Cryoradiolysis of oxygenated cytochrome P450 17A1 with lyase substrates generates expected products.

When subjected to γ-irradiation at cryogenic temperatures the oxygenated complexes of Cytochrome P450 CYP17A1 (CYP17A1) bound with either of the lyase substrates, 17α-Hydroxypregnenolone (17-OH PREG) or 17α-Hydroxyprogesterone (17-OH PROG) are shown to generate the corresponding lyase products, dehydroepiandrosterone (DHEA) and androstenedione (AD) respectively. The current study uses gas chromatography-mass spectrometry (GC/MS) to document the presence of the initial substrates and products in extracts of the processed samples. A rapid and efficient method for the simultaneous determination of residual substrate and products by GC/MS is described without derivatization of the products. It is also shown that no lyase products were detected for similarly treated control samples containing no nanodisc associated CYP17 enzyme, demonstrating that the product is formed during the enzymatic reaction and not by GC/MS conditions, nor the conditions produced by the cryoradiolysis process.

Steroid 17α-hydroxylase/17, 20-lyase (cytochrome P450 17A1).

Cytochrome P450 (P450) 17A1 plays a key role in steroidogenesis, in that this enzyme catalyzes the 17α-hydroxylation of both pregnenolone and progesterone, followed by a lyase reaction to cleave the C-20 land C-21 carbons from each steroid. The reactions are important in the production of both glucocorticoids and androgens. The enzyme is critical in humans but is also a drug target in treatment of prostate cancer. Detailed methods are described for the heterologous expression of human P450 17A1 in bacteria, purification of the recombinant enzyme, reconstitution of the enzyme system in the presence of cytochrome b5, and chromatographic procedures for sensitive analyses of reaction products. Historic assay approaches are reviewed. Some information is also provided about outstanding questions in the research field, including catalytic mechanisms and searches for selective inhibitors.

Friedelin, a novel inhibitor of CYP17A1 in prostate cancer from Cassia tora.

In prostate cancer (PC), drugs targeting CYP17A1 have shown great success in regulating PC progression. However, successful drug molecules show adverse side effects and therapeutic resistance in PC. Therefore, we proposed to discover the potent phytochemical-based inhibitor against CYP17A1 using virtual screening. In this study, a phytochemicals library of ∼13800 molecules was selected to screen the best possible inhibitors against CYP17A1. A molecular modelling approach investigated detailed intermolecular interactions, their structural stability, and binding affinity. Further, in vitro and in vivo studies were performed to confirm the anticancer activity of identified potential inhibitor against CYP17A1. Friedelin from Cassia tora (CT) is identified as the best possible inhibitor from the screened library. MD simulation study reveals stable binding of Friedelin to conserved binding pocket of CYP17A1 with higher binding affinity than studied control, that is, Orteronel. Friedelin was tested on hormone-sensitive (22Rv1) and insensitive (DU145) cell lines and the IC50 value was found to be 72.025 and 81.766 µg/ml, respectively. CT extract showed a 25.28% IC50 value against 22Rv1, ∼92.6% increase in late Apoptosis/Necrosis, and three folds decrease in early apoptosis in treated cells compared to untreated cells. Further, animal studies show a marked decrease in prostate weight by 39.6% and prostate index by 36.5%, along with a reduction in serum PSA level by 71.7% and testosterone level by 92.4% compared to the testosterone group, which was further validated with histopathological studies. Thus, we propose Friedelin and CT extract as potential leads, which could be taken further for drug development in PC.[Figure: see text]Communicated by Ramaswamy H. Sarma.

Amylin regulates testosterone levels via steroidogenesis-related enzymes in the central nervous system of male mice.

Amylin is a peripheral satiation signal polypeptide co-secreted with insulin by pancreatic ß-cells in response to nutrient ingestion. Amylin participates in the eating-inhibitory effect and regulates energy metabolism by acting on the central nervous system (CNS). However, the role of amylin in regulating the biosynthesis of steroid hormones, such as testosterone, through the hypothalamic-pituitary-gonadal axis (HPG) remains unexplored. However, only limited evidence is available on the involvement of amylin in steroid synthesis, we hypothesize that amylin regulates testosterone levels via steroidogenesis-related enzymes in the CNS. In this study, we elucidated the effect of intraperitoneal injection of amylin on the protein expression of steroidogenesis-related enzymes, including 3ß-hydroxysteroid dehydrogenase (3ß-HSD), cytochrome P450 17A1 (CYP17A1), and steroidogenic acute regulatory protein (StAR), and phospho-extracellular signal-regulated kinase (pERK). Additionally, the effect of amylin on testosterone levels in male mice was examined. Our results suggested that 3ß-HSD and CYP17A1 neurons were widely expressed in the CNS of male mice, whereas StAR neurons were mainly expressed in the zona incerta (ZI) and locus coeruleus (LC) regions. Intraperitoneal injection of amylin significantly reduced (p < 0.01) the expression of 3ß-HSD, CYP17A1, and StAR in ZI and other areas near the third ventricle (3 V) but increased (p < 0.01) pERK expression, brain testosterone levels, serum FSH, serum LH, and decreased (p < 0.01) serum testosterone levels in mice. In conclusion, amylin regulates testosterone levels via steroidogenesis-related enzymes in the central nervous system of male mice.

