Interaction of CYP11B1 (cytochrome P-45011 beta) with CYP11A1 (cytochrome P-450scc) in COS-1 cells.

The interactions of CYP11B1 (cytochrome P-45011beta), CYP11B2 (cytochrome P-450aldo) and CYP11A1 (cytochrome P-450scc) were investigated by cotransfection of their cDNA into COS-1 cells. The effect of CYP11A1 on CYP11B isozymes was examined by studying the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. It was shown that when human or bovine CYP11B1 and CYP11A1 were cotransfected they competed for the reducing equivalents from the limiting source contained in COS-1 cells; this resulted in a decrease of the CYP11B activities without changes in the product formation patterns. The competition of human CYP11A1 with human CYP11B1 and CYP11B2 could be diminished with excess expression of bovine adrenodoxin. However, the coexpression of bovine CYP11B1 and CYP11A1 in the presence of adrenodoxin resulted in a stimulation of 11beta-hydroxylation activity of CYP11B1 and in a decrease of the 18-hydroxycorticosterone and aldosterone formation. These results suggest that the interactions of CYP11A1 with CYP11B1 and CYP11B2 do not have an identical regulatory function in human and in bovine adrenal tissue.     

Steroid 11-beta-hydroxylase deficiency caused by compound heterozygosity for a novel mutation, p.G314R, in one CYP11B1 allele, and a chimeric CYP11B2/CYP11B1 in the other allele

AIMS: Steroid 11beta-hydroxylase deficiency (11beta-OHD) is the second most common (5-8%) cause of congenital adrenal hyperplasia (CAH), and results from homozygous or compound heterozygous mutations or deletions of the responsible gene CYP11B1. In order to better understand the molecular basis causing 11beta-OHD, we performed detailed studies of CYP11B1 in a newly described patient diagnosed with the classical signs of 11beta-OHD. METHODS:CYP11B1 of the patient was investigated by polymerase chain reaction (PCR), sequencing, restriction fragment length polymorphism (RFLP) analysis, Southern blotting, and transient cell expression. RESULTS: We identified two new mutated alleles in CYP11B1. In one allele CYP11B1 has a g.940G-->C (p.G314R) missense mutation. On the other allele we found a chimeric gene that consists of part of the aldosterone synthase gene (CYP11B2) at exons 1-3 and part of the 11beta-hydroxylase gene (CYP11B1) at exons 4-9. Inin vitro studies, the g.940G-->C (p.G314R) mutation abolished all hydroxylase activity in comparison with the wild-type 11beta-hydroxylase. The chimeric CYP11B2/CYP11B1 protein retained 11beta-hydroxylase enzymatic activity in vitro. CONCLUSION: This case is caused by compound heterozygosity for a nonfunctional missense mutation and a chimeric CYP11B2/CYP11B1 gene with hydroxylase activity that is controlled by the CYP11B2 promoter. The most likely explanation is that the CYP11B2 promoter does not function in the zona fasciculata/reticularis where cortisol is exclusively synthesized.     

Development of a test system for inhibitors of human aldosterone synthase (CYP11B2): screening in fission yeast and evaluation of selectivity in V79 cells

Aldosterone synthase (CYP11B2) is a mitochondrial cytochrome P450 enzyme catalyzing the last steps of aldosterone production in the adrenal cortex. A new pharmacological approach for the treatment of the aldosterone induced effects in congestive heart failure and all forms of hyperaldosteronism could be the use of CYP11B2 inhibitors. In search for such compounds, it was our goal to develop a cellular enzyme assay suitable for screening high numbers of compounds. An assay procedure for the evaluation of inhibitors using the human CYP11B2 expressed in fission yeast Schizosaccharomyces pombe was established and a series of 10 compounds was tested in this whole cellular system. Human 11beta-hydroxylase (CYP11B1), which catalyzes the production of glucocorticoids, shows more than 90% homology compared to human CYP11B2. As this enzyme should not be affected, strong inhibitors of CYP11B2 have to be tested for selectivity. For that purpose, an assay procedure with V79MZ cells that express human CYP11B1 and CYP11B2, respectively, was integrated into the evaluation process. Using these screening procedures a potent and rather selective non-steroidal inhibitor of human CYP11B2 was detected with an IC(50) value of 59nM. We also identified a very potent inhibitor of both enzymes showing a stronger inhibitory activity against the cortisol producing CYP11B1.     

