Kinetics of Intermediate Release Enhances P450 11B2-Catalyzed Aldosterone Synthesis.

The mitochondrial enzyme cytochrome P450 11B2 (aldosterone synthase) catalyzes the 3 terminal transformations in the biosynthesis of aldosterone from 11-deoxycorticosterone (DOC): 11ß-hydroxylation to corticosterone, 18-hydroxylation, and 18-oxidation. Prior studies have shown that P450 11B2 produces more aldosterone from DOC than from the intermediate corticosterone and that the reaction sequence is processive, with intermediates remaining bound to the active site between oxygenation reactions. In contrast, P450 11B1 (11ß-hydroxylase), which catalyzes the terminal step in cortisol biosynthesis, shares a 93% amino acid sequence identity with P450 11B2, converts DOC to corticosterone, but cannot synthesize aldosterone from DOC. The biochemical and biophysical properties of P450 11B2, which enable its unique 18-oxygenation activity and processivity, yet are not also represented in P450 11B1, remain unknown. To understand the mechanism of aldosterone biosynthesis, we introduced point mutations at residue 320, which partially exchange the activities of P450 11B1 and P450 11B2 (V320A and A320V, respectively). We then investigated NADPH coupling efficiencies, binding kinetics and affinities, and product formation of purified P450 11B1 and P450 11B2, wild-type, and residue 320 mutations in phospholipid vesicles and nanodiscs. Coupling efficiencies for the 18-hydroxylase reaction with corticosterone as the substrate failed to correlate with aldosterone synthesis, ruling out uncoupling as a relevant mechanism. Conversely, corticosterone dissociation rates correlated inversely with aldosterone production. We conclude that intermediate dissociation kinetics, not coupling efficiency, enable P450 11B2 to synthesize aldosterone via a processive mechanism. Our kinetic data also suggest that the binding of DOC to P450 11B enzymes occurs in at least two distinct steps, favoring an induced-fit mechanism.

Combining virtual screening and in vitro evaluation for the discovery of potential CYP11B2 inhibitors.

Aim: To search for highly bioactive hits for CYP11B2 inhibitors by virtual screening and in vitro evaluation. Materials & methods: Virtual screening of potential CYP11B2 inhibitors was performed by molecular docking and molecular dynamics simulation. Compound activity was determined by in vitro evaluation using MTT and ELISA assays. Results & conclusion: Based on the results of molecular docking and molecular dynamics simulation, nine lead hits were selected for in vitro biochemical testing. All hits in in vitro experiments had lower inhibitory effects on cell proliferation and certain inhibitory effects on aldosterone secretion. These hits may be excellent candidates for CYP11B2 inhibitors.

In silico selectivity modeling of pyridine and pyrimidine based CYP11B1 and CYP11B2 inhibitors: A case study.

DESIGN: of selective drug candidates for highly structural similar targets is a challenging task for researchers. The main objective of this study was to explore the selectivity modeling of pyridine and pyrimidine scaffold towards the highly homologous targets CYP11B1 and CYP11B2 enzymes by in silico (Molecular docking and QSAR) approaches. In this regard, a big dataset (n = 228) of CYP11B1 and CYP11B2 inhibitors were gathered and classified based on heterocyclic ring and the exhaustive analysis was carried out for pyridine and pyrimidinescaffolds. The LibDock algorithm was used to explore the binding pattern, screening, and identify the structural feature responsible for the selectivity of the ligands towards the studied targets. Finally, QSAR analysis was done to explore the correlation between various binding parameters and structural features responsible for the inhibitory activity and selectivity of the ligands in a quantitative way. The docking and QSAR analysis clearly revealed and distinguished the importance of structural features, functional groups attached for CYP11B2 and CYP11B1 selectivity for pyridine and pyrimidine analogs. Additionally, the docking analysis highlighted the differentiating amino acids residues for selectivity for ligands for each of the enzymes. The results obtained from this research work will be helpful in designing the selective CYP11B1/CYP11B2 inhibitors.