Synthesis and Structure-Activity Relationships of Novel Non-Steroidal CYP17A1 Inhibitors as Potential Prostate Cancer Agents.

Twenty new compounds, targeting CYP17A1, were synthesized, based on our previous work on a benzimidazole scaffold, and their biological activity evaluated. Inhibition of CYP17A1 is an important modality in the treatment of prostate cancer, which remains the most abundant cancer type in men. The biological assessment included CYP17A1 hydroxylase and lyase inhibition, CYP3A4 and P450 oxidoreductase (POR) inhibition, as well as antiproliferative activity in PC3 prostate cancer cells. The most potent compounds were selected for further analyses including in silico modeling. This combined effort resulted in a compound (comp 2, IC50 1.2 µM, in CYP17A1) with a potency comparable to abiraterone and selectivity towards the other targets tested. In addition, the data provided an understanding of the structure-activity relationship of this novel non-steroidal compound class.

Associations among maternal perfluoroalkyl substance levels, fetal sex-hormone enzymatic gene polymorphisms, and fetal sex hormone levels in the Hokkaido study.

Prenatal sex hormones affect fetal growth; for example, prenatal exposure to low levels of androgen accelerates female puberty onset. We assessed the association of perfluoroalkyl substances (PFASs) in maternal sera and infant genotypes of genes encoding enzymes involved in sex steroid hormone biosynthesis on cord sera sex hormone levels in a prospective birth cohort study of healthy pregnant Japanese women (n = 224) recruited in Sapporo between July 2002 and October 2005. We analyzed PFAS and five sex hormone levels using liquid chromatography-tandem mass spectrometry. Cytochrome P450 (CYP) 17A1 (CYP17A1 rs743572), 19A1 (CYP19A1 rs10046, rs700519, and rs727479), 3ß-hydroxysteroid dehydrogenase type 1 (HSD3B1 rs6203), type 2 (HSD3B2 rs1819698, rs2854964, and rs4659175), 17ß-hydroxysteroid dehydrogenase type 1 (HSD17B1 rs605059, rs676387, and rs2676531), and type 3 (HSD17B3 rs4743709) were analyzed using real-time PCR. Multiple linear regression models were used to establish the influence of log10-transformed PFAS levels and infant genotypes on log10-transformed sex steroid hormone levels. When the interaction between perfluorooctanesulfonate (PFOS) levels and female infant genotype CYP17A1 (rs743572) on the androstenedione (A-dione) levels was considered, the estimated changes (95 % confidence intervals) in A-dione levels against PFOS levels, female infant genotype CYP17A1 (rs743572)-AG/GG, and interaction between them showed a mean increase of 0.445 (0.102, 0.787), mean increase of 0.392 (0.084, 0.707), and mean reduction of 0.579 (0.161, 0.997) (Pint = 0.007), respectively. Moreover, a female-specific interaction with testosterone levels was observed. A-dione and T levels showed positive main effects and negative interaction with PFOS levels and the female infant CYP17A1 genotype.

Steroidogenic cytochrome P450 17A1 structure and function.

Cytochrome P450 17A1 (CYP17A1) is a critical steroidogenic enzyme, essential for producing glucocorticoids and sex hormones. This review discusses the complex activity of CYP17A1, looking at its role in both the classical and backdoor steroidogenic pathways and the complex chemistry it carries out to perform both a hydroxylation reaction and a carbon-carbon cleavage, or lyase reaction. Functional and structural investigations have informed our knowledge of these two reactions. This review focuses on a few specific aspects of this discussion: the identities of reaction intermediates, the coordination of hydroxylation and lyase reactions, the effects of cytochrome b5, and conformational selection. These discussions improve understanding of CYP17A1 in a physiological setting, where CYP17A1 is implicated in a variety of steroidogenic diseases. This information can be used to improve ways in which CYP17A1 can be effectively modulated to treat diseases such as prostate and breast cancer, Cushing's syndrome, and glioblastoma.

