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Rolf W. Hartmann - One of the best experts on this subject based on the ideXlab platform.
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Modulation of Cytochromes P450 with Xanthone-Based Molecules: From Aromatase to Aldosterone Synthase and Steroid 11β-Hydroxylase Inhibition
2016Co-Authors: Silvia Gobbi, Matthias Negri, Christina Zimmer, Federica Belluti, Angela Rampa, Rolf W. Hartmann, Alessandra BisiAbstract:Imidazolylmethylflavones previously reported by us as aromatase inhibitors proved to be able to interact with Aldosterone Synthase (CYP11B2), a cytochrome P450 enzyme involved in the biosynthesis of the mineralcorticoid hormone Aldosterone, and were used to obtain a pharmacophore model for this enzyme. Here, in the search for potential ligands for CYP11B2 and the related CYP11B1, a virtual screening of a small compounds library of our earlier synthesized aromatase inhibitors was performed and, according to the results and the corresponding biological data, led to the design and synthesis of a series of xanthones derivatives carrying an imidazolylmethyl substituent in position 1 and different substituents in position 4. Some very potent inhibitors were obtained; in particular, the 4-chlorine derivative was active in the low nanomolar or subnanomolar range on CYP11B2 and CYP11B1, respectively, proving that xanthone can be considered as an excellent scaffold, whose activity can be directed to different targets when appropriately functionalized
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Inhibition of Aldosterone Synthase (CYP11B2) by torasemide prevents atrial fibrosis and atrial fibrillation in mice
2015Co-Authors: Oliver Adam, Christina Zimmer, Rolf W. Hartmann, Nina Hanke, Birgit Klemmer, Michael Böhm, Ulrich LaufsAbstract:Abstract Loop diuretics are used for fluid control in patients with heart failure. Furosemide and torasemide may exert differential effects on myocardial fibrosis. Here, we studied the effects of torasemide and furosemide on atrial fibrosis and remodeling during atrial fibrillation. In primary neonatal cardiac fibroblasts, torasemide (50 μM, 24 h) but not furosemide (50 μM, 24 h) reduced the expression of connective tissue growth factor (CTGF; 65 ± 6%) and the pro-fibrotic miR-21 (44 ± 23%), as well as the expression of lysyl oxidase (LOX; 57 ± 8%), a regulator of collagen crosslinking. Mineralocorticoid receptor (MR) expression and activity were not altered. Torasemide but not furosemide inhibited human Aldosterone Synthase (CYP11B2) activity in transfected lung fibroblasts (V79MZ cells) by 75 ± 1.8%. The selective CYP11B2 inhibitor SL242 mimicked the torasemide effects. Mice with cardiac overexpression of Rac1 GTPase (RacET), which develop atrial fibrosis and spontaneous AF with aging, were treated long-term (8 months) with torasemide (10 mg/kg/day), furosemide (40 mg/kg/day) or vehicle. Treatment with torasemide but not furosemide prevented atrial fibrosis in RacET as well as the up-regulation of CTGF, LOX, and miR-2, whereas MR expression and activity remained unaffected. These effects correlated with a reduced prevalence of atrial fibrillation (33% RacET + Tora vs. 80% RacET). Torasemide but not furosemide inhibits CYP11B2 activity and reduces the expression of CTGF, LOX, and miR-21. These effects are associated with prevention of atrial fibrosis and a reduced prevalence of atrial fibrillation in mice.
