Katharina Beck

Pharmacophore Models and Pharmacophore-Based Virtual Screening: Concepts and Applications Exemplified on Hydroxysteroid Dehydrogenases

Computational methods are well-established tools in the drug discovery process and can be employed for a variety of tasks. Common applications include lead identification and scaffold hopping, as well as lead optimization by structure-activity relationship analysis and selectivity profiling. In addition, compound-target interactions associated with potentially harmful effects can be identified and investigated. This review focuses on pharmacophore-based virtual screening campaigns specifically addressing the target class of hydroxysteroid dehydrogenases. Many members of this enzyme family are associated with specific pathological conditions, and pharmacological modulation of their activity may represent promising therapeutic strategies. On the other hand, unintended interference with their biological functions, e.g., upon inhibition by xenobiotics, can disrupt steroid hormone-mediated effects, thereby contributing to the development and progression of major diseases. Besides a general introduction to pharmacophore modeling and pharmacophore-based virtual screening, exemplary case studies from the field of short-chain dehydrogenase/reductase (SDR) research are presented. These success stories highlight the suitability of pharmacophore modeling for the various application fields and suggest its application also in futures studies.

Evaluation of tetrabromobisphenol A effects on human glucocorticoid and androgen receptors: A comparison of results from human- with yeast-based in vitro assays.

The incidence of immune-related diseases increased over the last years in industrialized countries, suggesting a contribution of environmental factors. Impaired glucocorticoid action has been associated with immune disorders. Thus, there is an increasing interest to identify chemicals disrupting glucocorticoid action. The widely used flame retardant tetrabromobisphenol A (TBBPA) was reported earlier to potently inhibit glucocorticoid receptor (GR) and moderately androgen receptor (AR) activity in yeast-based reporter gene assays. To further characterize possible GR disrupting effects of TBBPA, transactivation experiments using a human HEK-293 cell-based reporter gene assay and cell-free receptor binding experiments were performed in the present study. Both, transactivation and GR binding experiments failed to detect any activity of TBBPA on GR function. Molecular docking calculations supported this observation. Additionally, the current study could confirm the antiandrogenic activity of TBBPA seen in the yeast assay, although the effect was an order of magnitude less pronounced in the HEK-293 cell-based system. In conclusion, TBBPA does not directly affect GR function and, considering its rapid metabolism and low concentrations found in humans, it is unlikely to cause adverse effects by acting through AR. This study emphasizes the use of cell-free assays in combination with cell-based assays for the in vitro evaluation of endocrine disrupting chemicals.

Sex differences in prolactin levels in emerging psychosis: Indication for enhanced stress reactivity in women

BACKGROUND Hyperprolactinemia is a known side effect of antipsychotics. In recent reports it has also been shown in antipsychotic-naïve at-risk mental state (ARMS) and first-episode psychosis (FEP) patients. Prolactin is not only involved in reproduction and lactation, but is also synthesized in response to stress. As stress is thought to play an important role in the onset and relapse of schizophrenia, the aim of this study was to further elucidate the influence of prolactin in emerging psychosis. METHODS The data analysed in this study were collected within the prospective Früherkennung von Psychosen (FePsy) study. Blood sample collection took place under standardized conditions between 8 and 10am after an overnight fast and 30minutes of rest. All patients were antipsychotic-naïve and did not take any prolactin influencing medication. RESULTS Our sample consisted of 116 antipsychotic-naïve ARMS and 49 FEP patients. Hyperprolactinemia was shown in 32% of ARMS and 35% of FEP patients. After correction for the normal biological variation between the sexes, we still found higher average prolactin levels in female than in male patients (β=0.42; t=2.47; p=0.01) but no difference in prolactin levels between ARMS and FEP patients (β=-0.05; t=-0.30; p=0.76). The survival analysis revealed no significant predictive value for prolactin levels to predict transition to psychosis. CONCLUSION Our findings support a possible role of prolactin in emerging psychosis and it could be speculated that stress, which can induce hyperprolactinemia, has a stronger effect on women than on men in emerging psychosis.

Carbonyl reductase 1 catalyzes 20β-reduction of glucocorticoids, modulating receptor activation and metabolic complications of obesity

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20β-dihydrocortisol (20β-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20β-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20β-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.

The relationship between negative symptoms and cognitive functioning in patients at clinical high risk for psychosis

Negative symptoms and neurocognitive performance have been reported to be negatively associated in patients with emerging psychosis. However, most previous studies focused on patients with frank psychosis and did not differentiate between subdomains of negative symptoms. Hence, we aimed to elucidate the specific relationship between negative symptoms and cognitive functioning in patients at clinical high risk (CHR) for psychosis. Data from 154 CHR patients collected within the prospective Früherkennung von Psychosen (FePsy) study were analyzed. Negative symptoms were assessed with the Scale for the Assessment of Negative Symptoms (SANS) and cognitive functioning with an extensive neuropsychological test battery. Regression analyses revealed significant negative associations between negative symptoms and cognitive functioning, particularly in the domains of nonverbal intelligence and verbal fluency. When analyzing each negative symptom domain separately, alogia and asociality/anhedonia were significantly negatively associated with nonverbal intelligence and alogia additionally with verbal fluency. Overall, our results in CHR patients are similar to those reported in patients with frank psychosis. The strong negative association between verbal fluency and negative symptoms may be indicative of an overlap between these constructs. Verbal fluency might have a strong influence on the clinical impression of negative symptoms (particularly alogia) and vice versa.