Structural and functional insights into aldosterone synthase interaction with its redox partner protein adrenodoxin.

Aldosterone is the major mineralocorticoid in the human body controlling blood pressure and salt homeostasis. Overproduction of aldosterone leads to primary aldosteronism, which is the most common form of secondary hypertension with limited treatment options. Production of aldosterone by cytochrome P450 11B2 (CYP11B2, aldosterone synthase) requires two reduction events with the electrons delivered by the iron/sulfur protein adrenodoxin. Very limited information is available about the structural and functional basis of adrenodoxin/CYP11B2 interaction, which impedes the development of new treatment options for primary aldosteronism. A systematic study was carried out to determine if adrenodoxin interaction with CYP11B2 might also have an allosteric component in addition to electron transfer. Indeed, local increases in adrenodoxin concentration promote binding of the substrate 11-deoxycorticosterone and the inhibitor osilodrostat (LCI699) in the active site-over 17 Å away-as well as enhance the inhibitory effect of this latter drug. The CYP11B2 structure in complex with adrenodoxin identified specific residues at the protein-protein interface interacting via five salt bridges and four hydrogen bonds. Comparisons with cholesterol-metabolizing CYP11A1 and cortisol-producing CYP11B1, which also bind adrenodoxin, revealed substantial structural differences in these regions. The structural and functional differences between different P450 interactions with adrenodoxin may provide valuable clues for an orthogonal treatment approach for primary aldosteronism by specifically targeting the interaction between CYP11B2 and adrenodoxin.

Substrate-induced modulation of protein-protein interactions within human mitochondrial cytochrome P450-dependent system.

Steroidogenesis is strictly regulated at multiple levels, as produced steroid hormones are crucial to maintain physiological functions. Cytochrome P450 enzymes are key players in adrenal steroid hormone biosynthesis and function within short redox-chains in mitochondria and endoplasmic reticulum. However, mechanisms regulating supply of reducing equivalents in the mitochondrial CYP-dependent system are not fully understood. In the present work, we aimed to estimate how the specific steroids, substrates, intermediates and products of multistep reactions modulate protein-protein interactions between adrenodoxin (Adx) and mitochondrial CYP11 s. Using the SPR technology we determined that steroid substrates affect affinity and stability of CYP11s-Adx complexes in an isoform-specific mode. In particular, cholesterol induces a 4-fold increase in the rate of CYP11A1 - Adx complex formation without significant effect on dissociation (koff decreased ∼1.5-fold), overall increasing complex affinity. At the same time steroid substrates decrease the affinity of both CYP11B1 - Adx and CYP11B2 - Adx complexes, predominantly reducing their stability (4-7 fold). This finding reveals differentiation of protein-protein interactions within the mitochondrial pool of CYPs, which have the same electron donor. The regulation of electron supply by the substrates might affect the overall steroid hormones production. Our experimental data provide further insight into protein-protein interactions within CYP-dependent redox chains involved in steroidogenesis.

Aldosterone synthase inhibitors for cardiovascular diseases: A comprehensive review of preclinical, clinical and in silico data.

Aldosterone, the main mineralocorticoid hormone, plays a fundamental role in maintaining blood pressure (BP)and volume under hypovolemic conditions. However, in numerous diseases, where it is produced in excess, it plays a detrimental role and contributes to cardiovascular events and ultimately to death in a multitude of patients. The seminal observation that the fungicide-derivative fadrozole blunted steroidogenesis has led to develop several agents to inhibit aldosterone synthase (AS, CYP11B2), the mitochondrial NADH-dependent enzyme that is necessary for aldosterone biosynthesis. Aldosterone synthase inhibitors (ASI) have, thereafter, been conceived and investigated in phase I and phase II studies. We herein reviewed the ASIs available so far considering their chemical structure, the related aldosterone synthase binding and pharmacodynamic properties. We also examined the promising results obtained with ASIs that have already been tested in phase II human studies.

