Comparative genetic analysis of different forms of low-renin arterial hypertension

High level of clinical and genetic heterogeneity is a characteristic of arterial hypertension (AH) that is one of the most wide-spread cardiovascular diseases. In most cases (excluding a few monogenic forms), AH is a polygenic disease and genes of renin-angiotensin-aldosterone system play an important role in AH predisposition. 20-25% AH cases occur during low activity of renin in blood plasma (low-renin form of AH) while aldosterone production can be increased (hyperaldosteronism, HA) or normal. We examined polymorphism of genes that code the renin-angiotensin-aldosterone system components in the groups of low-renin forms of AH, namely, primary HA, idiopathic HA and AH with normal level of aldosterone. For all HA cases, the absence of chimeric CYP11B2/CYP11B1 gene that is a cause for monogenic disease--amilial HA of first type, was shown. A comparison of distributions of alleles and genotypes of polymorphous regions of genes: CYP11B2 (C-344T), REN (C-5434T, C-5312T and A BglI G), AGT (Thr174Met), ACE (I/D), CMA (G-1903A), AT2R1 (A1166C) and of their combinations is the groups described above was done. The analysis of carriership of the alleles and genotypes combinations of the polymorphous regions has shown that genes CYP11B2, REN, ACE, CMA andA T2R1 participate in development of low-renin HA. The results are evidence of similarities and some definite differences in genetic nature of the different forms of low-renin AH and, to say more widely, argue that the investigation of genetic predisposition for clinically heterogeneous forms of polygene diseases by comparison of groups of patients, separated in accordance with peculiarities of disease course, holds much promise for their hereditary background understanding.     

New aspects on primary aldosteronism

The adrenal cortex synthesizes and releases steroid hormones, mainly mineralocorticoids and glucocorticoids. There is a functional zonation of the adrenal cortex and steroid synthesis is thoroughly regulated. Overproduction of aldosterone, primary aldosteronism, may be much more common than previously known and may be responsible for 10% of essential hypertension. Primary aldosteronism is characterized by autonomous production of aldosterone, suppressed renin activity, hypokalemia, and hypertension. The two most common forms are unilateral adenoma and bilateral hyperplasia. In spite of thorough clinical workup and careful histopathology it is often difficult to differentiate between adenoma and hyperplasia. The gene CYP11B2 encodes the steroid synthesizing enzymes for aldosterone production, while the genes CYP17 and CYP11B1 are needed for cortisol production. Most normal controls show expression of CYP11B2 in zona glomerulosa. Expression of CYP11B1 and CYP17 is seen in zona fasciculata and reticularis, whereas the expression of CYP21 is present in all three cortical layers. Adenomas from patients with primary aldosteronism show considerable variation in the expression of CYP11B2. Adenomas from patients with Cushing's syndrome have a strong expression of CYP11B1 and CYP17. In a patient material of 29 cases of primary aldosteronism, 4 patients had small nodules detected with expression of CYP11B2 gene. These nodules were not visualized on CT, whereas adrenal masses seen on CT in these patients showed CYP11B1 and CYP17 gene expression. This suggests that these small nodules are responsible for the aldosterone production and this is characteristic of nodular hyperplasia in patients with primary aldosteronism. In conclusion, this method to visualize mRNA gene expression of steroidogenic enzymes, and especially expression of CYP11B2, has increased the knowledge of adrenal pathophysiology. The results emphasize the value to include functional studies (venous sampling and/or scintigraphy) in the preoperative work up of patients with primary aldosteronism.     

Dihydrobenzisoxazole-4-one compounds are novel selective inhibitors of aldosterone synthase (CYP11B2) with in vivo activity

6,7-Dihydro-5H-2,1-benzisoxazol-4-one analogs are potent inhibitors of aldosterone synthase (CYP11B2) with selectivity over the highly homologous enzyme cortisol synthase (CYP11B1). These compounds are unique among inhibitors of CYP11B2 in their lack of a strong-heme binding group such as a pyridine or imidazole. Poor metabolic stability in hepatocyte incubations was found to proceed via a reduction of the isoxazole ring. While the enzyme responsible for the reductive metabolism remains unknown, the rate of metabolism could be attenuated by the addition of polar functionality. The in vitro CYP11B2 potency and selectivity were confirmed in vivo in a cynomolgus monkey model by the inhibition of ACTH stimulated aldosterone production without impacting plasma cortisol concentrations.     

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.     

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.     

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.     