Evaluating verbal learning and memory in patients with an at-risk mental state or first episode psychosis using structural equation modelling

Background Verbal learning and memory are impaired not only in patients with a first episode of psychosis (FEP) but also–to a lower extent–in those with an at-risk mental state for psychosis (ARMS). However, little is known about the specific nature of these impairments. Hence, we aimed to study learning and memory processes in ARMS and FEP patients by making use of structural equation modelling. Methods Verbal learning was assessed with the California Verbal Learning Test (CVLT) in 98 FEP patients, 126 ARMS patients and 68 healthy controls (HC) as part of the Basel early detection of psychosis (FePsy) study. The four-factorial CFA model of Donders was used to estimate test performance on latent variables of the CVLT and growth curve analysis was used to model the learning curve. The latter allows disentangling initial recall, which is strongly determined by attentional processes, from the learning rate. Results The CFA model revealed that ARMS and FEP patients were impaired in Attention Span, Learning Efficiency and Delayed Memory and that FEP patients were additionally impaired in Inaccurate Memory. Additionally, ARMS-NT, but not ARMS-T, performed significantly worse than HC on Learning Efficiency. The growth curve model indicated that FEP patients were impaired in both initial recall and learning rate and that ARMS patients were only impaired in the learning rate. Conclusions Since impairments were more pronounced in the learning rate than the initial recall, our results suggest that the lower scores in the CVLT reported in previous studies are more strongly driven by impairments in the rate of learning than by attentional processes.

Exploring the predictive power of the unspecific risk category of the Basel Screening Instrument for Psychosis

Ultrahigh risk (UHR) criteria, consisting of brief limited intermittent psychotic symptoms (BLIPS), attenuated psychotic symptoms (APS) and genetic risk and deterioration (GRD) syndrome are the most widely used criteria for assessing the clinical high‐risk state for psychosis (CHR‐P). The Basel Screening Instrument for Psychosis (BSIP) includes a further risk category, the unspecific risk category (URC). However, little is known about the predictive power of this risk category compared to other risk categories.

Virtual screening applications in short-chain dehydrogenase/reductase research

Several members of the short-chain dehydrogenase/reductase (SDR) enzyme family play fundamental roles in adrenal and gonadal steroidogenesis as well as in the metabolism of steroids, oxysterols, bile acids, and retinoids in peripheral tissues, thereby controlling the local activation of their cognate receptors. Some of these SDRs are considered as promising therapeutic targets, for example to treat estrogen-/androgen-dependent and corticosteroid-related diseases, whereas others are considered as anti-targets as their inhibition may lead to disturbances of endocrine functions, thereby contributing to the development and progression of diseases. Nevertheless, the physiological functions of about half of all SDR members are still unknown. In this respect, in silico tools are highly valuable in drug discovery for lead molecule identification, in toxicology screenings to facilitate the identification of hazardous chemicals, and in fundamental research for substrate identification and enzyme characterization. Regarding SDRs, computational methods have been employed for a variety of applications including drug discovery, enzyme characterization and substrate identification, as well as identification of potential endocrine disrupting chemicals (EDC). This review provides an overview of the efforts undertaken in the field of virtual screening supported identification of bioactive molecules in SDR research. In addition, it presents an outlook and addresses the opportunities and limitations of computational modeling and in vitro validation methods.

DHRS7 (SDR34C1) - A new player in the regulation of androgen receptor function by inactivation of 5α-dihydrotestosterone?

DHRS7 (SDR34C1) has been associated with potential tumor suppressor effects in prostate cancer; however, its function remains largely unknown. Recent experiments using purified recombinant human DHRS7 suggested several potential substrates, including the steroids cortisone and Δ4-androstene-3,17-dione (androstenedione). However, the substrate and cofactor concentrations used in these experiments were very high and the physiological relevance of these observations needed to be further investigated. In the present study, recombinant human DHRS7 was expressed in intact HEK-293 cells in order to investigate whether glucocorticoids and androgens serve as substrates at sub-micromolar concentrations and at physiological cofactor concentrations. Furthermore, the membrane topology of DHRS7 was revisited using redox-sensitive green-fluorescent protein fusions in living cells. The results revealed that (1) cortisone is a substrate of DHRS7; however, it is not reduced to cortisol but to 20β-dihydrocortisone, (2) androstenedione is not a relevant substrate of DHRS7, (3) DHRS7 catalyzes the oxoreduction of 5α-dihydrotestosterone (5αDHT) to 3α-androstanediol (3αAdiol), with a suppressive effect on androgen receptor (AR) transcriptional activity, and (4) DHRS7 is anchored in the endoplasmic reticulum membrane with a cytoplasmic orientation. Together, the results show that DHRS7 is a cytoplasmic oriented enzyme exhibiting 3α/20β-hydroxysteroid dehydrogenase activity, with a possible role in the modulation of AR function. Further research needs to address the physiological relevance of DHRS7 in the inactivation of 5αDHT and AR regulation.