Analyses of the CYP11B gene family in the guinea pig suggest the existence of a primordial CYP11B gene with aldosterone synthase activity.

In this study we describe the isolation of three genes of the CYP11B family of the guinea pig. CYP11B1 codes for the previously described 11beta-hydroxylase [Bülow, H.E.,M?bius, K., B?hr, V. & Bernhardt, R. (1996) Biochem. Biophys. Res. Commun. 221, 304-312] while CYP11B2 represents the aldosterone synthase gene. As no expression for CYP11B3 was detected this gene might represent a pseudogene. Transient transfection assays show higher substrate specificity for its proper substrate for CYP11B1 as compared to CYP11B2, which could account for the zone-specific synthesis of mineralocorticoids and glucocorticoids, respectively. Thus, CYP11B2 displayed a fourfold higher ability to perform 11beta-hydroxylation of androstenedione than CYP11B1, while this difference is diminished with the size of the C17 substituent of the substrate. Furthermore, analyses with the electron transfer protein adrenodoxin indicate differential sensitivity of CYP11B1 and CYP11B2 as well as the three hydroxylation steps catalysed by CYP11B2 to the availability of reducing equivalents. Together, both mechanisms point to novel protein intrinsic modalities to achieve tissue-specific production of mineralocorticoids and glucocorticoids in the guinea pig. In addition, we conducted phylogenetic analyses. These experiments suggest that a common CYP11B ancestor gene that possessed both 11beta-hydroxylase and aldosterone synthase activity underwent a gene duplication event before or shortly after the mammalian radiation with subsequent independent evolution of the system in different lines. Thus, a differential mineralocorticoid and glucocorticoid synthesis might be an exclusive achievement of mammals.     

Modulation of aldosterone biosynthesis by adrenodoxin mutants with different electron transport efficiencies.

Aldosterone biosynthesis is highly regulated on different levels by hormones, potassium, lipid composition of the membrane and the molecular structure of its gene. Here, the influence of the electron transport efficiency from adrenodoxin (Adx) to CYP11B1 on the activities of bovine CYP11B1 has been investigated using a liposomal reconstitution system with truncated mutants of Adx. It could be clearly demonstrated that Adx mutants Adx 4-114 and Adx 4-108, possessing enhanced electron transfer abilities, produce increases in corticosterone and aldosterone biosynthesis. Based on the Vmax values of corticosterone and aldosterone formation, Adx 4-108 and Adx 4-114 enhance corticosterone synthesis 1.3-fold and aldosterone formation threefold and twofold, respectively. The production of 18-hydroxycorticosterone was changed only slightly in these Adx mutants. The effect of Adx 1-108 on the product patterns of bovine CYP11B1, human CYP11B1 and human CYP11B2 was confirmed in COS-1 cells by cotransfection of CYP11B- and Adx-containing expression vectors. It could be shown that Adx 1-108 enhances the formation of aldosterone by bovine CYP11B1 and by human CYP11B2, and stimulates the production of corticosterone by bovine CYP11B1 and human CYP11B1 and CYP11B2 also.     

Exploring QSAR for CYP11B2 binding affinity and CYP11B2/CYP11B1 selectivity of diverse functional compounds using GFA and G/PLS techniques

A data set of a series of 132 structurally diverse compounds with cytochrome 11B2 and 11B1 (CYP11B2 and CYP11B1) enzyme inhibitory activities was subjected to molecular shape analysis to explore contributions of shape features as well as electronic, structural, and physicochemical parameters toward enzyme inhibitory activities, in search of appropriate molecular scaffolds with optimum substitutions for highly potent CYP11B2 inhibitors. Genetic function approximation (GFA) and genetic partial least squares (G/PLS) were used as chemometric tools for modeling, and the derived equations were of acceptable statistical quality considering both internal and external validation parameters (Q²: 0.514–0.659, R²_pred: 0.510–0.734). The G/PLS models with spline option for CYP11B2 and CYP11B1 inhibition and selectivity modeling appeared to be the best models based on r_m²_(overall) criterion. The study indicates the importance of the pyridinylnaphthalene and pyridylmethylene-indane scaffolds with less polar and electrophilic substituents for optimum CYP11B2 inhibitory activity and CYP11B2/CYP11B1 selectivity.     

