Cysteine-10 on 17 β-Hydroxysteroid dehydrogenase 1 has stabilizing interactions in the cofactor binding region and renders sensitivity to sulfhydryl modifying chemicals

17 β-Hydroxysteroid dehydrogenase type 1 (17 β -HSD1) catalyzes the conversion of estrone to the potent estrogen estradiol. 17 β -HSD1 is highly expressed in breast and ovary tissues and represents a prognostic marker for the tumor progression and survival of patients with breast cancer and other estrogen-dependent tumors. Therefore, the enzyme is considered a promising drug target against estrogen-dependent cancers. For the development of novel inhibitors, an improved understanding of the structure-function relationships is essential. In the present study, we examined the role of a cysteine residue, Cys(10), in the Rossmann-fold NADPH binding region, for 17 β -HSD1 function and tested the sensitivity towards sulfhydryl modifying chemicals. 3D structure modeling revealed important interactions of Cys(10) with residues involved in the stabilization of amino acids of the NADPH binding pocket. Analysis of enzyme activity revealed that 17 β -HSD1 was irreversibly inhibited by the sulfhydryl modifying agents N-ethylmaleimide (NEM) and dithiocarbamates. Preincubation with increasing concentrations of NADPH protected 17 β -HSD1 from inhibition by these chemicals. Cys(10)Ser mutant 17 β -HSD1 was partially protected from inhibition by NEM and dithiocarbamates, emphasizing the importance of Cys(10) in the cofactor binding region. Substitution of Cys(10) with serine resulted in a decreased protein half-life, without significantly altering kinetic properties. Despite the fact that Cys(10) on 17 β -HSD1 seems to have limited potential as a target for new enzyme inhibitors, the present study provides new insight into the structure-function relationships of this enzyme

Hexose-6-phosphate dehydrogenase modulates the effect of inhibitors and alternative substrates of 11[beta]-hydroxysteroid dehydrogenase 1

Intracellular glucocorticoid reactivation is catalyzed by 11[beta]-hydroxysteroid dehydrogenase 1 (11[beta]-HSD1), which functions predominantly as a reductase in cells expressing hexose-6-phosphate dehydrogenase (H6PDH). We recently showed that the ratios of cortisone to cortisol and 7-keto- to 7-hydroxy-neurosteroids are regulated by 11[beta]-HSD1 and very much depend on co-expression with H6PDH, providing cosubstrate NADPH. Here, we investigated the impact of H6PDH on the modulation of 11[beta]-HSD1-dependent inter-conversion of cortisone and cortisol by inhibitors and alternative substrates. Using HEK-293 cells expressing 11[beta]-HSD1 or co-expressing 11[beta]-HSD1 and H6PDH, we observed significant differences of 11[beta]-HSD1 inhibition by natural and pharmaceutical compounds as well as endogenous hormone metabolites. Furthermore, we show potent and dose-dependent inhibition of 11[beta]-HSD1 by 7-keto-DHEA in differentiated human THP-1 macrophages and in HEK-293 cells over-expressing 11[beta]-HSD1 with or without H6PDH. In contrast, 7-ketocholesterol (7-KC) did not inhibit 11[beta]-HSD1 in HEK-293 cells, even in the presence of H6PDH, but inhibited 11[beta]-HSD1 reductase activity in differentiated THP-1 macrophages (IC~50~ = 8.1 +/- 0.9 [mu]M). 7-keto-DHEA but not 7-KC inhibited 11[beta]-HSD1 in HEK-293 cell lysates. In conclusion, cellular factors such as H6PDH can significantly modulate the effect of inhibitors and alternative 7-oxygenated substrates on intracellular glucocorticoid availability

Copper pumping ATPases: Common concepts in bacteria and man

AbstractRecently, four genes encoding putative copper pumping ATPases have been cloned from widely different sources: two genes from Enterococcus hirae that are involved in copper metabolism and two human genes that are defective in the copper-related Wilson and Menkes disease. The predicted gene products are P-type ATPases. They exhibit extensive sequence similarity and appear to be members of a new class of ATP driven copper pumps involved in the regulation of cellular copper

Gynecomastia and hypertension in a patient treated with posaconazole

Posaconazole therapy may lead to increased serum estradiol levels and development of gynecomastia. Early detection by endocrine hormone measurements may help preventing gynecomastia

Drug-induced endocrine blood pressure elevation

Patients with uncontrolled hypertension are at risk for cardiovascular complications. The majority of them suffers from unidentified forms of hypertension and a fraction has so-called secondary hypertension with an identifiable cause. The patient's medications, its use of certain herbal supplements and over-the-counter agents represent potential causal factors for secondary hypertension that are often overlooked. The current review focuses on drugs that are likely to elevate blood pressure by affecting the human endocrine system at the level of steroid synthesis or metabolism, mineralocorticoid receptor activity, or by affecting the catecholaminergic system. Drugs with known adverse effects but where benefits outweigh their risks, drug candidates and market withdrawals are reviewed. Finally, potential therapeutic strategies are discussed

