Time-of-Day Adrenal Modulation of Corticosterone Synthesis is Affected by Sex and Diet but Not by Proanthocyanidins in Rat.

SCOPE: The aim of this study is to investigate the effect of time-of-day on serum hormones and gene expression in adrenal glands, studying the impact of sex, obesogenic diet, and timing of proanthocyanidins administration, with a focus on glucocorticoids synthesis by this gland. METHODS AND RESULTS: Female and male rats, assigned to a standard chow or a cafeteria diet-fed group, receive a daily oral dose of a grape seed proanthocyanidin extract (GSPE), or a vehicle (when light is turned on, or when light is turned off). Corticosterone, estradiol, and testosterone serum levels, and the expression analysis of clock genes and genes related to corticosterone synthesis pathway, are assessed. Serum hormone levels exhibited a marked time-of-day effect also see in the expression of scavenger receptor class B member 1 (Scarb1) and cyp11b genes. The correlation between these two genes and period circadian regulator 2 (Per2) is also extended to other clock genes, although to a lesser extent: cryptochrome (Cry) and nuclear receptor subfamily 1 group D member 1 (Rev-erba). CONCLUSION: The strong correlations found suggest an important role of local Per2 (but also of Cry and Rev-erbA) in regulating the expression of the enzymes involved in the corticosterone synthesis pathway. The expression of clock genes in adrenals is influenced by sex and diet but not by GSPE.

Double CYP11B1/CYP11B2 Immunohistochemistry and Detection of KCNJ5 Mutations in Primary Aldosteronism.

CONTEXT: The search for somatic mutations in adrenals resected from patients with primary aldosteronism (PA) is performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. OBJECTIVE: To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next-generation sequencing (NGS). METHODS: We investigated 127 consecutive adrenal aldosterone-producing adenomas from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, by Sanger sequencing and NGS. RESULTS: Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2 = 22.492, P < .001). CONCLUSION: NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of patients with aldosterone-producing adenomas.

[Analysis of a family with 11ß-hydroxylase deficiency due to a mutation in the CYP11B1 gene].

This study reports a family of patients with 11ß-hydroxylase deficiency (11ß-OHD) caused by a novel mutation in the CYP11B1 gene, and analyzes its clinical and genetic characteristics. The clinical data of a patient with intractable hypertension at Air Force Medical Center on May 16, 2014 were retrospectively analyzed. The patient was clinically diagnosed with congenital adrenal cortical hyperplasia. The clinical data of the patient were further collected and the peripheral blood samples of the patient, his parents and his sister were collected for CYP11B1(NM_000497) gene sequencing, suggesting that the patient had compound heterozygous mutations in exon 1:c.199delG, p.Glu67Lysfs*9 and exon 5:c.905_907 delATGinsTT, p.Asp302Valfs*23, both of which were pathogenic variants. The patient's father and sister carried heterozygous mutations in exon 1:c.199delG, p.Glu67Lysfs*9, and the mother carried heterozygous mutations in exon 5:c.905_907delATGinsTT, p.Asp302Valfs*23. This study is the first to report a new compound heterozygous mutation in exon 1:c.199delG and exon 5 c.905_907 delATGinsTT of CYP11B1 gene, enriching the database of 11ß-OHD mutations and providing information to further understand the genetic mechanism of the disease.

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.

Non-classical 11ß-hydroxylase deficiency caused by a novel heterozygous mutation: a case report and review of the literature.