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heteroatom insertion into 3 4 dihydro 1h quinolin 2 ones leads to potent and selective inhibitors of human and rat Aldosterone Synthase
2015Co-Authors: Cornelia M Grombein, Christina Zimmer, Sabrina Rau, Rolf W. HartmannAbstract:Abstract Aldosterone Synthase (CYP11B2) catalyzes the conversion of 11-deoxycorticosterone to Aldosterone via corticosterone and 18-hydroxycorticosterone. CYP11B2 is regarded as a new target for several cardiovascular diseases which are associated with chronically elevated Aldosterone levels such as hypertension, congestive heart failure and myocardial fibrosis. In this paper, we optimized heterocycle substituted 3,4-dihydropyridin-2(1H)-ones as CYP11B inhibitors by systematic introduction of heteroatoms and by bioisosteric exchange of the lactame moiety by a sultame moiety. The most promising compounds regarding inhibition of human CYP11B2 and selectivity versus human enzymes CYP11B1, CYP17, and CYP19 were tested for inhibition of rat CYP11B2. Thus, we discovered compounds 4 and 9 which show potent inhibition of hCYP11B2 (IC50
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novel pyridyl or isoquinolinyl substituted indolines and indoles as potent and selective Aldosterone Synthase inhibitors
2014Co-Authors: Lina Yin, Juliette Emmerich, Edward Metzger, Amjad Ali, Rolf W. HartmannAbstract:Pathologically, high levels of Aldosterone are associated with severe cardiovascular diseases such as congestive heart failure, hypertension, and myocardial fibrosis. The inhibition of Aldosterone Synthase (CYP11B2) to reduce Aldosterone levels has been proposed as a promising treatment for diseases related to CYP11B2 because it is the crucial enzyme in the biosynthesis of Aldosterone. A series of novel pyridyl- or isoquinolinyl-substituted indolines and indoles was designed via a ligand-based approach. The synthesized compounds were tested and found to be strong CYP11B2 inhibitors. The most potent ones showed IC50 values of less than 3 nM, being similarly potent as fadrozole and LCI699. Among them, compounds 14 and 23 showed good selectivity over the highly homologous CYP11B1, with selectivity factors (SF = IC50 CYP11B1/IC50 CYP11B2) around 170; thus, they are superior to fadrozole and LCI699 (SFs 50 μM) of a panel of hepatic CYP enzy...
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Aldosterone Synthase inhibitors as promising treatments for mineralocorticoid dependent cardiovascular and renal diseases
2014Co-Authors: Lina Yin, Rolf W. HartmannAbstract:Besides the well-known roles of Aldosterone as a mineralocorticoid in regulating homeostasis of electrolytes and volume, recent studies revealed that it is also a potent proinflammation factor inducing reactive oxygen species and up-regulating a panel of fibrosis related genes. Under pathological circumstances, excessive Aldosterone is involved in a lot of chronic diseases, including hypertension, cardiac fibrosis, congestive heart failure, ventricular remodeling, and diabetic nephropathy. Therefore, the inhibition of Aldosterone Synthase (CYP11B2), which is the pivotal enzyme in Aldosterone biosynthesis, was proposed as a superior approach. Expected pharmacodynamic effects have been demonstrated in both animal models and clinical trials after the application of CYP11B2 inhibitors. The importance of selectivity over other steroidogenic CYP enzymes, in particular 11β-hydroxylase (CYP11B1), was also revealed. Recently, much more selective CYP11B2 inhibitors have been reported, which could be promising drug ...
Simon Lucas - One of the best experts on this subject based on the ideXlab platform.
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fine tuning the selectivity of Aldosterone Synthase inhibitors structure activity and structure selectivity insights from studies of heteroaryl substituted 1 2 5 6 tetrahydropyrrolo 3 2 1 ij quinolin 4 one derivatives
2011Co-Authors: Simon Lucas, Matthias Negri, Christina Zimmer, Ralf Heim, Rolf W. HartmannAbstract:Pyridine substituted 3,4-dihydro-1H-quinolin-2-ones (e.g., 1−3) constitute a class of highly potent and selective inhibitors of Aldosterone Synthase (CYP11B2), a promising target for the treatment ...
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Selective Aldosterone Synthase inhibitors reduce Aldosterone formation in vitro and in vivo.
2009Co-Authors: Christina Ries, Simon Lucas, Ralf Heim, Barbara Birk, Rolf W. HartmannAbstract:Abstract Aldosterone plays a crucial role in salt and water homeostasis but in case of pathologically increased plasma Aldosterone levels it is also involved in the development and the progression of severe cardiovascular diseases like heart failure and myocardial fibrosis. For the treatment of these diseases we propose inhibition of the Aldosterone forming enzyme CYP11B2 as a new pharmacological strategy. We recently developed in vitro highly potent and selective inhibitors of human CYP11B2, but the evidence of their in vivo activity is still missing. For this purpose, rat Aldosterone Synthase gene was cloned and expressed in V79MZ cells to establish a new screening assay for the identification of “rat-active” substances. Compound 7 from the class of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones showed a moderate inhibitory effect (65% at 2 μM) on rat CYP11B2 in vitro. Furthermore, it diminished the conversion of deoxycorticosterone to Aldosterone in rat adrenals and significantly reduced plasma Aldosterone levels in vivo.