The adrenocortical tumor cell line NCI-H295R as an in vitro screening system for the evaluation of CYP11B2 (aldosterone synthase) and CYP11B1 (steroid-11beta-hydroxylase) inhibitors

Aldosterone plays a key role in salt and water homeostasis but is also involved in the development and progression of congestive heart failure and myocardial fibrosis. As a new pharmacological strategy for the treatment of these diseases, we propose the inhibition of the key enzyme of mineralcorticoid formation, CYP11B2 (aldosterone synthase). For studies of the effects of CYP11B2 inhibitors on the adrenal cortex, we selected the NCI-H295R cell line which expresses most of the key enzymes necessary for steroidogenesis. To evaluate this cell line as a test system for effects and side effects of CYP inhibitors, we established assays using radiolabeled substrates of CYP11B2 and CYP11B1 and subsequently tested a series of CYP11B2 inhibitors including the CYP19 inhibitor fadrozole. Fadrozole and compounds 6, 9 and 10 were more potent towards CYP11B2 compared to CYP11B1 with IC(50) values in the nanomolar range. To analyze their overall effect, the formation of steroids in the cell culture supernatant was monitored. All compounds led to a concentration-dependent reduction of the aldosterone secretion but also reduced the formation of cortisol and androgens. In conclusion, the H295R cell line is a suitable tool for the prediction of overall side effects of CYP11B2 inhibitors on steroidogenesis.     

Fission yeast Schizosaccharomyces pombe as a new system for the investigation of corticosterone methyloxidase deficiency-causing mutations

The aldosterone synthase, CYP11B2, catalyses the conversion of 11-deoxycorticosterone to aldosterone, a process that requires three steps: a hydroxylation at position 11β to form corticosterone, another one at position 18 to produce 18-hydroxycorticosterone, and, finally, an oxidation at position 18 to form aldosterone. Aldosterone synthase deficiency usually finds its expression in infancy as a life-threatening electrolyte imbalance, caused by mutations in the CYP11B2 gene. Therefore, in depth studies of mutations and their enzymatic activities will provide information for the diagnosis and management of hypoaldosteronism caused by CYP11B2 deficiencies. Here, we report the development of a fast and cheap whole-cell technology for the enzymatic characterisation of CYP11B2 mutations. The principle of the new system is the heterologous expression of the mutants of CYP11B2 in fission yeast (Schizosaccharomyces pombe) followed by steroid bioconversion assays for the enzymatic characterisation of the investigated mutants. The new system was validated and 10 known mutations of CYP11B2 have been investigated, two of them for the first time concerning their effect on the CYP11B2 three-step reaction. The results of the fission yeast system were in good agreement with the cell culture results presenting this new system as an alternative non radioactive method that can be applied for the enzymatic characterisation of CYP11B2 mutations.     

Separate induction of the two isozymes of cytochrome P-45011β in rat adrenal zona glomerulosa cells

In the rat adrenal cortex, two isozymes of cytochrome P-450_11β (CYP11B1 and CYP11B2) have been identified. They are encoded by two different genes with a homology much higher in their coding than in their 5′-flanking regions. CYP11B1 is found in all the zones of the gland and catalyzes a single hydroxylation of deoxycorticosterone (DOC) in the 11β- or the 18-position. CYP11B2 is produced exclusively in the zona glomerulosa and catalyzes all three reactions involved in the conversion of DOC to aldosterone. In vivo and in vitro, the expression of the genes encoding CYP11B1 and CYP11B2 is regulated by two separate control systems which appear to operate both independently and interdependently. In vivo, zona glomerulosa expression of CYP11B1 was enhanced by ACTH treatment or potassium depletion and was lowered by potassium repletion. CYP11B2 expression disappeared upon potassium depletion or ACTH treatment, but reappeared during potassium repletion. In vitro, only CYP11B1 activity was detectable and responsive to ACTH treatment in zona glomerulosa cells cultured at a potassium concentration of 6.4 mmol/1. Aldosterone biosynthetic activity and mRNA encoding CYP11B2 could be detected only after at least 1 day of exposure to a high extracellular potassium concentration ( 12 mmol/1).     

Molecular identity and gene expression of aldosterone synthase cytochrome P450

11Beta-hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11beta-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression.     

Ordered chimerogenesis applied to CYP2B P450 enzymes

BackgroundStructural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11.MethodsA series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast.ResultsMost of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F′/G′ cassette role and substrate access channels.ConclusionsSome sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity.General significanceThe result provides a new basis for determining the structural elements that control functions in complex enzymes.     

Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.     