PURPOSE: 11ß-hydroxylase deficiency (11ß-OHD) constitutes a rare form of congenital adrenal hyperplasia (CAH), typically accounting for ~5-8% of CAH cases. Non-classical 11ß-OHD is reported even more rarely and frequently results in misdiagnosis or underdiagnosis due to its mild clinical symptoms. METHODS: A clinical, biochemical, radiological, and genetic study was conducted on a 9-year-old girl presenting with mild breast development, axillary hair growth, and advanced bone age. Additionally, a comprehensive review and synthesis of the literature concerning 11ß-OHD were conducted. RESULTS: The patient presented with breast enlargement, axillary hair development, and accelerated growth over the past year. Laboratory tests revealed levels of cortisol, luteinizing hormone, testosterone, and progesterone that were below normal. A gonadotropin-releasing hormone (GnRH) stimulation test suggested the possibility of central precocious puberty. Radiologic examination revealed a 2-year advance in bone age, while bilateral adrenal ultrasonography showed no abnormalities. Her mother exhibited hirsutism, while her father's physical examination revealed no abnormalities. Whole-exon genetic testing of the child and her parents indicated a heterozygous mutation of c.905_907delinsTT in exon 5 of the 11ß-hydroxylase gene (CYP11B1) in the child and her mother. This mutation resulted in a substitution of aspartic acid with valine at amino acid position 302 of the coding protein. This frameshift resulted in a sequence of 23 amino acids, culminating in a premature stop codon (p.Asp302ValfsTer23). A review of the previous literature revealed that the majority of heterozygous mutations in 11ß-OHD were missense mutations, occurring primarily in exons 2, 6, 7, and 8. The most common mutation among 11ß-OHD patients was the change of Arg-448 to His (R448H) in CYP11B1. Furthermore, bioinformatics analyses revealed that heterozygous mutation of c.905_907delinsTT had deleterious effects on the function of CYP11B1 and affected the stability of the protein, presumably leading to a partial impairment of enzyme activity. The results of the in vitro functional study demonstrated that the missense mutant (p.Asp302ValfsTer23) exhibited partial enzymatic activity. CONCLUSIONS: We report a novel heterozygous mutation of CYP11B1 (c.905_907delinsTT), enriching the spectrum of genetic variants of CYP11B1. This finding provides a valuable case reference for early diagnosis of non-classical patients with 11ß-OHD.

Chimeric Genes Causing 11ß-Hydroxylase Deficiency: Implications in Clinical and Molecular Diagnosis.

Deficiency of 11ß-hydroxylase (11ß-OHD) is the second most common cause of congenital adrenal hyperplasia (CAH), accounting for 0.2-8% of all cases. The disease is transmitted as an autosomal recessive trait and the underlying genetic causes of 11ß-OHD are primarily small pathogenic variants affecting the CYP11B1 gene coding the 11ß-hydroxylase enzyme. However, special events complicate the molecular diagnosis of 11ß-OHD such as an unequal crossing over between the CYP11B2 (coding aldosterone synthase enzyme) and CYP11B1 genes. The resulting allele contains a hybrid gene, with a CYP11B2 5'-end and a CYP11B1 3'-end, where the CYP11B1 gene is under the control of the CYP11B2 promoter and thus not responding to the adrenocorticotropin (ACTH) but to angiotensin II and K+. This leads a reduction of cortisol production in 11ß-OHD. In particular, CYP11B2/CYP11B1 chimeric genes can be distinguished into two groups depending on the breakpoint site: chimeras with breakpoint after the exon 5 of CYP11B2 preserve the aldosterone synthase activity, the others with breakpoint before exon 5 lose this function. In the last case, a more severe phenotype is expected. The aim of this review was to explore the setting of CYP11B2/CYP11B1 chimeras in 11ß-OHD, performing a careful review of clinical literature cases.

The intrafollicular concentrations of biologically active cortisol in women rise abruptly shortly before ovulation and follicular rupture.