Predominance of interaction among wild-type alleles of CYP11B2 in Himalayan natives associates with high-altitude adaptation

Sojourners visiting high-altitude (HA) (>2500 m) are susceptible to HA disorders; on the contrary, HA natives are well adapted to the extreme hypoxic environment. High aldosterone levels are believed to be involved in HA disorders, we, therefore, envisaged role of CYP11B2 gene variants in HA adaptation and therefore investigated the -344T/C, intron-2 conversion (Iw/Ic), K173R, and A5160C polymorphisms. In addition, polymorphisms in AGT, AT1R, ATP1A1, ADRB2, and GSTP1 genes were also investigated. The study comprised of 662 subjects, comprising of 426 Himalayan highlanders (HLs) and 236 lowlanders (LLs). The -344T/C and K173R polymorphisms were found to be in complete linkage disequilibrium. The wild-type allele -344T and combination of wild-type homozygous genotypes between -344T/C, Iw/Ic, and A5160C polymorphisms, containing all the six wild-type alleles were over-represented in the HLs (p < 0.0001, and p = 0.008, respectively). The wild-type haplotypes -344T-Iw, -344T-5160A, and -344T-Iw-5160A also showed over-representation in the HLs (p < 0.0001). Furthermore, greater the number of wild-type alleles, lower was the ARR (p < 0.05). The genotype distribution in remaining genes did not differ. To conclude, the over-representation of wild-type -344T allele, genotype combinations and haplotypes of CYP11B2, and their correlation with lower aldosterone levels associate with HA adaptation in the HLs. Such an allelic presentation in sojourners may help them cope with adverse HA environment.     

Clinical characteristics of primary hyperaldosteronism due to adrenal microadenoma

An increasing number of patients are being diagnosed with primary aldosteronism (PA) due to aldosterone-producing macroadenoma (APA). However, there are only limited data available on the clinical characteristics of PA that are associated with adrenal microadenoma. Of the 55 patients that were diagnosed with PA in our study, 22 patients showed a unilateral adrenal over-production of aldosterone. The histopathology of the surgically removed adrenal tissues led to six patients being diagnosed with microadenoma, and the clinical features of microadenoma, macroadenoma and idiopathic hyperaldosteronism (IHA) were studied. The expression levels of CYP11B2, CYP17, CYP21 and 3β-hydroxysteroid dehydrogenase 2 (HSD3B2) mRNA in the adrenal cortices (n = 5 and 6, respectively) that remained attached to the adrenal microadenomas or macroadenomas were examined by real time-PCR and then compared to the expression levels in the adrenal cortices (n = 5) of non-functioning adrenal adenomas (NF). The patients with microadenoma (n = 6) had significantly higher diastolic blood pressure than the patients with macroadenoma (n = 16) or IHA (n = 33) (p < 0.05). The systolic blood pressure, plasma aldosterone concentration, serum potassium level and renal function did not differ between the PA sub-groups. The levels of CYP11B2 and CYP17 mRNA were significantly increased in the adjacent tissues of microadenomas, as compared with macroadenomas or NF (p < 0.05), whereas no significant differences in the CYP21 and HSD3B2 mRNA levels were found between the PA sub-groups. The tumor size did not influence the clinical characteristics of APA. The non-tumor portions of the microadenomas showed marked and sustained CYP11B2 mRNA expression under the suppressed renin-angiotensin system. We suggest that an increased number of microadenomas should be sampled, and the immunohistochemistry for steoridogenic enzymes should be investigated to clarify the etiology of microadenoma.     

ACE、CYP11B2基因多态性与慢性心力衰竭患者醛固酮脱逸的关系

目的:研究CYP11B2-344C/T(醛固酮合成酶)及ACEI/D(血管紧张素转化酶)基因多态性与慢性心力衰竭(CHF)患者实施ACEI治疗后出现醛固酮脱逸表现的关系。方法:回顾分析2008年10月至2012年10月我科收治的252例CHF患者,全部患者应用ACEI治疗3月,醛固酮在基线以上为醛固酮脱逸,依据此标准将患者分为研究组(脱逸组,n=86)与对照组(非脱逸组,n=166),依据PCR(聚合酶链反应)及RFLP(片段长度限制多态性)等方法分别检测两组CYP11B2及ACE基因型,比较两组基因型频率的分布。结果:252例患者中,共86例出现醛固酮脱逸,发生率为34.1%。全部受试患者CYP11B2基因型及ACE基因型频率与Weinberg-Hardy平衡均相符(P均0.05)。研究组ACE I/D三种基因型的组间分布与对照组相较,无统计学差异(P0.05);CYP11B2基因TT型的频率与对照组相较,呈明显统计学差异(P0.05),等位基因C/T频率的组间分布同对照组相较,亦呈明显差异(P0.05)。研究组ACEI/D的基因多态性及CYP11B2-344C/T的多态性中,基因型联合组间分布与对照组相较,无统计学差异(P0.05)。结论:ACE基因多态性与CHF患者ACEI治疗后出现醛固酮脱逸无关,CYP11B2基因T等位基因及TT基因型多态性可能是CHF患者ACEI治疗后发生醛固酮脱逸的高危因素。醛固酮脱逸时,ACE、CYP11B2基因不具有协同效果。     