N-(Pyridin-3-yl)benzamides as selective inhibitors of human aldosterone synthase (CYP11B2)

A series of 23 N-(Pyridin-3-yl)benzamides was synthesized and evaluated for their potential to inhibit human steroid-11β-hydroxylase (CYP11B1) and human aldosterone synthase (CYP11B2). The most potent and selective CYP11B2 inhibitors (IC₅₀ values 53-166 nM) were further evaluated for their potential to inhibit human CYP17 and CYP19, and no inhibition was observed. Clear evidence was shown for N-(Pyridin-3-yl)benzamides to be a highly selective class of CYP11B2 inhibitors in vitro.     

Mutations in aldosterone synthase gene of Milan hypertensive rats: phenotypic consequences

Using in vitro and in vivo methods, we have demonstrated increased sensitivity of adrenocortical steroidogenesis to ACTH in Milan hypertensive (MHS) compared with normotensive (MNS) rats and have investigated whether this is caused by mutations of steroidogenic enzymes. Genes encoding aldosterone synthase (CYP11B2) and 11beta-hydroxylase (CYP11B1) in MHS and MNS have been cloned and sequenced. Nucleotide 752 (G) in exon 4 of MHS CYP11B2 differs from that of MNS (A); CYP11B1 sequences were identical. The nucleotide 752 mutation caused a Q251R substitution in the amino acid sequence of MHS CYP11B2. The phenotype of MHS CYP11B2 alleles, when expressed in COS-1 cells, differed from that of MNS alleles. The relative activities of the three reactions catalyzed by CYP11B2 (11beta-hydroxylation of deoxycorticosterone, 18-hydroxylation of corticosterone, and dehydrogenation of 18-hydroxycorticosterone) were estimated after incubation of transfected cells with [(14)C]deoxycorticosterone and analysis of radioactivity associated with deoxycorticosterone, corticosterone, 18 hydroxycorticosterone, and aldosterone. Both 11- and 18-hydroxylase activities were lower (19 and 12%, respectively; P < 0.01 and P < 0.05) in cells transfected with MHS compared with MNS alleles, whereas 18-oxidase activity was 42% higher (P < 0.01). To assess the significance of the CYP11B2 mutation in vivo, DNA from F2 hybrid MHS x MNS rats was genotyped. MHS alleles were associated with lower urine volumes in both sexes, lower ventricle weights in male rats, but no difference in systolic or diastolic blood pressures between the sexes. We conclude that a mutation in CYP11B2 may affect aldosterone secretion in MHS; however, under normal environmental circumstances, we were unable to demonstrate any influence of this mutation on blood pressure.     

Benzophenones as xanthone-open model CYP11B1 inhibitors potentially useful for promoting wound healing

The inhibition of steroidogenic cytochrome P450 enzymes has been shown to play a central role in the management of life-threatening diseases such as cancer, and indeed potent inhibitors of CYP19 (aromatase) and CYP17 (17α hydroxylase/17,20 lyase) are currently used for the treatment of breast, ovarian and prostate cancer. In the last few decades CYP11B1 (11-β-hydroxylase) and CYP11B2 (aldosterone synthase), key enzymes in the biosynthesis of cortisol and aldosterone, respectively, have been also investigated as targets for the identification of new potent and selective agents for the treatment of Cushing's syndrome, impaired wound healing and cardiovascular diseases.In an effort to improve activity and synthetic feasibility of our different series of xanthone-based CYP11B1 and CYP11B2 inhibitors, a small series of imidazolylmethylbenzophenone-based compounds, previously reported as CYP19 inhibitors, was also tested on these new targets, in order to explore the role of a more flexible scaffold for the inhibition of CYP11B1 and -B2 isoforms. Compound 3 proved to be very potent and selective towards CYP11B1, and was thus selected for further optimization via appropriate decoration of the scaffold, leading to new potent 4′-substituted derivatives. In this second series, 4 and 8, carrying a methoxy group and a phenyl ring, respectively, proved to be low-nanomolar inhibitors of CYP11B1, despite a slight decrease in selectivity against CYP11B2. Moreover, unlike the benzophenones of the first series, the 4′-substituted derivatives also proved to be selective for CYP11B enzymes, showing very weak inhibition of CYP19 and CYP17.Notably, the promising result of a preliminary scratch test performed on compound 8 confirmed the potential of this compound as a wound-healing promoter.     

