Profiling withanolide A for therapeutic targets in neurodegenerative diseases.

To identify new potential therapeutic targets for neurodegenerative diseases, we initiated activity-based protein profiling studies with withanolide A (WitA), a known neuritogenic constituent of Withania somnifera root with unknown mechanism of action. Molecular probes were designed and synthesized, and led to the discovery of the glucocorticoid receptor (GR) as potential target. Molecular modeling calculations using the VirtualToxLab predicted a weak binding affinity of WitA for GR. Neurite outgrowth experiments in human neuroblastoma SH-SY5Y cells further supported a glucocorticoid-dependent mechanism, finding that WitA was able to reverse the outgrowth inhibition mediated by dexamethasone (Dex). However, further GR binding and transactivation assays found no direct interference of WitA. Further molecular modeling analysis suggested that WitA, although forming several contacts with residues in the GR binding pocket, is lacking key stabilizing interactions as observed for Dex. Taken together, the data suggest that WitA-dependent induction of neurite outgrowth is not through a direct effect on GR, but might be mediated through a closely related pathway. Further experiments should evaluate a possible role of GR modulators and/or related signaling pathways such as ERK, Akt, NF-κB, TRα, or Hsp90 as potential targets in the WitA-mediated neuromodulatory effects.

Steroid profiling in H295R cells to identify chemicals potentially disrupting the production of adrenal steroids.

The validated OECD test guideline 456 based on human adrenal H295R cells promotes measurement of testosterone and estradiol production as read-out to identify potential endocrine disrupting chemicals. This study aimed to establish optimal conditions for using H295R cells to detect chemicals interfering with the production of key adrenal steroids. H295R cells' supernatants were characterized by liquid chromatography-mass spectrometry (LC-MS)-based steroid profiling, and the influence of experimental conditions including time and serum content was assessed. Steroid profiles were determined before and after incubation with reference compounds and chemicals to be tested for potential disruption of adrenal steroidogenesis. The H295R cells cultivated according to the OECD test guideline produced progestins, glucocorticoids, mineralocorticoids and adrenal androgens but only very low amounts of testosterone. However, testosterone contained in Nu-serum was metabolized during the 48h incubation. Thus, inclusion of positive and negative controls and a steroid profile of the complete medium prior to the experiment (t=0h) was necessary to characterize H295R cells' steroid production and indicate alterations caused by exposure to chemicals. Among the tested chemicals, octyl methoxycinnamate and acetyl tributylcitrate resembled the corticosteroid induction pattern of the positive control torcetrapib. Gene expression analysis revealed that octyl methoxycinnamate and acetyl tributylcitrate enhanced CYP11B2 expression, although less pronounced than torcetrapib. Further experiments need to assess the toxicological relevance of octyl methoxycinnamate- and acetyl tributylcitrate-induced corticosteroid production. In conclusion, the extended profiling and appropriate controls allow detecting chemicals that act on steroidogenesis and provide initial mechanistic evidence for prioritizing chemicals for further investigations.

Formation of threohydrobupropion from bupropion is dependent on 11ß-hydroxysteroid dehydrogenase 1.

Bupropion is widely used for treatment of depression and as a smoking-cessation drug. Despite more than 20 years of therapeutic use, its metabolism is not fully understood. While CYP2B6 is known to form hydroxybupropion, the enzyme(s) generating erythro- and threohydrobupropion have long remained unclear. Previous experiments using microsomal preparations and the nonspecific inhibitor glycyrrhetinic acid suggested a role for 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1) in the formation of both erythro- and threohydrobupropion. 11ß-HSD1 catalyzes the conversion of inactive glucocorticoids (cortisone, prednisone) to their active forms (cortisol, prednisolone). Moreover, it accepts several other substrates. Here, we used for the first time recombinant 11ß-HSD1 to assess its role in the carbonyl reduction of bupropion. Furthermore, we applied human, rat, and mouse liver microsomes and a selective inhibitor to characterize species-specific differences and to estimate the relative contribution of 11ß-HSD1 to bupropion metabolism. The results revealed 11ß-HSD1 as the major enzyme responsible for threohydrobupropion formation. The reaction was stereoselective and no erythrohydrobupropion was formed. Human liver microsomes showed 10 and 80 times higher activity than rat and mouse liver microsomes, respectively. The formation of erythrohydrobupropion was not altered in experiments with microsomes from 11ß-HSD1-deficient mice or upon incubation with 11ß-HSD1 inhibitor, indicating the existence of another carbonyl reductase that generates erythrohydrobupropion. Molecular docking supported the experimental findings and suggested that 11ß-HSD1 selectively converts R-bupropion to threohydrobupropion. Enzyme inhibition experiments suggested that exposure to bupropion is not likely to impair 11ß-HSD1-dependent glucocorticoid activation but that pharmacological administration of cortisone or prednisone may inhibit 11ß-HSD1-dependent bupropion metabolism.

Species-specific differences in the inhibition of human and zebrafish 11ß-hydroxysteroid dehydrogenase 2 by thiram and organotins.

Dithiocarbamates and organotins can inhibit enzymes by interacting with functionally essential sulfhydryl groups. Both classes of chemicals were shown to inhibit human 11ß-hydroxysteroid dehydrogenase 2 (11ß-HSD2), which converts active cortisol into inactive cortisone and has a role in renal and intestinal electrolyte regulation and in the feto-placental barrier to maternal glucocorticoids. In fish, 11ß-HSD2 has a dual role by inactivating glucocorticoids and generating the major androgen 11-ketotestosterone. Inhibition of this enzyme may enhance glucocorticoid and diminish androgen effects in fish. Here, we characterized 11ß-HSD2 activity of the model species zebrafish. A comparison with human and mouse 11ß-HSD2 revealed species-specific substrate preference. Unexpectedly, assessment of the effects of thiram and several organotins on the activity of zebrafish 11ß-HSD2 showed weak inhibition by thiram and no inhibition by any of the organotins tested. Sequence comparison revealed the presence of an alanine at position 253 on zebrafish 11ß-HSD2, corresponding to cysteine-264 in the substrate-binding pocket of the human enzyme. Substitution of alanine-253 by cysteine resulted in a more than 10-fold increased sensitivity of zebrafish 11ß-HSD2 to thiram. Mutating cysteine-264 on human 11ß-HSD2 to serine resulted in 100-fold lower inhibitory activity. Our results demonstrate significant species differences in the sensitivity of human and zebrafish 11ß-HSD2 to inhibition by thiram and organotins. Site-directed mutagenesis revealed a key role of cysteine-264 in the substrate-binding pocket of human 11ß-HSD2 for sensitivity to sulfhydryl modifying agents.