Inhibitory effect of central ghrelin on steroid synthesis affecting reproductive health in female mice.

Ghrelin is a 28-amino acid peptide hormone that regulates ovarian steroid hormone synthesis; however, there is limited evidence regarding the regulation of this pathway by ghrelin in mice ovary. Thus, we aimed to investigate whether central ghrelin action plays a role in murine reproductive health by inhibiting steroid synthesis. Further, we sought to examine the mechanism of central ghrelin action in ovarian steroid hormone synthesis. After the administration of intracerebroventricular ghrelin (1 nmol), we found reduced serum concentrations of oestradiol and progesterone and reduced secretion of follicle-stimulating hormone and luteinising hormone. Although ghrelin reduced 3ß-hydroxysteroid dehydrogenase mRNA and protein levels in the hypothalamus, it did not affect the expression of steroidogenic acute regulatory protein and cytochrome P450 17A1. In the ovary, central ghrelin regulation indirectly inhibited the mRNA and protein levels of steroidogenic acute regulatory protein, cytochrome P450 17A1, and 3ß-hydroxysteroid dehydrogenase. Moreover, no changes were observed in the expression of proliferating cell nuclear antigen and phosphorylation of extracellular signal-regulated kinase. We hypothesised that central ghrelin regulation suppressed serum oestradiol and progesterone levels by indirectly inhibiting the expression of steroidogenic acute regulatory protein, cytochrome P450 17A1, and 3ß-hydroxysteroid dehydrogenase in the ovary. In this regulation, the suppressed secretion of the follicle-stimulating hormone and luteinising hormone in the pituitary by ghrelin could be involved. Furthermore, hypothalamic 3ß-hydroxysteroid dehydrogenase expression is reduced by ghrelin injection.

Discovery of Novel Non-Steroidal Cytochrome P450 17A1 Inhibitors as Potential Prostate Cancer Agents.

The current study presents the design, synthesis, and evaluation of novel cytochrome P450 17A1 (CYP17A1) ligands. CYP17A1 is a key enzyme in the steroidogenic pathway that produces androgens among other steroids, and it is implicated in prostate cancer. The obtained compounds are potent enzyme inhibitors (sub µM) with antiproliferative activity in prostate cancer cell lines. The binding mode of these compounds is also discussed.

Effect of mutations in porcine CYB5A and CYP17A1 on the metabolism of pregnenolone.

Cytochrome P450 17A1 (CYP17A1) catalyses the 17α-hydroxylation and 17,20 lyase reactions to convert pregnenolone to 17α-hydroxypregnenolone (17OHP) and subsequently the androgen dehydroepiandrosterone (DHEA). In pigs and humans, CYP17A1 also catalyses the delta-16-synthase reaction to produce the 16-androstene steroid 5,16-androstadien-3ß-ol (16A), which is then further metabolised to the sex pheromone androstenone. Cytochrome b5A (CYB5A) stimulates the 17,20 lyase reaction and is required for the delta 16-synthase reaction. We have identified and mutated residues in porcine CYP17A1 and CYB5A that may alter the synthesis of DHEA and 16A. This included residues in the steroid binding pocket of CYP17A1 and residues on the surface of CYP17A1 and CYB5A that are involved in binding of CYP17A1 to CYB5A. We then expressed the various mutations of CYB5A and CYP17A1 along with porcine cytochrome P450 oxidoreductase (POR) and cytochrome b5 reductase (CYB5R3) in HEK293 cells and measured the formation of metabolites 16A, 17OHP and DHEA from radiolabelled pregnenolone by high performance liquid chromatography (HPLC). Mutations were identified in both CYP17A1 and CYB5A that affected the production of the different metabolites and also affected the overall production of metabolites. Several combinations of mutations decreased the production of both 16A and DHEA and increased production of 17OHP, while the N62S mutation of CYB5A with wild type CYP17A1 increased production of both 16A and DHEA. The best combination of mutations to reduce the production of 16A, while maintaining the production of DHEA and the overall conversion similar to wild type are the N21K, L28V, N21K/L28V and the R52 M/N62S mutations of CYB5A with the D103S mutation of CYP17A1.

Comparative study of the binding mode between cytochrome P450 17A1 and prostate cancer drugs in the absence of haem iron.