STUDY QUESTION: What is the temporal activity and the concentration in follicular fluid (FF) of the anti-inflammatory steroid cortisol during the ovulatory process in humans? SUMMARY ANSWER: Intrafollicular concentrations of cortisol become massively upregulated close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. WHAT IS KNOWN ALREADY: Ovulation has been described as a local, controlled inflammatory process resulting in the degeneration of the follicle wall which facilitate oocyte extrusion. Ovulation also affects the glucocorticoid metabolism of granulosa cells (GCs) and although de novo synthesis of cortisol only occurs in the adrenal cortex, the mid-cycle surge has been shown to induce a change from high expression of HSD11B2, inactivating cortisol to cortisone, to high expression of HSD11B1 which reversibly catalyses cortisol production from cortisone. Furthermore, high concentrations of progesterone and 17OH-progesterone within follicles may cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women donated FF and GCs from one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles: T = 0 h, T = 12 h, T = 17 h, T = 32 h. A second sample was collected at oocyte pick up at T = 36 h. The concentration of cortisol and cortisone together with a range of sex steroids was measured by LC-MS/MS in FF collected at the five time points mentioned above. Whole genome microarray data, validated by q-PCR analysis, was used to evaluate gene expression of CYP11B1, CYP21A2, HSD11B1, HSD11B2, and NR3C1 in GCs at the same time points. MAIN RESULTS AND THE ROLE OF CHANCE: The concentration of cortisol was significantly increased from a few nM at 0 h to around 100-140 nM (P ≤ 0.0001) at 32-36 h, whilst cortisone was almost constant from 0 to 17 h at a concentration of between 90 and 100 nM being significantly reduced to 25-40 nM (P ≤ 0.0001) at 32-36 h. This was paralleled by a 690-fold upregulation of HSD11B1 from 0 to 12 h increasing to a more than 20.000-fold change at 36 h. HSD11B2 was quickly downregulated 15- to 20-fold after ovulation induction. Concentrations of progesterone and 17OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CBP due to similar binding affinities. Furthermore, a significant decrease in 11-deoxycortisol expression was seen, but CYP11B1 expression was below detection limit in GCs. LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and results may differ from the natural cycle. More observations at each specific time point may have strengthened the conclusions. Furthermore, we have not been able to measure the actual active biological concentration of cortisol. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study collectively evaluated the temporal pattern of cortisol and cortisone concentrations during human ovulation, rendering a physiological framework for understanding potential dysregulations in the inflammatory reaction of ovulation. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, and Novo Nordisk Foundation grant number NNF21OC00700556. Interreg V ÔKS through ReproUnion (www.reprounion.eu); Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Identification and functional characterization of compound heterozygous CYP11B1 gene mutations.

PURPOSE: 11ß-Hydroxylase deficiency (11ß-OHD) is the second leading cause of congenital adrenal hyperplasia (CAH), a rare autosomal recessive disease caused by mutations in the CYP11B1 gene. We previously reported the case of a male Chinese patient with typical 11ß-OHD symptoms. Sanger sequencing revealed that the patient carried a splice-site mutation, c.595+1G>A in the CYP11B1 gene. His mother and sister harbored the heterozygous mutation, c.595+1G>A. Paradoxically, Sanger sequencing did not detect any abnormality in the CYP11B1 gene of his father and brother. Therefore, in this study, we aimed to further explore the exact genetic etiology of 11ß-OHD in this pedigree and analyze the functional consequence of the c.595+1G>A mutation. METHODS: Gemomic DNA was extracted from the peripheral blood leukocytes of the family members and normal control individuals, followed by quantitative real-time polymerase chain reaction (qPCR) to detect the copy number of the target CYP11B1 gene fragment. Mutation analysis was also performed via whole-exome sequencing (WES) followed by Sanger sequencing validation. In vitro minigene assay was also performed to investigate the impact of the c.595+1G>A mutation on pre-mRNA splicing. RESULTS: qPCR results suggested a heterozygous deletion encompassing position c.595+1 along with flanking exonic and intronic sequences in the CYP11B1 gene of the patient and his father. WES followed by Sanger sequencing verified that the patient carried compound heterozygous mutations in the CYP11B1 gene, including a novel 2840-bp deletion (c.395+661_c.1121+180del) and c.595+1G>A, while his father carried the heterozygous c.395+661_c.1121+180del mutation. No other novel CYP11B1 mutations were found in the rest of the family members. Furthermore, minigene assay revealed that the c.595+1G>A mutation resulted in a 70-bp deletion of exon 3 in the mRNA, and this altered the reading frame at amino acid 176 and created a premature stop codon at amino acid 197. CONCLUSION: We identified a novel 2840-bp-sized large deletion and confirmed that the c.595+1G>A mutation disrupts normal pre-mRNA splicing. Either mutation could significantly alter the reading frame and abolish CYP11B1 enzyme activity. Therefore, our findings widen the mutation spectrum of CYP11B1 and provide an accurate diagnosis of 11ß-OHD at a molecular genetic level.