MiR-138-5p is downregulated in patients with atrial fibrillation and reverses cardiac fibrotic remodeling via repressing CYP11B2.

OBJECTIVE: To investigate the connection between atrial fibrillation (AF) and miR-138-5p and to further explore the possible mechanism. PATIENTS AND METHODS: MiR-138-5p expression of right atrial appendage (RAA) tissues in 28 patients with AF and 22 patients with sinus rhythm (SR) was detected by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Moreover, cell proliferation assay was conducted in AC16 cells which were transfected by miR-138-5p inhibitors or mimics. Furthermore, Western blot assay, luciferase assay, and RNA immunoprecipitation assay were performed to uncover the mechanism. RESULTS: In the present research, miR-138-5p expression in RAA samples decreased significantly in AF patients than that in SR ones. Moreover, in AC16 cells, higher miR-138-5p expression level suppressed cell growth, while cell growth was promoted after miR-138-5p was knockdown. In addition, further experiments showed that CYP11B2 acted as the main target of miR-138-5p and its expression in AF tissues negatively correlated to miR-138-5p expression. CONCLUSIONS: All the results above elucidated that cell proliferation of AF could be inhibited by miR-138-5p via suppressing CYP11B2, which may offer a new vision for interpreting the mechanism of AF development.

Discovery of Novel Pyrazole-Based Selective Aldosterone Synthase (CYP11B2) Inhibitors: A New Template to Coordinate the Heme-Iron Motif of CYP11B2.

It is necessary for aldosterone synthase (CYP11B2) inhibitors to have both high potency and high selectivity over 11ß-hydroxylase (CYP11B1), a critical enzyme for cortisol synthesis. Previous studies have reported a number of CYP11B2 inhibitors, most of which have an imidazole or pyridine ring to coordinate the heme-iron motif of CYP11B2; however, highly selective inhibitors of human CYP11B2 are still needed. To expand the selectivity in humans, we explored alternative templates and found that pyrazoles were suitable templates for CYP11B2 inhibitors. Investigation of pyrazoles, especially N-alkyl pyrazoles, as a new template to coordinate the heme-iron motif led to a potent and highly selective CYP11B2 inhibitor 28 with an aldosterone-lowering effect at 1 mg/kg dosing in cynomolgus monkeys.

MiR-4421 regulates the progression of preeclampsia by regulating CYP11B2.

OBJECTIVE: Preeclampsia is a serious disease that affects maternal and fetal health in pregnancy. Mechanism of miRNA in preeclampsia has gradually been explored. This study mainly investigated the mechanism of miR-4421 in preeclampsia. PATIENTS AND METHODS: The expression of miR-4421 in 42 preeclampsia tissues and 42 normal pregnancy placentas tissues was detected by qRT-PCR. The relationship between the miR-4421 level and clinicopathological features of preeclampsia was analyzed. After miR-4421 was overexpressed, cell proliferation, cell cycle, and apoptosis were examined. The target gene CYP11B2 of miR-4421 was detected by luciferase reporter assay. The protein expressions were accessed by Western blot. RESULTS: miR-4421 was highly expressed in the placenta of preeclampsia. Clinical data analysis revealed higher systolic blood pressure, diastolic blood pressure, and urinary protein level in preeclampsia patients with high expression of miR-4421 compared with those in low expression group. Birth weight of fetuses was significantly lower than those born from normal pregnant women. After overexpression of miR-4421, trophoblast proliferation was significantly inhibited and cell cycle was significantly blocked. Luciferase reporter assay and Western blot showed that CYP11B2 can be served as a target gene of miR-4421. CONCLUSIONS: MiR-4421 was highly expressed in preeclampsia, which may promote the progression of preeclampsia by down-regulating the expression of CYP11B2.