Imidazopyridyl compounds as aldosterone synthase inhibitors

The inhibition of aldosterone synthase (CYP11B2) may be an effective treatment of hypertension and heart failure, among other ailments. Previously reported benzimidazole CYP11B2 inhibitors led the way for bioisosteric imidazopyridines that are both potent and selective over CYP11B1.     

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.     

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.     

Efficient conversion of 11-deoxycortisol to cortisol (hydrocortisone) by recombinant fission yeast Schizosaccharomyces pombe

Genetically engineered microorganisms are being increasingly used for the industrial production of complicated chemical compounds such as steroids; however, there have been few reports on the use of the fission yeast Schizosaccharomyces pombe for this purpose. We previously have demonstrated that this yeast is a unique host for recombinant expression of human CYP11B2 (aldosterone synthase), and here we report the functional production of human CYP11B1 (steroid 11beta-hydroxylase) in S. pombe using our new integration vector pCAD1. In the human adrenal, the mitochondrial cytochrome P450 enzyme CYP11B1 catalyses the conversion of 11-deoxycortisol to cortisol, a key reaction in cortisol biosynthesis that in addition is of fundamental interest for the technical synthesis of glucocorticoids. We observed that the endogenous mitochondrial electron transport system detected previously by us is capable of supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. Under optimised cultivation conditions the transformed yeasts show in vivo the inducible ability to efficiently and reliably convert deoxycortisol to cortisol at an average rate of 201 microM d(-1) over a period of 72h, the highest value published to date for this biotransformation.     

Novel homozygous p.Y395X mutation in the CYP11B1 gene found in a Vietnamese patient with 11β-hydroxylase deficiency

Context

The deficiency of steroid 11β-hydroxylase is caused by mutations in the CYP11B1 gene and is the second major form of congenital adrenal hyperplasia associated with hypertension.

Objective

The objective of this study was to screen the CYP11B1 gene for mutations in one Vietnamese male suffering from congenital adrenal hyperplasia.

Patient

The patient (46,XY) had congenital adrenal hyperplasia. The clinical manifestations presented precocious puberty, hyper-pigmentation and high blood pressure at 4 years.

Results

The patient was a homozygous carrier of a novel mutation located in exon 7 containing a premature stop codon instead of tyrosine at 395 (p.Y395X).

Conclusion

We have identified a novel mutant of the CYP11B1 gene in one Vietnamese family associated with phenotypes of congenital adrenal hyperplasia. The mutant gene p.Y395X produces a truncated form of the polypeptide and abolishes the enzyme activities, leading to a severe phenotype of congenital adrenal hyperplasia.     

Discovery of indazole aldosterone synthase (CYP11B2) inhibitors as potential treatments for hypertension

We report the discovery and hit-to-lead optimization of a structurally novel indazole series of CYP11B2 inhibitors. Benchmark compound 34 from this series displays potent inhibition of CYP11B2, high selectivity versus related steroidal and hepatic CYP targets, and lead-like physical and pharmacokinetic properties. On the basis of these and other data, the indazole series was progressed to lead optimization for further refinement.     