Histopathological analysis of tumor microenvironment in adrenocortical carcinoma: Possible effects of in situ disorganized glucocorticoid production on tumor immunity.

Adrenocortical carcinoma (ACC) patients with glucocorticoid excess have been reported to be associated with decreased tumor-infiltrating immune cells, but the effects of in situ glucocorticoid production on tumor immunity have remained unknown. In addition, ACC was also known to harbor marked intra-tumoral heterogeneity of steroidogenesis or disorganized steroidogenesis. Therefore, in this study, we immune-profiled tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs) and pivotal steroidogenic enzymes of glucocorticoid biosynthesis (CYP17A and CYP11B1) to explore the potential effects of in situ glucocorticoid production and intra-tumoral heterogeneity/disorganized steroidogenesis on tumor immunity of ACC. We also studied the correlations of the status of tumor immunity with that of angiogenesis and tumor grade to further explore the tumor tissue microenvironment of ACC. TILs (CD3, CD4, CD8, and FOXP3), TAMs (CD68 and CD163), key steroidogenic enzymes of glucocorticoid (CYP17A and CYP11B1), angiogenesis (CD31 and vasohibin-1 (VASH-1)), tumor grade (Ki-67 and Weiss score) were immunohistochemically evaluated in 34 ACCs. Increased CYP17A immunoreactivity in the whole tumor area was significantly positively correlated with FOXP3-positive TILs (p = 0.021) and negatively with CD4/CD3 ratio (p = 0.001). Increased CYP11B1 immunoreactivity in the whole tumor area was significantly positively correlated with CD8/CD3 (p = 0.039) and CD163/CD68 ratios (p = 0.006) and negatively with CD4-positive TILs (p = 0.036) and CD4/CD3 ratio (p = 0.001). There were also significant positive correlations between CYP17A and CD8 (r = 0.334, p < 0.001) and FOXP3-positive TILs (r = 0.414, p < 0.001), CD8/CD3 ratio (r = 0.421, p < 0.001), and CD68-positive TAMs (r = 0.298, p < 0.001) in randomly selected areas. Significant positive correlations were also detected between CYP11B1 and CD8/CD3 ratio (r = 0.276, p = 0.001) and negative ones detected between CYP11B1 and CD3- (r = -0.259, p = 0.002) and CD4-positive TILs (r = -0.312, p < 0.001) in those areas above. Increased micro-vessel density (MVD) -VASH-1 was significantly positively correlated with CD68- (p = 0.015) and CD163-positive TAMs (p = 0.009) and CD163/CD68 ratio and the high VASH-1 with CD163-positive TAMs (p = 0.042). Ki-67 labeling index was significantly positively correlated with MAD-VASH-1 (p = 0.006) and VASH-1 (p = 0.006) status. Results of our present study indicated that in situ glucocorticoid production did influence the status of tumor immunity in ACC. In particular, increased levels of CYP17A and CYP11B1, both involved in glucocorticoid producing immunoreactivity played different effects on tumor immunity, i.e., reflecting the involvement of intra-tumoral heterogeneity and disorganized steroidogenesis of ACC, which also did indicate the importance of in situ approaches when analyzing tumor immunity of ACC.

Prolonged adrenal insufficiency following discontinuation of osilodrostat treatment for intense hypercortisolism.