Molecular Recognition in Mitochondrial Cytochromes P450 That Catalyze the Terminal Steps of Corticosteroid Biosynthesis.

The mitochondrial cytochromes P450 11B1 and P450 11B2 are responsible for the final stages of cortisol and aldosterone synthesis, respectively. Dysregulation of both enzymes has been implicated in secondary forms of hypertension. Molecular recognition of the cytochromes P450 with their corresponding redox partner is a key step in the catalytic cycle, yet the precise nature of the interaction of P450 11B1 or P450 11B2 with their proximal partner, adrenodoxin (Adx), is still unknown. Here, we obtained P450 11B1·Adx2 and P450 11B2·Adx2 complexes using the zero-length cross-linker ethyl-3-[3-(dimethylamino)propyl]carbodiimide, which formed best under low-ionic strength conditions. R-to-K mutations were introduced into the P450s at residues predicted to form salt bridges with Adx and allow cross-linking with the carbodiimide reagent. Mass spectrometric analysis of the chymotrypsin-digested ternary complexes identified seven cross-linked peptide pairs. Consistent with the electrostatic interaction of K370 in P450 11B1-WT and K366 in P450 11B2-R366K with D79 of Adx, Adx mutation L80K abolished complex formation. Using these sites of interaction as constraints, protein docking calculations based on the crystal structures of the two proteins yielded a structural model of the P450 11B1·Adx2 complex. The appositional surfaces include R/K366, K370, and K357 of P450 11B1, which interact with D79, D76, and D113 (second molecule) of Adx, respectively. Similar to P450 11B1, P450 11B2 also forms a complex with the Adx dimer via three lysine residues. We describe similarities and differences in our models of the P450 11B1·Adx2 and P450 11B2·Adx2 complexes with the structure of the P450 11A1-Adx fusion protein.

The impact of the clinical CYP11B2 mutation V386A strongly depends on the enzyme's genetic background.

Human cytochrome P450 11B2 (CYP11B2) is an essential enzyme in the steroid hormone biosynthesis, which catalyzes the last three reaction steps of the aldosterone synthesis. These reactions comprise a hydroxylation at position C11 of the steroid intermediate deoxycorticosterone yielding corticosterone, followed by a hydroxylation at position C18 yielding 18-hydroxy-corticosterone and a subsequent oxidation of the hydroxyl group at C18, which results in the formation of aldosterone. Alterations in the amino acid sequence of CYP11B2 often cause severe disease patterns. We previously described a procedure for expression and purification of human CYP11B2 employing recombinant E. coli, which allows the rapid characterization of CYP11B2 mutants on a molecular level. This system was now utilized for the examination of the influence of the polymorphism at position 173 in combination with the mutation V386A on the activity of CYP11B2. Our in vitro findings show that the combination of the V386A mutation with the variant CYP11B2 173Arg only slightly reduces the 18-hydroxylase and 18-oxidase activity, whereas the V386A mutation with the CYP11B2 173Lys variant almost abolishes the 18-hydroxylation and 18-oxidation. In both cases the 11-hydroxylase activity is not affected. These findings highlight the importance of the genetic background of an enzyme when regarding the effect of clinical mutations.

Discovery of Potential Inhibitors of Aldosterone Synthase from Chinese Herbs Using Pharmacophore Modeling, Molecular Docking, and Molecular Dynamics Simulation Studies.