The discovery of potent inhibitors of aldosterone synthase that exhibit selectivity over 11-β-hydroxylase

Aldosterone, the final component of the renin–angiotensin–aldosterone system, plays an important role in the pathophysiology of hypertension and congestive heart failure. Aldosterone synthase (CYP11B2) catalyzes the last three steps of aldosterone biosynthesis, and as such appears to be a target for the treatment of these disorders. A sulfonamide–imidazole scaffold has proven to be a potent inhibitor of CYP11B2. Furthermore, this scaffold can achieve high levels of selectivity for CYP11B2 over CYP11B1, a key enzyme in the biosynthesis of cortisol.     

Stable expression of rat cytochrome P450 11β-Hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) in MA-10 cells

Glucocorticoids and mineralocorticoids are synthesized in the adrenal cortex through the action of two different cytochrome 11β-hydroxylases, CYP11B1 (11β-hydroxylase) and CYP11B2 (aldosterone synthase) which are distributed in the zona fasciculata and glomerulosa, respectively. We have created stably transfected cell lines using the Leydig tumor cell line MA-10 with CYP11B1 and CYP11B2 cDNA-containing plasmids which have a selectable gene to confer resistance to geneticin. The expression of the transfected cDNA in the cells was characterized by Northern-blot and measurement of enzymatic activity. The cell lines express the enzymes stably for many generations. CYP11B1 transfected cells converted DOC into corticosterone, 18-OH-DOC and small amounts of 18-OH-corticosterone, in a time and concentration dependent manner. Incubation of the cells with corticosterone generated 18-OH-corticosterone especially at concentrations of 30 and 100 μM. The production of 18-OH-corticosterone from corticosterone at these doses was significantly higher than incubations with similar concentrations of DOC. CYP11B2 transfected cells converted DOC into corticosterone, 18-OH-corticosterone, aldosterone and small amounts of 18-OH-DOC in a time and concentration dependent manner. They converted corticosterone into 18-OH-corticosterone and aldosterone in a time and concentration dependent manner. The absolute and relative production of aldosterone from DOC was significantly higher than when cells were incubated with corticosterone, and the ratio of aldosterone to 18-OH-corticosterone was higher at all concentrations of DOC compared to corticosterone. CYP11B2 transfected cells (but not the CYP11B1 transfected cells) transform 18-OH-DOC into 18-OH-corticosterone, but can not convert 18-OH-DOC into aldosterone. In conclusion, stably transfected MA-10 cells with the cDNAs for the CYP11B1 and CYP11B2 enzymes were prepared and their enzymatic activity studied. These cells are useful in the study of inhibitors of the specific enzymes, as well as determining the roles that each enzyme plays in zone-specific steroidogenesis in the adrenal cortex.     

Coexpression of CYP11B2 or CYP11B1 with adrenodoxin and adrenodoxin reductase for assessing the potency and selectivity of aldosterone synthase inhibitors

Excessive production of aldosterone has been implicated in the pathogenesis of hypertension and heart failure. One approach to ameliorate the deleterious effects of aldosterone is to suppress its biosynthesis. The enzyme aldosterone synthase (CYP11B2) is responsible for the final step of aldosterone synthesis. It requires electron transfer from the adrenodoxin/adrenodoxin reductase system to catalyze the production of aldosterone. A stable cell line simultaneously overexpressing recombinant human CYP11B2 as well as human adrenodoxin and adrenodoxin reductase was established to help maximize the enzyme activity. The homogenate of these cells was used to develop an in vitro CYP11B2 assay using 11-deoxycorticosterone as a substrate. By the same strategy, another stable cell line simultaneously overexpressing human 11β-hydroxylase (CYP11B1), an enzyme responsible for the final step of cortisol biosynthesis, and the two electron transfer proteins was also established, and an in vitro CYP11B1 assay using 11-deoxycortisol as a substrate was likewise developed to assess the selectivity of CYP11B2 inhibitors. FAD286, a reference CYP11B2 inhibitor, inhibited CYP11B2 and CYP11B1 activities with IC₅₀ values of 1.6 ± 0.1 and 9.9 ± 0.9 nM (mean ± SEM, n = 3–6), respectively. Kinetics studies revealed that the compound inhibited the activity of both enzymes competitively with respective K_i values of 0.8 ± 0.04 and 2.2 ± 0.2 nM (n = 3–4). These assays can be used for assessing the potency and selectivity of CYP11B2 inhibitors for the treatment of hypertension and heart failure.