The cases of 3 patients with Cushing's disease who developed long-term adrenal insufficiency after discontinuation of prolonged osilodrostat therapy were recently described for the first time. We report 2 additional cases of persistent prolonged adrenal insufficiency after discontinuation of osilodrostat treatment for intense hypercortisolism due to Cushing's disease and ectopic ACTH syndrome. In addition, we show for that adrenal insufficiency in these patients was associated with low/normal 11-deoxycortisol concentrations despite high plasma ACTH concentrations. These results suggest that CYP11B1 is not the only target of osilodrostat and that, in vivo, osilodrostat has other prolonged and strong inhibitory effect on adrenal steroidogenesis upstream of CYP11B1. Knowledge of this remnant effect is important for the care of patients with Cushing's syndrome treated with osilodrostat. Further studies are needed to clarify the frequency and the mechanisms of this remnant effect.

Clinical Features of Unrecognized Congenital Adrenal Hyperplasia Due to 17α-hydroxylase Deficiency Since Adolescence: A Case Report.

The majority of patients with congenital adrenal hyperplasia (CAH) present with a deficiency of 21-hydroxylase or 11-beta-hydroxylase, which account for 90% and 7% of cases, respectively. However, CAH due to 17α-hydroxylase deficiency (17OHD) is an extremely rare form of CAH (<1% of all CAH cases) that leads to a deficiency of cortisol and sex steroids, along with features of aldosterone excess. This is a case of a 51-year-old single female who was referred to us for the evaluation of new-onset hypertension and hypokalaemia of one-year duration. She was born out of a second-degree consanguineous marriage and reared as a female. She was diagnosed to have testicular feminization syndrome when she presented with a history of primary amenorrhea, absence of secondary sexual characteristics, and bilateral labial swellings at pubertal age. Subsequently, she underwent gonadectomy at the age of 16. Due to the presence of hypertension, metabolic alkalosis and bilaterally enlarged adrenals on CT scan, 46, XY disorders of sexual development (DSD) was considered. A karyotype confirmed the presence of 46, XY chromosomal sex, and genetic analysis revealed a mutation in the CYP17A1 gene, thus confirming the diagnosis of 17α-hydroxylase deficiency.

A common germline variant in CYP11B1 is associated with adverse clinical outcome of treatment with abiraterone or enzalutamide.

Extragonadal androgens play a pivotal role in prostate cancer disease progression on androgen receptor signaling inhibitors (ARSi), including abiraterone and enzalutamide. We aimed to investigate if germline variants in genes involved in extragonadal androgen synthesis contribute to resistance to ARSi and may predict clinical outcomes on ARSi. We included ARSi naive metastatic prostate cancer patients treated with abiraterone or enzalutamide and determined 18 germline variants in six genes involved in extragonadal androgen synthesis. Variants were tested in univariate and multivariable analysis for the relation with overall survival (OS) and time to progression (TTP) by Cox regression, and PSA response by logistic regression. A total of 275 patients were included. From the investigated genes CYP17A1, HSD3B1, CYP11B1, AKR1C3, SRD5A1 and SRD5A2, only rs4736349 in CYP11B1 in homozygous form (TT), present in 54 patients (20%), was related with a significantly worse OS (HR = 1.71, 95% CI 1.09 - 2.68, p = 0.019) and TTP (HR = 1.50, 95% CI 1.08 - 2.09, p = 0.016), and was related with a significantly less frequent PSA response (OR = 0.48, 95% CI 0.24 - 0.96, p = 0.038) on abiraterone or enzalutamide in a multivariable analysis. The frequent germline variant rs4736349 in CYP11B1 is, as homozygote, an independent negative prognostic factor for treatment with abiraterone or enzalutamide in ARSi naive metastatic prostate cancer patients. Our findings warrant prospective investigation of this potentially important predictive biomarker.

Case Report: A combination of chimeric CYP11B2/CYP11B1 and a novel p.Val68Gly CYP11B1 variant causing 11ß-Hydroxylase deficiency in a Chinese patient.