According to the X-ray crystal structures of CYP17A1 (including its complexes with inhibitors), it is shown that a hydrogen bond exists between CYP17A1 and its inhibitors (such as abiraterone and TOK-001). Previous short MD simulations (50 ns) suggested that the binding of abiraterone to CYP17A1 is stronger than that of TOK-001. In this work, by carrying out long atomistic MD simulations (200 ns) of CYP17A1 and its complexes with abiraterone and TOK-001, we observed a binding mode between CYP17A1 and abiraterone, which is different from the binding mode between CYP17A1 and TOK-001. In the case of abiraterone binding, the unfilled volume in the active site cavity increases the freedom of movement of abiraterone within CYP17A1, leading to the collective motions of the helices G and B' as well as the breaking of hydrogen bond existing between the 3ß-OH group of abiraterone and N202 of CYP17A1. However, the unfilled volume in the active site cavity can be occupied by the benzimidazole ring of TOK-001, restraining the motion of TOK-001. By pulling the two inhibitors (abiraterone and TOK-001) out of the binding pocket in CYP17A1, we discovered that abiraterone and TOK-001 were moved from their binding sites to the surface of protein similarly through the channels formed by the helices G and B'. In addition, based on the free energy calculations, one can see that it is energetically favorable for the two inhibitors (abiraterone and TOK-001) to enter into the binding pocket in CYP17A1.

Alterations in the steroid biosynthetic pathways in the human prefrontal cortex in mood disorders: A post-mortem study.

Altered levels of steroids have been reported in the brain, cerebral spinal fluid and plasma of patients with mood disorders. Neuroimaging studies have reported both functional and structural alterations in mood disorders, for instance in the anterior cingulate cortex (ACC) and dorsolateral prefrontal cortex (DLPFC). In order to determine whether the endogenous production of steroids is altered in the ACC and DLPFC of patients with major depressive disorder (MDD) or bipolar disorder (BPD), quantitative real-time PCR was performed to detect mRNA expression level of key enzymes in the steroid biosynthetic pathways. In MDD, a significant decrease in mRNA level of cytochrome P450 17A1 (CYP17A1, synthesizing C19 ketosteroids) in the ACC and a significant increase in mRNA levels of hydroxysteroid sulfotransferase 2A1 [SULT2A1, catalyzing the sulfate conjugation of dehydroepiandrosterone (DHEA)] were observed in the DLPFC, suggesting alterations in DHEA and its sulfate metabolite DHEAS levels. Decreased intensity and distribution of CYP17A1 immunohistochemical staining was found in the ACC of MDD patients. Interestingly, there was a significant positive correlation between the mRNA levels of CYP17A1 and tyrosine-related kinase B (TrkB) full length isoform. In a unique post-mortem human brain slice culture paradigm, BDNF mRNA expression was found to be significantly increased following incubation with DHEA. Together, these data indicate a close relationship between DHEA and BDNF-TrkB pathways in depression. Furthermore, in the DLPFC, higher mRNA levels of 11ß-hydroxysteroid dehydrogenase-1 (HSD11B1, reducing cortisone to the active hormone cortisol) and steroidogenic acute regulatory protein (STAR, facilitating the shuttle of cholesterol through the intermembrane space) were found in the MDD patients and BPD patients, respectively. In conclusion, this study suggests the presence of a disturbance in the endogenous synthesis of DHEA and DHEAS in mood disorders, which has a close relationship with BDNF-TrkB signaling.

Phase 1b study of orteronel in postmenopausal women with hormone-receptor positive (HR+) metastatic breast cancer.

Introduction Suppressing both androgens and estrogens may circumvent hormone receptor resistance in breast cancer by reducing androgen receptor stimulation. Selective inhibition of the 17, 20-lyase enzyme by orteronel leads to decreased androgen production in men and would be anticipated to reduce estrogen and androgen production in women. Thus, we conducted a phase 1b study of orteronel in postmenopausal women with hormone-receptor positive (HR+) metastatic breast cancer. Methods The primary objective was to identify the recommended phase 2 dose (R2PD) of orteronel in women; escalation was via standard 3 + 3 design. The initial dose was 300 mg BID and escalated to 400 mg BID. Cycle length was 28 days. Enrolled patients had HR+ metastatic breast cancer and were evaluated every 8 weeks for disease progression. Results Eight heavily pre-treated women enrolled [median age: 57 yo (range 47-73)]. Four received 300 mg BID at dose level 1; 4 received 400 mg BID at dose level 2. No dose limiting toxicities (DLTs) were observed. Adverse events (AE) at least possibly related to orteronel included grade 1-2 nausea (n = 4) and bone pain (n = 3), and grade 1 hypokalemia, hot flashes, myalgia and AST elevation (n = 2). The only grade 3 AE was hypertension (n = 2) with 8 patients receiving 34 cycles of treatment. No objective responses were seen; clinical benefit was seen in 2 patients with stable disease for more than 6 months. Serum estrogens and testosterone were suppressed from baseline on both doses of orteronel. Conclusions Orteronel 400 mg BID is well tolerated in postmenopausal women, and significantly suppresses serum estrogens and testosterone. Clinical benefit was seen among heavily pretreated postmenopausal women with HR+ metastatic breast cancer.