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in vivo active Aldosterone Synthase inhibitors with improved selectivity lead optimization providing a series of pyridine substituted 3 4 dihydro 1h quinolin 2 one derivatives
2008Co-Authors: Simon Lucas, Christina Ries, Ralf Heim, Barbara Birk, Katarzyna E Schewe, Rolf W. HartmannAbstract:Pyridine substituted naphthalenes (e.g., I−III) constitute a class of potent inhibitors of Aldosterone Synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1−11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12−26), with 12, a bioisostere of 9, being nontoxic up to 200 μM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline der...
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novel Aldosterone Synthase inhibitors with extended carbocyclic skeleton by a combined ligand based and structure based drug design approach
2008Co-Authors: Simon Lucas, Silvia Gobbi, Matthias Negri, Alessandra Bisi, Christina Ries, Ralf Heim, Iris Antes, Katarzyna E Schewe, Rolf W. HartmannAbstract:Pharmacophore modeling of a series of Aldosterone Synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)−N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors...
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overcoming undesirable cyp1a2 inhibition of pyridylnaphthalene type Aldosterone Synthase inhibitors influence of heteroaryl derivatization on potency and selectivity
2008Co-Authors: Ralf Heim, Matthias Negri, Christina Ries, Simon Lucas, Barbara Birk, Ursula Mullervieira, Cornelia M Grombein, Katarzyna E Schewe, Rolf W. HartmannAbstract:Recently, we reported on the development of potent and selective inhibitors of Aldosterone Synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats.
William E Rainey - One of the best experts on this subject based on the ideXlab platform.
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abstract 359 modulation of Aldosterone Synthase by estrogens evidence for an interaction of gper 1 and estrogen b receptors and relevance for the gender dimorphism of blood pressure
2013Co-Authors: Brasilina Caroccia, William E Rainey, Teresa Maria Seccia, Livia Lenzini, Maniselvan Kuppusamy, Abril Gonzalez Campos, Sara Mareso, Francesca Gioco, Ambrogio Fassina, Gian Paolo RossiAbstract:Background. Fertile women have lower blood pressure and are held to be at lower risk for cardiovascular events than age-matched man. However, whether this gender dimorphism involves a modulation of Aldosterone synthesis by 17 beta-estradiol (E2) is unknown. Aims. i) To investigate estrogen receptor subtypes gene expression in the normal human adrenal cortex (NAC), in Aldosterone producing adenoma (APA) and in a human adrenocortical carcinoma cell line (HAC15); ii) To assess the effect of E2 on Aldosterone Synthase (CYP11B2) gene expression and to identify the receptor subtypes involved in this effect. Methods. We measured the expression of alpha (ERa), beta (ERb) and of G protein-coupled receptor (GPER-1)-1 in NAC and in APA tissue, and in HAC15 cells by real time RT-PCR. After demonstration that HAC15 cells express ERa, ERb, and GPER-1 we stimulated cells with 10-7M E2 alone, or after ERb selective blockade with 10-5M tetrahydrochrysenediol (THC), ERa selective blockade with 10-5M MPP dihydrochloride (MPP), non selective ERa and ERb blockade with 10-5M ICI 182.780, or after selective GPER-1 receptor blockade with 10-5M G-15. The cells were also exposed to the GPER-1 agonist G-1, alone or in the presence of MPP, THC, or Fulvestrant, and/or G-15. Changes of expression of CYP11B2 mRNA, measured with RT RT-PCR, was the experimental endpoint. Results. The quantitative expression of estrogen receptor subtypes was ERb > GPER-1 >> ERα in NAC, GPER-1 > ERb> ERa in APA, and ERb>ERa=GPER-1 in HAC15 cells. E2 alone or on top of selective ERa antagonism did not alter CYP11B2 expression. By contrast, E2 significantly increased CYP11B2 expression (+500 to + 700% from baseline, p Conclusion. E2 potently stimulates Aldosterone Synthase expression via GPER-1 subtype receptor activation when the ERb is blocked. The ERb-mediated tonic inhibition of Aldosterone Synthase could contribute to explaining both the lower BP and CV risk of fertile women and the increase of BP after when this tonic ERb-mediated inhibition wanes during menopause or estrogen-modulation treatment.
