefore, 11beta-HSD1 represents an important therapeutic target for the treatment of these diseases. To facilitate the iterative design of inhibitors, we have crystallized and determined the three-dimensional structures of a binary complex of murine 11beta-HSD1 with NADP(H) to a resolution of 2.3 A and of a ternary complex with corticosterone and NADP(H) to a resolution of 3.0 A by X-ray crystallography. The enzyme forms a homodimer in the crystal and has a fold similar to those of other members of the family of short chain steroid dehydrogenases/reductases (SDRs). The structure shows a novel folding feature at the C-terminus of the enzyme. The C-terminal helix insertions provide additional dimer contacts, exert an influence on the conformations of the substrate binding loops, and present hydrophobic regions for potential membrane attachment. The structure also reveals how 11beta-HSD1 achieves its selectivity for its substrate.