Structural insights into the function of steroidogenic cytochrome P450 17A1.

Cytochrome P450 17A1 (CYP17A1) operates at the core of human steroidogenesis, directing precursors into mineralocorticoids, glucocorticoids, or sex steroids. Although the 17α-hydroxylase and 17,20-lyase activities of this dual function enzyme have been investigated extensively, until recently no CYP17A1 structures were available to inform our understanding. Structures of CYP17A1 with a range of steroidal inhibitors and substrates are now available. This review relates functional knowledge of this enzyme to structural features defining the selective differentiation between its various substrates. While both hydroxylase and lyase substrates have similar orientations with respect to the heme, subtle differences in hydrogen bonding between CYP17A1 and the C3 substituent at the opposite end of ligands appear to correlate with differential substrate utilization and product formation. Complementary structural information from solution NMR supports cytochrome b5 allosteric modulation of the lyase reaction, implicating regions involved in ligand access to the otherwise buried active site.

[Interaction of novel oxazoline derivatives of 17(20)e-pregna-5,17(20)-diene with cytochrome P450 17A1].

In order to find novel inhibitors of 17a-hydroxylase-17,20-lyase (cytochrome P450 17A1, CYP17A1), a key enzyme of biosynthesis of androgens, molecular docking of six new oxazoline-containing derivatives 17(20)E-pregna-5,17(20)-diene has been carried out to the active site of the crystal structure of CYP17A1 (pdb 3ruk). Results of this study indicate that: 1) complex formation of docked compounds with CYP17A1 causes their isomerization in energetically less favorable 17(20)Z-isomer; 2) the localization of the steroid moiety of all compounds in the active site is basically the same; 3) the structure of the oxazoline moiety significantly influences its position relative to heme as well as the energy of complex formation; 4) coordination of the nitrogen atom of the oxazoline moiety and the heme iron is only possible in the 17(20)Z-conformation with anti oriented double bonds 17(20), and C=N; 5) the presence of two substituents at C4' of the oxazoline moiety significantly impairs ligand binding; 6) oxazoline--and benzoxazole-containing derivatives 17(20)E-pregna-5,17(20)-diene can effectively inhibit the catalytic activity CYP17A1 and may be of interest as a basis for the development of new drugs for the treatment of androgen-dependent cancer.

A Single Nucleotide Polymorphism near the CYP17A1 Gene Is Associated with Left Ventricular Mass in Hypertensive Patients under Pharmacotherapy.

Cytochrome P450 17A1 (CYP17A1) catalyses the formation and metabolism of steroid hormones. They are involved in blood pressure (BP) regulation and in the pathogenesis of left ventricular hypertrophy. Therefore, altered function of CYP17A1 due to genetic variants may influence BP and left ventricular mass. Notably, genome wide association studies supported the role of this enzyme in BP control. Against this background, we investigated associations between single nucleotide polymorphisms (SNPs) in or nearby the CYP17A1 gene with BP and left ventricular mass in patients with arterial hypertension and associated cardiovascular organ damage treated according to guidelines. Patients (n = 1007, mean age 58.0 ± 9.8 years, 83% men) with arterial hypertension and cardiac left ventricular ejection fraction (LVEF) ≥ 40% were enrolled in the study. Cardiac parameters of left ventricular mass, geometry and function were determined by echocardiography. The cohort comprised patients with coronary heart disease (n = 823; 81.7%) and myocardial infarction (n = 545; 54.1%) with a mean LVEF of 59.9% ± 9.3%. The mean left ventricular mass index (LVMI) was 52.1 ± 21.2 g/m2.7 and 485 (48.2%) patients had left ventricular hypertrophy. There was no significant association of any investigated SNP (rs619824, rs743572, rs1004467, rs11191548, rs17115100) with mean 24 h systolic or diastolic BP. However, carriers of the rs11191548 C allele demonstrated a 7% increase in LVMI (95% CI: 1%-12%, p = 0.017) compared to non-carriers. The CYP17A1 polymorphism rs11191548 demonstrated a significant association with LVMI in patients with arterial hypertension and preserved LVEF. Thus, CYP17A1 may contribute to cardiac hypertrophy in this clinical condition.

Substrate-modulated cytochrome P450 17A1 and cytochrome b5 interactions revealed by NMR.

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site.