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regulation of Aldosterone Synthase by activator transcription factor camp response element binding protein family members
2010Co-Authors: Edson F Nogueira, William E RaineyAbstract:Aldosterone synthesis is regulated by angiotensin II (Ang II) and K(+) acting in the adrenal zona glomerulosa, in part through the regulation of Aldosterone Synthase (CYP11B2). Here, we analyzed the role of cAMP response element (CRE)-binding proteins (CREBs) in the regulation of CYP11B2. Expression analysis of activator transcription factor (ATF)/CREB family members, namely the ATF1 and ATF2, the CREB, and the CRE modulator, in H295R cells and normal human adrenal tissue was performed using quantitative real-time PCR. Ang II-induced phosphorylation of ATF/CREB members was analyzed by Western blot analysis, and their subsequent binding to the CYP11B2 promoter using chromatin immunoprecipitation assay. Aldosterone production and CYP11B2 expression were measured in small interfering RNA-transfected cells to knockdown the expression of ATF/CREB members. CYP11B2 promoter activity was measured in H295R cells cotransfected with NURR1 (NR4A2) alone or with constitutively active vectors for ATF/CREB members. Ang II induced phosphorylation of ATF1, ATF2, and CRE modulator in a time-dependent manner. Based on chromatin immunoprecipitation analysis, there was an increased association of these proteins with the CYP11B2 promoter after Ang II and K(+) treatment. Phosphorylated ATF/CREB members also bound the CYP11B2 promoter. Knockdown of ATF/CREB members reduced Ang II and K(+) induction of adrenal cell CYP11B2 mRNA expression and Aldosterone production. The constitutively active ATF/CREB vectors increased the promoter activity of CYP11B2 and had a synergistic effect with NURR1. In summary, these results suggest that ATF/CREB and NGFI-B family members play a crucial role in the transcriptional regulation of CYP11B2 and adrenal cell capacity to produce Aldosterone.
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the regulation of Aldosterone Synthase expression
2004Co-Authors: Mary H Bassett, Perrin C White, William E RaineyAbstract:Abstract Aldosterone, the primary human mineralocorticoid, is a major regulator of intravascular volume and blood pressure. The capacity of the adrenal gland to produce Aldosterone is controlled, in large part, by the regulated transcription of CYP11B2 , the gene encoding Aldosterone Synthase. Aldosterone Synthase is responsible for the conversion of 11-deoxycorticosterone to Aldosterone and is expressed only within the zona glomerulosa of the adrenal cortex. The development of new systems for in vitro studies of expression has helped define molecular mechanisms that regulate this enzyme and thus the capacity of the adrenal gland to produce Aldosterone. Both potassium and angiotensin II (ANG II) increase intracellular calcium levels, which regulate expression of CYP11B2 through transcription factors that interact with defined sites in the 5′-flanking region of the gene.
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calmodulin dependent kinase i regulates adrenal cell expression of Aldosterone Synthase
2002Co-Authors: Jennifer C Condon, Vincenzo Pezzi, Brad M Drummond, Su Yin, William E RaineyAbstract:Aldosterone Synthase (CYP11B2) is expressed in the adrenal glomerulosa and controls the capacity of the adrenal glomerulosa to produce Aldosterone. Herein, human NCI-H295R (H295R) adrenocortical cells were used to define the calcium-dependent mechanisms regulating CYP11B2 gene transcription using reporter constructs containing CYP11B2 gene 5′-flanking DNA. Treatment of H295R cells with calcium/calmodulin-dependent protein kinase (CaMK) inhibitor (KN93) or calmodulin inhibitor (calmidazolium) blocked angiotensin II and potassium (K+) stimulation of CYP11B2 reporter gene expression. To determine which CaMK regulates CYP11B2, vectors containing the complete coding sequences for CaMKI, CaMKII, and CaMKIV were transfected with the CYP11B2 reporter construct. CaMKI augmented reporter expression when cellular calcium was elevated by ionomycin, whereas CaMKIV had a small effect, and CaMKII had no effect. To further study the role of CaMKs, constitutively active forms of CaMKI (CaMKI-295), II (CaMKII-290), and IV ...