Introduction: 11ß-Hydroxylase deficiency (11ß-OHD, OMIM#202010) is the second most common form of congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the CYP11B1 gene. Both single nucleotide variations (SNV)/small insertion and deletion and genomic rearrangements of CYP11B1 are important causes of 11ß-OHD. Among these variant types, pathogenic CYP11B2/CYP11B1 chimeras only contribute to a minority of cases. Heterozygote cases (chimera combined with SNV) are very rare, and genetic analysis of these cases can be challenging. Case presentation: We presented a suspected 11ß-OHD female patient with incomplete virilization, adrenal hyperplasia, and hypokalemia hypertension. Whole exome sequencing (WES) revealed that the patient carried both a chimeric CYP11B2/CYP11B1 and a novel missense variant, NM_000497.4: c.203T>G, p.Val68Gly (chr8:143961027) in CYP11B1, which were confirmed by CNVplex and Sanger sequencing, respectively. The patient's manifestations and genetic findings confirmed the diagnosis of 11ß-OHD, and oral dexamethasone was administered as a subsequent treatment. Conclusion: This report showed a rare CYP11B2/CYP11B1 chimera combined with a novel missense variant in a 11ß-OHD female patient. The result expands variant spectrum of CYP11B1 and suggests that both chimera and CYP11B1 variant screening should be performed simultaneously in suspected cases of 11ß-OHD. To our knowledge, this is the first report about CYP11B2/CYP11B1 chimera detected by WES analysis. WES combined with CNV analysis is an efficient method in the genetic diagnosis of this rare and complex disorder.

Structural and clinical characterization of CYP11B2 inhibition by dexfadrostat phosphate.

Aldosterone synthase (CYP11B2) represents a promising drug target because its genetic dysregulation is causally associated with cardiovascular disease, its autonomous activity leads to primary aldosteronism, and its deficiency leads to salt wasting syndromes. The serendipitous discovery that the dextro-rotatory stereoisomer of the racemic aromatase (CYP19A1) inhibitor CGS16949A mediates potent CYP11B2 inhibition led to the purification and clinical development of dexfadrostat phosphate. To characterize the pharmacophore of dexfadrostat phosphate, structure-based enzyme coordination with CYP11B2, CYP11B1 and CYP19A1 was combined with steroid turnover upon in vitro and clinical treatment. Dexfadrostat, but not its 5S-enantiomer (5S-fadrozole), precisely coordinates with the catalytic heme moiety in the space of the CYP11B2 substrate binding pocket forming a tight and stable complex. Conversely, neither rigid nor flexible docking led to a plausible coordination geometry for dexfadrostat in steroid 11ß-hydroxylase (CYP11B1 - orthologue to CYP11B2) or in CYP19A1. The inhibitory preference of dexfadrostat was confirmed in vitro using an adrenal cortex-derived cell line. Dexfadrostat phosphate treatment of healthy subjects in the context of a clinical phase 1 study led to a dose-dependent decrease in urinary aldosterone secretion, accompanied by an increase in urinary corticosterone and deoxycorticosterone metabolites. Increased urinary corticosterone metabolites are indicative of CYP11B2 (18-oxidase) inhibition with clinical features reminiscent of patients with inborn corticosterone methyloxidase type II deficiency. An off-target effect on CYP19A1 was not observed as indicated by no clinical changes in testosterone and estradiol levels. Therefore, dexfadrostat exhibits the ideal structural features for binding and catalytic inhibition of CYP11B2 but not CYP11B1. Clinically, treatment with dexfadrostat phosphate leads to suppression of aldosterone levels by inhibiting predominantly one or both final CYP11B2-mediated reactions.

Fluorinated aldosterone synthase (CYP11B2)-inhibitors for differential diagnosis between bilateral and unilateral conditions of primary aldosteronism.

The enzyme aldosterone synthase (CYP11B2) is specifically expressed in aldosterone-producing tissue of the adrenal cortex and is overexpressed in aldosterone-producing adenomas (APA). It therefore represents an ideal target for molecular imaging, particularly for the differential diagnosis between bilateral hyperplasia and unilateral APA in primary aldosteronism. However, the presence of the cortisol-producing enzyme 11ß-hydroxylase (CYP11B1) in the adrenal cortex remains very challenging owing to its high homology to CYP11B2. Within this study, we efficiently synthesized a variety of disubstituted fluorinated pyridines and pyrazines by Suzuki coupling reactions. These compounds were evaluated for their ability to inhibit CYP11B1 and CYP11B2 in transfected Y1 cells and in NCI-h295 cells. Several compounds were found to exhibit excellent affinity (IC50 < 10 nM) to CYP11B2 as well as strong selectivity (up to 125-fold) over CYP11B1. These findings support the further development of an analogous 18F-labelled PET tracer.