Characterization of Vemurafenib Phototoxicity in a Mouse Model

Vemurafenib is a first-in-class, small molecule B-Raf kinase inhibitor for the treatment of patients with unresectable or metastatic melanoma carrying the BRAFV600E mutation, commercially available since 2011. A general phototoxic potential was identified early during development; however, based on results of an animal study in hairless rats, it was concluded that there would exist no relevant risk for humans. Surprisingly, signs of clinical photosensitivity were reported in many patients during clinical development. Therefore, it became a fundamental question to understand this discrepancy. An established mouse model (oral UV-Local Lymph Node Assay, UV-LLNA) for the assessment of in vivo photosafety was used to investigate the impact of formulations, dose levels, duration of treatment, and timing of irradiation. Moreover, a basic pharmacokinetic profile was established within the same mouse strain. We were able to demonstrate dose- and time-dependent phototoxicity of vemurafenib using commercially available tablets (stabilized amorphous material). The lowest phototoxic dose was 350mg/kg administrated for 3 consecutive days followed by exposure to UV-visible irradiation at a UVA-normalized dose of 10 J/cm2. In comparison, pure vemurafenib, which easily forms crystalline variants and is known to have poor bioavailability, was tested at 350mg/kg, and no signs of phototoxicity could be seen. The most apparent difference between the early study in hairless rats and this study in mice was the spectral range of the irradiation light source (350-400nm vs 320-700nm). Because vemurafenib does not absorb sufficiently light above 350nm, this difference can easily explain the negative earlier study result in hairless rat

Species-specific differences in the inhibition of 11β-hydroxysteroid dehydrogenase 2 by itraconazole and posaconazole

11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) converts active 11β-hydroxyglucocorticoids to their inactive 11-keto forms, thereby preventing inappropriate mineralocorticoid receptor activation by glucocorticoids. Disruption of 11β-HSD2 activity by genetic defects or inhibitors causes the syndrome of apparent mineralocorticoid excess (AME), characterized by hypokalemia, hypernatremia and hypertension. Recently, the azole antifungals itraconazole and posaconazole were identified to potently inhibit human 11β-HSD2, and several case studies described patients with acquired AME. To begin to understand why this adverse drug effect was missed during preclinical investigations, the inhibitory potential of itraconazole, its main metabolite hydroxyitraconazole (OHI) and posaconazole against 11β-HSD2 from human and three commonly used experimental animals was assessed. Whilst human 11β-HSD2 was potently inhibited by all three compounds (IC; 50; values in the nanomolar range), the rat enzyme was moderately inhibited (1.5- to 6-fold higher IC; 50; values compared to human), and mouse and zebrafish 11β-HSD2 were very weakly inhibited (IC; 50; values above 7 μM). Sequence alignment and application of newly generated homology models for human and mouse 11β-HSD2 revealed significant differences in the C-terminal region and the substrate binding pocket. Exchange of the C-terminus and substitution of residues Leu170,Ile172 in mouse 11β-HSD2 by the corresponding residues His170,Glu172 of the human enzyme resulted in a gain of sensitivity to itraconazole and posaconazole, resembling human 11β-HSD2. The results provide an explanation for the observed species-specific 11β-HSD2 inhibition by the studied azole antifungals. The obtained structure-activity relationship information should facilitate future assessments of 11β-HSD2 inhibitors and aid choosing adequate animal models for efficacy and safety studies

Selection of peptide ligands binding to the basolateral cell surface of proximal convoluted tubules

Selection of peptide ligands binding to the basolateral cell surface of proximal convoluted tubules.BackgroundRecently, we have reported a novel approach of screening phage-display peptide libraries on microdissected intact renal tubular segments and identified an RGD-containing peptide ligand that specifically binds to the basolateral membrane of cortical collecting ducts (CCD). However, screening phage libraries on proximal convoluted tubules (PCT) did not yield a tubule segment-specific ligand. Here, we describe the successful modification of our previously developed phage-display approach and the identification of two distinct ligands that bind specifically to receptors expressed at the basolateral membrane of PCT.MethodsEx vivo screening of phage-display peptide libraries for specific ligands was adapted for PCT. The previously developed method was significantly extended by applying it to a distinct tubular segment, varying the number of rounds of biopanning and incubating phage libraries with absorber cells prior to biopanning. Binding specificity and cellular localization of selected peptide-displaying phage or the corresponding synthetic peptide were analyzed using various epithelial cell lines as well as competition assays and confocal immunofluorescence microscopy.ResultsScreening phage-display peptide libraries, depleted of ligands binding to ubiquitously expressed receptors by preincubation with HEK-293 cells, led to the identification of two PCT-specific ligands. Phage expressing peptides with the consensus sequence GV(K/R)GX3(T/S) or RDXR mediated 15-fold and 13-fold higher binding to PCT than control phage, and binding to PCT was 13-fold and 21-fold higher than binding to CCD, respectively. Neither phage mediated significant binding to various epithelial cell lines, and binding of both ligands was abolished by the addition of the corresponding synthetic peptide. Immunofluorescence experiments revealed a submembrane localization of both ligands upon incubation with PCT.ConclusionsExploiting the versatility of phage-display and biopanning allowed the identification of two distinct peptide ligands that bind specifically to the basolateral membrane of PCT. Tubule segment-specific ligands, such as the described PCT ligands, may be useful for the analysis of cell-extracellular matrix interactions and may contribute to the development of new therapeutic strategies for renal diseases