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differential regulation of Aldosterone Synthase and 11beta hydroxylase transcription by steroidogenic factor 1
2002Co-Authors: Mary H Bassett, Yin Zhang, Colin Clyne, Perrin C White, William E RaineyAbstract:11β-Hydroxylase (hCYP11B1) and Aldosterone Synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and Aldosterone production respectively. Aldosterone Synthase catalyzes the final step in Aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11 β-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2, Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17a-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.
Perrin C White - One of the best experts on this subject based on the ideXlab platform.
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Aldosterone Synthase promoter polymorphism and cardiovascular phenotypes in a large multiethnic population based study
2015Co-Authors: James Brian Byrd, Richard J Auchus, Perrin C WhiteAbstract:Background A single-nucleotide polymorphism in the Aldosterone Synthase gene (CYP11B2) promoter [−344C/T, rs1799998] has been reported to associate with cardiovascular phenotypes. Methods The Dallas Heart Study is a large, multiethnic cohort with a high prevalence of hypertension. We genotyped 3452 Dallas Heart Study participants for −344C/T. Generalized linear models were used to assess whether variation at −344C/T associated with plasma Aldosterone concentration (PAC), systolic and diastolic blood pressure (SBP and DBP), plasma glucose (in persons with no diabetes), HOMA IR (Homeostasis Model Assessment as an Index of Insulin Resistance), and left ventricular (LV) mass indexed to height. Systolic blood pressure and DBP were significantly higher in blacks compared with whites (P Conclusions We were unable to reproduce previously reported associations between −344C/T and PAC, blood pressure, plasma glucose, or LV mass. Methodological differences might explain the differences between our findings and those previously reported.
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the regulation of Aldosterone Synthase expression
2004Co-Authors: Mary H Bassett, Perrin C White, William E RaineyAbstract:Abstract Aldosterone, the primary human mineralocorticoid, is a major regulator of intravascular volume and blood pressure. The capacity of the adrenal gland to produce Aldosterone is controlled, in large part, by the regulated transcription of CYP11B2 , the gene encoding Aldosterone Synthase. Aldosterone Synthase is responsible for the conversion of 11-deoxycorticosterone to Aldosterone and is expressed only within the zona glomerulosa of the adrenal cortex. The development of new systems for in vitro studies of expression has helped define molecular mechanisms that regulate this enzyme and thus the capacity of the adrenal gland to produce Aldosterone. Both potassium and angiotensin II (ANG II) increase intracellular calcium levels, which regulate expression of CYP11B2 through transcription factors that interact with defined sites in the 5′-flanking region of the gene.
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Aldosterone Synthase deficiency and related disorders
2004Co-Authors: Perrin C WhiteAbstract:Aldosterone's main actions are to regulate intravascular volume and serum electrolytes by controlling sodium absorbtion and potassium excretion in the distal nephron. Inherited defects in Aldosterone biosynthesis thus cause hypovolemia, hyponatremia and hyperkalemia. Defective Aldosterone biosynthesis may be caused by congenital adrenal hyperplasia due to 21-hydroxylase (CYP21) deficiency, in which case cortisol biosynthesis is also affected, or as an isolated defect termed Aldosterone Synthase (corticosterone methyloxidase, CYP11B2) deficiency. Many mutations have been documented in each of these genes; in general enzymatic activity must be reduced to <1% of normal for Aldosterone biosynthesis to be impaired. An additional form of familial hyperreninemic hypoaldosteronism has been described that is not due to mutations in CYP11B2, but its etiology remains to be elucidated.
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differential regulation of Aldosterone Synthase and 11beta hydroxylase transcription by steroidogenic factor 1
2002Co-Authors: Mary H Bassett, Yin Zhang, Colin Clyne, Perrin C White, William E RaineyAbstract:11β-Hydroxylase (hCYP11B1) and Aldosterone Synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and Aldosterone production respectively. Aldosterone Synthase catalyzes the final step in Aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11 β-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2, Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17a-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.