Aldosterone synthase (CYP11B2) is a key enzyme for the biosynthesis of aldosterone, which plays a significant role for the regulation of blood pressure. Excess aldosterone can cause the dysregulation of the renin-angiotensin-aldosterone system (RAAS) and lead to hypertension. Therefore, research and development of CYP11B2 inhibitor are regarded as a novel approach for the treatment of hypertension. In this study, the pharmacophore models of CYP11B2 inhibitors were generated and the optimal model was used to identify potential CYP11B2 inhibitors from the Traditional Chinese Medicine Database (TCMD, Version 2009). The hits were further refined by molecular docking and the interactions between compounds and CYP11B2 were analyzed. Compounds with high Fitvalue, high docking score, and expected interactions with key residues were selected as potential CYP11B2 inhibitors. Two most promising compounds, ethyl caffeate and labiatenic acid, with high Fitvalue and docking score were reserved for molecular dynamics (MD) study. All of them have stability of ligand binding which suggested that they might perform the inhibitory effect on CYP11B2. This study provided candidates for novel drug-like CYP11B2 inhibitors by molecular simulation methods for the hypertension treatment.

Synthesis of annulated pyridines as inhibitors of aldosterone synthase (CYP11B2).

A series of cyclopenta[c]pyridine aldosterone synthase (AS) inhibitors were conveniently accessed using batch or continuous flow Kondrat'eva reactions. Preparation of the analogous cyclohexa[c]pyridines led to the identification of a potent and more selective AS inhibitor. The structure-activity-relationship (SAR) in this new series was rationalized using binding mode models in the crystal structure of AS.

Computational analysis of functional single nucleotide polymorphisms associated with the CYP11B2 gene.

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variations in humans and play a major role in the genetics of human phenotype variation and the genetic basis of human complex diseases. Recently, there is considerable interest in understanding the possible role of the CYP11B2 gene with corticosterone methyl oxidase deficiency, primary aldosteronism, and cardio-cerebro-vascular diseases. Hence, the elucidation of the function and molecular dynamic behavior of CYP11B2 mutations is crucial in current genomics. In this study, we investigated the pathogenic effect of 51 nsSNPs and 26 UTR SNPs in the CYP11B2 gene through computational platforms. Using a combination of SIFT, PolyPhen, I-Mutant Suite, and ConSurf server, four nsSNPs (F487V, V129M, T498A, and V403E) were identified to potentially affect the structure, function, and activity of the CYP11B2 protein. Furthermore, molecular dynamics simulation and structure analyses also confirmed the impact of these nsSNPs on the stability and secondary properties of the CYP11B2 protein. Additionally, utilizing the UTRscan, MirSNP, PolymiRTS and miRNASNP, three SNPs in the 3'UTR region were predicted to exhibit a pattern change in the upstream open reading frames (uORF), and eight microRNA binding sites were found to be highly affected due to 3'UTR SNPs. This cataloguing of deleterious SNPs is essential for narrowing down the number of CYP11B2 mutations to be screened in genetic association studies and for a better understanding of the functional and structural aspects of the CYP11B2 protein.

Structural insights into aldosterone synthase substrate specificity and targeted inhibition.

Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11ß-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.

A case of primary selective hypoaldosteronism carrying three mutations in the aldosterone synthase (Cyp11b2) gene.

An infant with a clinical phenotype of early onset hypoaldosteronism has been screened for mutation analysis of the Cyp11b2 gene encoding aldosterone synthase enzyme. We have described a novel nonsense mutation in exon 3 (c.508C>T) that gave rise to a shorter protein (Q170X) and two known concurrent missense mutations (c.594A>C in exon 3 and c.1157T>C in exon 7) that led to substitution of glutamic acid for aspartic acid at amino acid position 198 (E198D) and of valine for alanine at amino acid position 386 (V386A). The father, who carried E198D plus V386A mutations, showed a fractional sodium excretion of 1.25% that was unmodified by dietary salt restriction, suggesting a mild haploinsufficiency. We examined by in silico analysis the effect of the mutations on the secondary and tertiary structures of aldosterone synthase to explain the inefficient enzymatic activity. The Q170X mutation produced a truncated protein, which was consequently associated with a loss of catalytic activity. As predicted by JPred web system and Dock 6.3 software, the concurrent expression of E198D and V386A mutations induced a significant secondary structure rearrangement and a shift of the heme group and the 18-hydroxycorticosterone substrate from their optimal placement.