Unequal crossing over between CYP11B2 and CYP11B1 causes 11 ß -hydroxylase deficiency in a consanguineous family.

Cytochrome P450 (CYP) family CYP11B2/CYP11B1 chimeric genes have been shown to arise from unequal crossing over of the genes encoding aldosterone synthase (CYP11B2) and 11ß-hydroxylase (CYP11B1) during meiosis. The activity deficiency or impaired activity of aldosterone synthase and 11ß-hydroxylase resulting from these chimeric genes are important reasons for 11ß-hydroxylase deficiency (11ß-OHD). Here，two patients with pseudoprecocious puberty and hypokalemia hypertension and three carriers in a consanguineous marriage family were studied. A single CYP11B2/CYP11B1 chimera consisting of the promoter and exons 1 through 5 of CYP11B2, exons 8 and 9 of CYP11B1, and a breakpoint consisting of part of exon 6 of CYP11B2 and part of exon 6, intron 6, and exon 7 of CYP11B1 were detected in the patients and carriers. At the breakpoint of the chimera, a c 0.1086 G > C ( p.Leu.362 =) synonymous mutation in exon 6 of CYP11B2, a c 0.1157 C＞G(p. A386V) missense mutation in exon 7 of CYP11B1, and an intronic mutation in intron 6 were detected. The allele model of the CYP11B2/CYP11B1 chimera demonstrated homozygosity and heterozygosity in the patients and the carriers, respectively. Molecular docking and enzymatic activity analyses indicated that the CYP11B2/CYP11B1 chimeric protein interacted with the catalytic substrate of aldosterone synthase and had similar enzymatic activity to aldosterone synthase. Our study indicated that deletion of CYP11B1 and CYP11B2 abolished the enzymatic activity of 11 ß-hydroxylase and aldosterone synthase; however, the compensation of the enzymatic activity of aldosterone synthase by the CYP11B2/CYP11B1 chimeric protein maintained normal aldosterone levels in vitro. All of the above findings explained the 11ß-OHD phenotypes of the proband and patients in the family.

Aire drives steroid hormone biosynthesis by medullary thymic epithelial cells.

Thymic epithelial cells (TECs) produce glucocorticoids, which antagonize negative selection of autoreactive thymocytes and promote a competent T cell antigen-specific repertoire. To characterize their source, we generated a knock-in reporter mouse in which endogenous Cyp11b1, the final enzyme in de novo production of active glucocorticoids, was fluorescently tagged with mScarlet. Here, we find that Cyp11b1 is expressed in medullary TECs (mTECs) but not cortical TECs or other cells in the thymus. A distinct characteristic of mTECs is the presence of Aire, a transcription factor that drives expression of tissue-restricted antigens (TRAs) important for establishing immune tolerance. Cyp11b1 expression was highest in Aire+ mTECs, lower in post-Aire mTECs, and absent in mTECs of Aire-deficient mice. Transcriptomic analyses found that multiple enzymatic biosynthetic pathways are expressed specifically in mTECs and are also Aire dependent. In particular, we found that the thymus expresses messenger RNA for enzymes that catalyze production of many bioactive steroids and that glucocorticoids and sex steroids were secreted by cultured thymi. Expression of the transcripts for these genes and production of their final steroid products were markedly reduced in the absence of Aire. Thus, in addition to its well-established role in inducing TRAs that promote negative selection, Aire has an additional and contrary function of inducing glucocorticoids that antagonize negative selection, which together may expand and enhance the TCR repertoire. Furthermore, because Aire drives expression of multiple enzymes responsible for production of other non-gene-encoded bioactive molecules, it might have yet other roles in thymus development and function.