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differential regulation of Aldosterone Synthase and 11beta hydroxylase transcription by steroidogenic factor 1
2002Co-Authors: Mary H Bassett, Yin Zhang, Colin Clyne, Perrin C White, William E RaineyAbstract:11beta-Hydroxylase (hCYP11B1) and Aldosterone Synthase (hCYP11B2) are closely related isozymes with distinct roles in cortisol and Aldosterone production respectively. Aldosterone Synthase catalyzes the final step in Aldosterone biosynthesis and is expressed only in the zona glomerulosa of the normal adrenal. 11beta-Hydroxylase catalyzes the final reaction in the production of cortisol and is expressed at higher levels in the zona fasciculata. The mechanisms causing differential expression of these genes are not well defined. Herein, we demonstrate contrasting roles for the orphan receptor steroidogenic factor-1 (SF-1) in the regulation of human (h) CYP11B1 and hCYP11B2. Human NCI-H295R (H295R) or mouse Y-1 cells were transiently transfected with luciferase reporter constructs containing 5'-flanking regions of hCYP11B1, hCYP11B2, human 17alpha-hydroxylase (hCYP17), human cholesterol side-chain cleavage (hCYP11A1) or mouse (m) cyp11b2 (mcyp11b2). Co-transfection of vectors encoding SF-1 increased expression of hCYP11B1, hCYP11A1 and hCYP17 constructs, but inhibited hCYP11B2 reporter activity. Murine, bovine and human SF-1 were unable to increase transcription of hCYP11B2 in H295R cells. Both hCYP11B2 and mcyp11b2 promoter constructs were inhibited similarly by human SF-1. In mouse Y-1 cells, reporter expression of hCYP11B2 and mcyp11b2 was very low compared with hCYP11B1 constructs, suggesting that this adrenal cell model may not be appropriate for studies of CYP11B2. Electrophoretic mobility shift assay demonstrated that SF-1 interacted with an element from both hCYP11B1 and hCYP11B2. However, mutation of this element, termed Ad4, did not prevent agonist stimulation of hCYP11B2 by angiotensin II or forskolin but blocked activity of hCYP11B1. In some, but not all, reports of genetic linkage analysis, a naturally occurring single nucleotide polymorphism within the Ad4 element of hCYP11B2 (-344C/T) has been associated with cardiovascular disease. Herein, we have demonstrated that this polymorphism influenced binding of SF-1 in electrophoretic mobility shift assays, with the C allele binding SF-1 more strongly than the T allele. However, when hCYP11B2 constructs containing these alleles were transfected into H295R cells, there was no difference in agonist-stimulated expression or the response of either reporter construct to co-expression with human SF-1. Taken together, these data suggest that SF-1 and the Ad4 element are not major regulators of adrenal hCYP11B2 gene expression. Thus far, hCYP11B2 is the first steroid hydroxylase gene which is not positively regulated by SF-1.
Ralf Heim - One of the best experts on this subject based on the ideXlab platform.
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fine tuning the selectivity of Aldosterone Synthase inhibitors structure activity and structure selectivity insights from studies of heteroaryl substituted 1 2 5 6 tetrahydropyrrolo 3 2 1 ij quinolin 4 one derivatives
2011Co-Authors: Simon Lucas, Matthias Negri, Christina Zimmer, Ralf Heim, Rolf W. HartmannAbstract:Pyridine substituted 3,4-dihydro-1H-quinolin-2-ones (e.g., 1−3) constitute a class of highly potent and selective inhibitors of Aldosterone Synthase (CYP11B2), a promising target for the treatment ...
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Selective Aldosterone Synthase inhibitors reduce Aldosterone formation in vitro and in vivo.
2009Co-Authors: Christina Ries, Simon Lucas, Ralf Heim, Barbara Birk, Rolf W. HartmannAbstract:Abstract Aldosterone plays a crucial role in salt and water homeostasis but in case of pathologically increased plasma Aldosterone levels it is also involved in the development and the progression of severe cardiovascular diseases like heart failure and myocardial fibrosis. For the treatment of these diseases we propose inhibition of the Aldosterone forming enzyme CYP11B2 as a new pharmacological strategy. We recently developed in vitro highly potent and selective inhibitors of human CYP11B2, but the evidence of their in vivo activity is still missing. For this purpose, rat Aldosterone Synthase gene was cloned and expressed in V79MZ cells to establish a new screening assay for the identification of “rat-active” substances. Compound 7 from the class of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones showed a moderate inhibitory effect (65% at 2 μM) on rat CYP11B2 in vitro. Furthermore, it diminished the conversion of deoxycorticosterone to Aldosterone in rat adrenals and significantly reduced plasma Aldosterone levels in vivo.