Alteration in accumulated aldosterone synthesis as a result of N-terminal cleavage of aldosterone synthase.

Aldosterone synthase (AS) regulates blood volume by synthesizing the mineralocorticoid aldosterone. Overproduction of aldosterone in the adrenal gland can lead to hypertension, a major cause of heart disease and stroke. Aldosterone production depends upon stimulation of AS expression by the renin-angiotensin system, which takes 12 h to reach full effect, and then 24 h to subside. However, this promoter-dependent regulation of aldosterone production fails to explain phenomena such as rapid-onset hypertension that occurs quickly and then subsides. Here, we investigate the fate of AS after expression and how these events relate to aldosterone production. Using isolated mitochondria from steroidogenic cells and cell-free synthesized AS, we first showed that the precursor form of AS translocated into the matrix of the mitochondria, where it underwent cleavage by mitochondrial processing peptidase to a mature form approximately 54 kDa in size. Mature AS seemed to translocate across the inner mitochondrial membrane a second time to finally reside in the intermembrane space. Unprocessed N-terminal AS has 2-fold more activity than physiological levels. These results show how the subcellular mechanisms of AS localization relate to production of aldosterone and reveal a rapid, promoter-independent regulation of aldosterone production.

Regulation of human CYP11B1 and CYP11B2 promoters by transposable elements and conserved cis elements.

CYP11B1 and CYP11B2 responsible for the final steps of cortisol and aldosterone synthesis, respectively, are believed to be duplicate genes with distinctive promoters. Our sequence analysis uncovers that these two genes share great homology in the proximal upstream regions, but insertion of Alu and L1 elements drives promoters divergent. Each CYP11B promoter contains two Alu elements embedded in a truncated L1 element, breaking L1 into three disconnected fragments. Alu functions as an enhancer in both genes regardless of orientation and copy number. Insertion of Alu upstream of a SV40 promoter also elevates promoter activity. However, the effect of Alu on CYP11B1 is blocked by a second L1 element (CYP11B1-L1.2) inserted between the first one and the conserved proximal upstream region. Although CYP11B1-L1.2 is 5'-truncated and lacks a functional ORF, replacing it with a fluorescent gene demonstrates that the element can be transcribed from the CYP11B1 core promoter in an opposite direction and a smaller magnitude compared to CYP11B1. Deletion of CYP11B1-L1.2 greatly increases CYP11B1 promoter activity and restores the enhancing effect of Alu. The Ad5 and SF-1 binding elements conserved in the proximal core promoter play a role in basal expression of both genes. Mutation of the Ad5 site reduces promoter activity to the minimal level. ERRα is the transcription factor interacting with Ad5 during basal expression. The core promoters of both genes are also conserved in mouse and rat despite the fact that the sites corresponding to cre, Ad5, and SF-1 in rodent Cyp11b1 promoters deviate from consensus.

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. 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.

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.

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 of hyperaldosteronism, congestive heart failure, and myocardial fibrosis. Among these, ethyl-substituted 3 possesses high selectivity against CYP1A2. Rigidification of 3 by incorporation of the ethyl group into a 5- or 6-membered ring affords compounds with a pyrroloquinolinone or pyridoquinolinone molecular scaffold (e.g., 4 and 5). It was found that these molecules are even more potent and selective CYP11B2 inhibitors than their corresponding open-chain analogues. Moreover, pyrroloquinolinone 4 exhibits no inhibition of the six most important hepatic CYP enzymes as well as a bioavailability in the range of the marketed drug fadrozole. The SAR studies disclose that subtle changes in the heterocyclic moiety are responsible for either a strong or a weak inhibition of the highly homologous 11ß-hydroxylase (CYP11B1). These results are not only important for fine-tuning the selectivity of CYP11B2 inhibitors but also for the development of selective CYP11B1 inhibitors that are of interest for the treatment of Cushing's syndrome and metabolic syndrome.