Virtual screening and biological evaluation to identify pharmaceuticals potentially causing hypertension and hypokalemia by inhibiting steroid 11ß-hydroxylase.

Several drugs were found after their market approval to unexpectedly inhibit adrenal 11ß-hydroxylase (CYP11B1)-dependent cortisol synthesis. Known side-effects of CYP11B1 inhibition include hypertension and hypokalemia, due to a feedback activation of adrenal steroidogenesis, leading to supraphysiological concentrations of 11-deoxycortisol and 11-deoxycorticosterone that can activate the mineralocorticoid receptor. This results in potassium excretion and sodium and water retention, ultimately causing hypertension. With the risk known but usually not addressed in preclinical evaluation, this study aimed to identify drugs and drug candidates inhibiting CYP11B1. Two conceptually different virtual screening methods were combined, a pharmacophore based and an induced fit docking approach. Cell-free and cell-based CYP11B1 activity measurements revealed several inhibitors with IC50 values in the nanomolar range. Inhibitors include retinoic acid metabolism blocking agents (RAMBAs), azole antifungals, α2-adrenoceptor ligands, and a farnesyltransferase inhibitor. The active compounds share a nitrogen atom embedded in an aromatic ring system. Structure activity analysis identified the free electron pair of the nitrogen atom as a prerequisite for the drug-enzyme interaction, with its pKa value as an indicator of inhibitory potency. Another important parameter is drug lipophilicity, exemplified by etomidate. Changing its ethyl ester moiety to a more hydrophilic carboxylic acid group dramatically decreased the inhibitory potential, most likely due to less efficient cellular uptake. The presented work successfully combined different in silico and in vitro methods to identify several previously unknown CYP11B1 inhibitors. This workflow facilitates the identification of compounds that inhibit CYP11B1 and therefore pose a risk for inducing hypertension and hypokalemia.

Glucocorticoids are differentially synthesized along the murine and human respiratory tree.

BACKGROUND: Synthetic glucocorticoids (GC) are effective in the treatment of inflammatory diseases of the lung. However, long-term use leads to severe side effects. Endogenous GC can be synthesized locally, either de novo from cholesterol in a 11ß-hydroxylase (Cyp11b1)-dependent manner, or by reactivation from 11-dehydrocorticosterone/cortisone by 11ß-hydroxysteroid dehydrogenase 1 (Hsd11b1). We aimed to define the molecular pathways of endogenous GC synthesis along the respiratory tree to provide a basis for understanding how local GC synthesis contributes to tissue homeostasis. METHODS: Expression of steroidogenic enzymes in murine lung epithelium was analyzed by macroscopic and laser capture microdissection, followed by RT-qPCR. Flow cytometry analysis was performed to identify the cellular source of steroidogenic enzymes. Additionally, the induction of steroidogenic enzyme expression in the lung was analyzed after lipopolysaccharide (LPS) injection. mRNA and protein expression of steroidogenic enzymes was confirmed in human lung tissue by RT-qPCR and immunohistochemistry. Furthermore, GC synthesis was examined in ex vivo cultures of fresh tissue from mice and human lobectomy patients. RESULTS: We observed that the murine and human lung tissue differentially expresses synthesis pathway-determining enzymes along the respiratory tree. We detected Hsd11b1 expression in bronchial, alveolar, club and basal epithelial cells, whereas Cyp11b1 expression was detectable only in tracheal epithelial cells of mice. Accordingly, de novo synthesis of bioactive GC occurred in the large conducting airways, whereas reactivation occurred everywhere along the respiratory tree. Strikingly, Cyp11b1 but not Hsd11b1 expression was enhanced in the trachea upon LPS injection in mice. CONCLUSION: We report here the differential synthesis of bioactive GC along the murine and human respiratory tree. Thus, extra-adrenal de novo GC synthesis and reactivation may differentially contribute to the regulation of immunological and inflammatory processes in the lung.