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in vivo active Aldosterone Synthase inhibitors with improved selectivity lead optimization providing a series of pyridine substituted 3 4 dihydro 1h quinolin 2 one derivatives
2008Co-Authors: Simon Lucas, Christina Ries, Ralf Heim, Barbara Birk, Katarzyna E Schewe, Rolf W. HartmannAbstract:Pyridine substituted naphthalenes (e.g., I−III) constitute a class of potent inhibitors of Aldosterone Synthase (CYP11B2). To overcome the unwanted inhibition of the hepatic enzyme CYP1A2, we aimed at reducing the number of aromatic carbons of these molecules because aromaticity has previously been identified to correlate positively with CYP1A2 inhibition. As hypothesized, inhibitors with a tetrahydronaphthalene type molecular scaffold (1−11) exhibit a decreased CYP1A2 inhibition. However, tetralone 9 turned out to be cytotoxic to the human cell line U-937 at higher concentrations. Consequent structural optimization culminated in the discovery of heteroaryl substituted 3,4-dihydro-1H-quinolin-2-ones (12−26), with 12, a bioisostere of 9, being nontoxic up to 200 μM. The investigated molecules are highly selective toward both CYP1A2 and a wide range of other cytochrome P450 enzymes and show a good pharmacokinetic profile in vivo (e.g., 12 with a peroral bioavailability of 71%). Furthermore, isoquinoline der...
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novel Aldosterone Synthase inhibitors with extended carbocyclic skeleton by a combined ligand based and structure based drug design approach
2008Co-Authors: Simon Lucas, Silvia Gobbi, Matthias Negri, Alessandra Bisi, Christina Ries, Ralf Heim, Iris Antes, Katarzyna E Schewe, Rolf W. HartmannAbstract:Pharmacophore modeling of a series of Aldosterone Synthase (CYP11B2) inhibitors triggered the design of compounds 11 and 12 by extending a previously established naphthalene molecular scaffold (e.g., present in molecules 1 and 2) via introduction of a phenyl or benzyl residue in 3-position. These additional aromatic moieties have been hypothesized to fit into the newly identified hydrophobic pharmacophore feature HY3. Subsequent docking studies in our refined CYP11B2 protein model have been performed prior to synthesis to estimate the inhibitory properties of the proposed molecules. While phenyl-substituted compound 11 (IC50 > 500 nM) did not dock under the given pharmacophore constraint (i.e., the Fe(heme)−N(ligand) interaction), benzyl-substituted compound 12 (IC50 = 154 nM) was found to exploit a previously unexplored subpocket of the inhibitor binding site. By structural optimization based on the pharmacophore hypothesis, 25 novel compounds were synthesized, among them highly potent CYP11B2 inhibitors...
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overcoming undesirable cyp1a2 inhibition of pyridylnaphthalene type Aldosterone Synthase inhibitors influence of heteroaryl derivatization on potency and selectivity
2008Co-Authors: Ralf Heim, Matthias Negri, Christina Ries, Simon Lucas, Barbara Birk, Ursula Mullervieira, Cornelia M Grombein, Katarzyna E Schewe, Rolf W. HartmannAbstract:Recently, we reported on the development of potent and selective inhibitors of Aldosterone Synthase (CYP11B2) for the treatment of congestive heart failure and myocardial fibrosis. A major drawback of these nonsteroidal compounds was a strong inhibition of the hepatic drug-metabolizing enzyme CYP1A2. In the present study, we examined the influence of substituents in the heterocycle of lead structures with a naphthalene molecular scaffold to overcome this unwanted side effect. With respect to CYP11B2 inhibition, some substituents induced a dramatic increase in inhibitory potency. The methoxyalkyl derivatives 22 and 26 are the most potent CYP11B2 inhibitors up to now (IC50 = 0.2 nM). Most compounds also clearly discriminated between CYP11B2 and CYP11B1, and the CYP1A2 potency significantly decreased in some cases (e.g., isoquinoline derivative 30 displayed only 6% CYP1A2 inhibition at 2 microM concentration). Furthermore, isoquinoline derivative 28 proved to be capable of passing the gastrointestinal tract and reached the general circulation after peroral administration to male Wistar rats.