Association of Aldosterone Synthase Polymorphism (CYP11B2 -344T>C) and Genetic Ancestry with Atrial Fibrillation and Serum Aldosterone in African Americans with Heart Failure

The objective of this study was to examine the extent to which aldosterone synthase genotype (CYP11B2) and genetic ancestry correlate with atrial fibrillation (AF) and serum aldosterone in African Americans with heart failure. Clinical data, echocardiographic measurements, and a genetic sample for determination of CYP11B2 -344T>C (rs1799998) genotype and genetic ancestry were collected from 194 self-reported African Americans with chronic, ambulatory heart failure. Genetic ancestry was determined using 105 autosomal ancestry informative markers. In a sub-set of patients (n = 126), serum was also collected for determination of circulating aldosterone. The CYP11B2 −344C allele frequency was 18% among the study population, and 19% of patients had AF. Multiple logistic regression revealed that the CYP11B2 −344CC genotype was a significant independent predictor of AF (OR 12.7, 95% CI 1.60–98.4, p = 0.0150, empirical p = 0.011) while holding multiple clinical factors, left atrial size, and percent European ancestry constant. Serum aldosterone was significantly higher among patients with AF (p = 0.036), whereas increased West African ancestry was inversely correlated with serum aldosterone (r = −0.19, p = 0.037). The CYP11B2 −344CC genotype was also overrepresented among patients with extreme aldosterone elevation (≥90th percentile, p = 0.0145). In this cohort of African Americans with chronic ambulatory heart failure, the CYP11B2 −344T>C genotype was a significant independent predictor of AF while holding clinical, echocardiographic predictors, and genetic ancestry constant. In addition, increased West African ancestry was associated with decreased serum aldosterone levels, potentially providing an explanation for the lower risk for AF observed among African Americans.     

Study of the Nucleolar Organizer Regions in Palinurus elephas (Crustacea: Decapoda)

Using fluorescence in situ hybridization (FISH), chromomycin (CMA₃) staining and silver staining, we studied the nucleolar organizer regions in the spiny lobster Palinurus elephas in order to extend our knowledge on the karyology of this commercially important species. Multiple NORs have been detected by FISH, and CMA₃ showed a good correspondence between the localization of GC-rich heterochromatin and the ribosomal genes mapped by FISH. In contrast, the number of Ag-positive regions was higher than the number of FISH and CMA₃ signals, which may be explained by silver staining of the kinetochores. A variability in the number of FISH and CMA₃ signals has been detected in metaphases I and II which is probably due to the occurrence of rDNA cistrons on B chromosomes.     

Polymorphisms of CYP1B1 and COMT in Breast and Endometrial Cancer

CYP1B1 and COMT code for the key enzymes of catecholestrogen biosynthesis and metabolism, and their polymorphisms determine the variation of enzyme activities. RFLP analysis was used to study the allele and genotype frequency distributions of CYP1B1 polymorphisms Arg48Gly, Ala119Ser, and Val432Leu, and COMT polymorphism Val158Met among 210 breast cancer patients, 138 endometrial cancer patients, and 152 healthy women. The COMT polymorphism showed no significant association with breast or endometrial cancer. For the first time, such association was observed for the CYP1B1 polymorphisms. CYP1B1 allele C (Arg48), which codes for the enzyme more active in estradiol 4-hydroxylation, was associated with higher risk of breast (OR = 3.22, CI 2.34–4.43, P = 0.000) and endometrial (OR = 2.43, CI 1.72–3.44, P = 0.000) cancer. Similar data were obtained for CYP1B1 allele G (Ala119): OR = 2.18, CI 1.58–3.01, P = 0.000 in breast cancer and OR = 2.52, CI 1.78–3.56, P = 0.000 in endometrial cancer. Risk of endometrial but not breast cancer was significantly higher in carriers of CYP1B1 genotype Val432/Val. This was explained by stronger estrogen dependence and, consequently, higher estrogen responsiveness of the endometrium as compared with the mammary gland.     

Synthesis and biological evaluation of pyrrole-based chalcones as CYP1 enzyme inhibitors,for possible prevention of cancer and overcoming cisplatin resistance

Inhibitors of CYP1 enzymes may play vital roles in the prevention of cancer and overcoming chemo-resistance to anticancer drugs. In this letter, we report synthesis of twenty-three pyrrole based heterocyclic chalcones which were screened for inhibition of CYP1 isoforms. Compound 3n potently inhibited CYP1B1 with an IC₅₀ of ～0.2 μM in Sacchrosomes? and CYP1B1-expressing live human cells. However, compound 3j which inhibited both CYP1A1 and CYP1B1 with an IC₅₀ of ～0.9 µM, using the same systems, also potently antagonized B[a]P-mediated induction of AhR signaling in yeast (IC₅₀, 1.5 µM), fully protected human cells from B[a]P toxicity and completely reversed cisplatin resistance in human cells that overexpress CYP1B1 by restoring cisplatin’s cytotoxicity. Molecular modeling studies were performed to rationalize the observed potency and selectivity of enzyme inhibition by compounds 3j and 3n.