Construction of 3D models of the CYP11B family as a tool to predict ligand binding characteristics

Aldosterone is synthesised by aldosterone synthase (CYP11B2). CYP11B2 has a highly homologous isoform, steroid 11β-hydroxylase (CYP11B1), which is responsible for the biosynthesis of aldosterone precursors and glucocorticoids. To investigate aldosterone biosynthesis and facilitate the search for selective CYP11B2 inhibitors, we constructed three-dimensional models for CYP11B1 and CYP11B2 for both human and rat. The models were constructed based on the crystal structure of Pseudomonas Putida CYP101 and Oryctolagus Cuniculus CYP2C5. Small steric active site differences between the isoforms were found to be the most important determinants for the regioselective steroid synthesis. A possible explanation for these steric differences for the selective synthesis of aldosterone by CYP11B2 is presented. The activities of the known CYP11B inhibitors metyrapone, R-etomidate, R-fadrazole and S-fadrazole were determined using assays of V79MZ cells that express human CYP11B1 and CYP11B2, respectively. By investigating the inhibitors in the human CYP11B models using molecular docking and molecular dynamics simulations we were able to predict a similar trend in potency for the inhibitors as found in the in vitro assays. Importantly, based on the docking and dynamics simulations it is possible to understand the enantioselectivity of the human enzymes for the inhibitor fadrazole, the R-enantiomer being selective for CYP11B2 and the S-enantiomer being selective for CYP11B1

Involvement of insulin-like factor 3 (Insl3) in diethylstilbestrol-induced cryptorchidism

Recently, it has been shown that targeted inactivation of the Insl3 gene in male mice results in cryptorchidism. The Insl3 gene encodes insulin-like factor 3 (Insl3), which is expressed in fetal Leydig cells. The testicular factor Insl3 appears to play an important role in the transabdominal phase of testis descent, which involves development of the gubernaculum. Other studies have demonstrated that in utero exposure to diethylstilbestrol (DES), a synthetic estrogen, can lead to cryptorchidism both in humans and in animal models. The present study was undertaken to investigate whether prenatal DES-exposure might interfere with testicular Insl3 mRNA expression. Furthermore, the effect of DES on steroidogenic factor 1 (SF-1) mRNA expression lev

Application of a Ligand-Based Theoretical Approach to Derive Conversion Paths and Ligand Conformations in CYP11B2-Mediated Aldosterone Formation

The biosynthesis of the mineralocorticoid hormone aldosterone involves a multistep hydroxylation of 11-deoxycorticosterone at the 11- and 18-positions, resulting in the formation of corticosterone and 18-hydroxycorticosterone, the final precursor of aldosterone. Two members of the cytochrome P450 11B family, CYP11B1 and CYP11B2, are known to catalyze these 11- and 18-hydroxylations, however, only CYP11B2 can oxidize 18-hydroxycorticosterone to aldosterone. It is unknown what sequence of hydroxylations leads to the formation of 18-hydroxycorticosterone. In this study we have investigated which of the possible conversion paths towards formation of 18-hydroxycorticosterone and aldosterone are most likely from the ligand perspective. Therefore, we combined quantum mechanical investigations on the steroid conformations of 11-deoxycorticosterone and its ensuing reaction intermediates with Fukui indices calculations to predict the reactivity of their carbon atoms for an attack by the iron-oxygen species. Both F(-) and F(0) were calculated to account for different mechanisms of substrate conversion. We show which particular initial conformations of 11-deoxycorticosterone and which conversion paths are likely to result in the successful synthesis of aldosterone, and thereby may be representative for the mechanism of aldosterone biosynthesis by CYP11B2. Moreover, we found that the most likely path for aldosterone synthesis coincides with the substrate conformation proposed in an earlier publication (Ref. (2)). To summarize, we show that on a theoretical and strictly ligand-directed basis only a limited number of reaction paths in the conversion of 11-deoxycorticosterone to aldosterone is possible. Despite its theoretical nature, this knowledge may help to understand the catalytic function of CYP11B1 and CYP11B2

Synthesis, Biological Evaluation, and Molecular Modeling of 1-Benzyl-1H-imidazoles as Selective Inhibitors of Aldosterone Synthase (CYP11B2)

Reducing aldosterone action is beneficial in various major diseases such as heart failure. Currently, flits is achieved with mineralocorticoid receptor antagonists, however, aldosterone synthase (CYP11B2) inhibitors may offer a promising alternative. In this study, WC used three-dimensional modeling of CYP11B2 to model the binding modes of the natural substrate 18-hydroxycorticosterone and the recently published CYP11B2 inhibitor R-fadrozole as a rational guide to design 44 structurally simple and achiral 1-benzyl-1H-imidazoles. Their syntheses, in vitro inhibitor potencies, and in silico docking are described. Some promising CYP11B2 inhibitors were identified, with our novel lead MOERAS115 (4-((5-phenyl-1H-imidazol-1-y1)methyl)benzonitrile) displaying an IC50 for CYP11B2 of 1.7 nM, and a CYP11B2 (versus CYP11B1) selectivity of 16.5, comparable to R-fadrozole (IC50 for CYP11B2 6.0 nM. Selectivity 19.8). Molecular docking of the Inhibitors in the models enabled us to generate posthoc hypotheses oil their binding modes, providing a Valuable basis for future Studies and further design of CYP11B2 inhibitors

Circulating adipokine levels and COVID-19 severity in hospitalized patients

BACKGROUND: Obesity is a risk factor for adverse outcomes in COVID-19, potentially driven by chronic inflammatory state due to dysregulated secretion of adipokines and cytokines. We investigated the association between plasma adipokines and COVID-19 severity, systemic inflammation, clinical parameters, and outcome of COVID-19 patients. METHODS: In this multi-centre prospective cross-sectional study, we collected blood samples and clinical data from COVID-19 patients. The severity of COVID-19 was classified as mild (no hospital admission), severe (ward admission), and critical (ICU admission). ICU non-COVID-19 patients were also included and plasma from healthy age, sex, and BMI-matched individuals obtained from Lifelines. Multi-analyte profiling of plasma adipokines (Leptin, Adiponectin, Resistin, Visfatin) and inflammatory markers (IL-6, TNFα, IL-10) were determined using Luminex multiplex assays. RESULTS: Between March and December 2020, 260 SARS-CoV-2 infected individuals (age: 65 [56-74] BMI 27.0 [24.4-30.6]) were included: 30 mild, 159 severe, and 71 critical patients. Circulating leptin levels were reduced in critically ill patients with a high BMI yet this decrease was absent in patients that were administered dexamethasone. Visfatin levels were higher in critical COVID-19 patients compared to non-COVID-ICU, mild and severe patients (4.7 vs 3.4, 3.0, and 3.72 ng/mL respectively, p < 0.05). Lower Adiponectin levels, but higher Resistin levels were found in severe and critical patients, compared to those that did not require hospitalization (3.65, 2.7 vs 7.9 µg/mL, p < 0.001, and 18.2, 22.0 vs 11.0 ng/mL p < 0.001). CONCLUSION: Circulating adipokine levels are associated with COVID-19 hospitalization, i.e., the need for oxygen support (general ward), or the need for mechanical ventilation and other organ support in the ICU, but not mortality