Inhibition and stimulation of activity of purified recombinant CYP11A1 by therapeutic agents

doi:10.1016/j.mce.2012.10.013

Molecular and Cellular Endocrinology

Volume 371, Issues 1–2, 22 May 2013, Pages 100-106

https://doi.org/10.1016/j.mce.2012.10.013 Get rights and content

Abstract

In vertebrates, the biosynthesis of steroid hormones is initiated by cytochrome P450 CYP11A1 which converts cholesterol to pregnenolone. We investigated whether some of the experimental and FDA-approved therapeutic agents alter the activity of CYP11A1 in the reconstituted system in vitro. We found that under the experimental conditions used and when phospholipids are included, ketoconazole, posaconazole, carbenoxolone, and selegiline inhibit CYP11A1-mediated production of pregnenolone by at least 67%. Conversely, pemirolast, clobenpropit, desogestrel, dexmedetomidine, and tizanidine stimulate the enzyme activity by up to 70%. We then evaluated the identified inhibitors and activators for spectral binding to CYP11A1 and their effect on enzyme activity in the absence of phospholipids. The data obtained provide insight into how different drugs interact with CYP11A1 and demonstrate that P450 association with the lipid bilayer determines, in many cases, a drug’s effect on enzyme activity.

Highlights

► Structurally distinct pharmaceuticals were found to alter the activity of CYP11A1. ► The effect on CYP11A1 activity was not only inhibitory but also stimulatory. ► CYP11A1-membrane interactions contribute to a drug’s effect on enzyme activity.

Introduction

Cytochrome P450 11A1 (CYP11A1) or cholesterol side chain cleavage enzyme, is a mitochondrial monooxygenase catalyzing the conversion of cholesterol to pregnenolone, the precursor of all steroid hormones. CYP11A1 has been intensively studied during the last 40 years (reviewed in Guengerich, 2005, Pikuleva, 2006, Miller et al., 2011), yet little is currently known about the effect of marketed drugs on activity of CYP11A1, both in vivo and in vitro. Our knowledge in this area is mainly limited to earlier studies in the field showing that CYP11A1, as well as some other steroidogenic P450s, are inhibited by the antifungal agent ketoconazole and anticonvulsant aminoglutethimide (withdrawn from the US market in 1986), whose administration leads to adrenocortical dysfunction (Bossche, 1992, Harvey et al., 2003). Recently, we determined the crystal structure of CYP11A1 (Mast et al., 2011) and compared it to the structure of CYP46A1, another P450 which utilizes cholesterol as the endogenous substrate. CYP46A1, or cholesterol 24-hydroxylase, is a microsomal enzyme expressed predominantly in the brain whether it initiates the major pathway of cholesterol removal (Lutjohann et al., 1996, Russell et al., 2009). We found that in CYPs 11A1 and 46A1, enzymes that share <25% of amino acid sequence identity, the shape of the active site is similar, a long curved tube, as is the positioning of cholesterol (Mast et al., 2011). The major difference is that the active site in CYP11A1 is longer and more narrow, providing an explanation for the more strict substrate specificity of CYP11A1 as compared to that of CYP46A1 which may bind compounds structurally unrelated to cholesterol (Mast et al., 2003, Mast et al., 2008, Mast et al., 2010). Similar architecture of the active sites in CYP11A1 and CYP46A1 gave impetus to the present work in which we investigated whether the pharmaceuticals that modulate CYP46A1 activity in vitro, also affect the activity of purified recombinant CYP11A1. We identified several strong CYP11A1 inhibitors, and unexpectedly found that CYP11A1 activity could also be stimulated. The latter is a novel finding which enhances our understanding of CYP11A1 and opens new directions in studies of this enzyme as a target for therapeutic agents.

Section snippets

Materials

Pharmaceuticals for screening were purchased from one of the following sources: Sigma–Aldrich Co (St. Louis, MO), Cayman Chemical Company (Ann Arbor, MI), Alfa Aesar (Ward Hill, MA), Waterstone Technology LLC (Carmel, IN), and Toronto Research Chemicals Inc. (North York, Ontario, Canada). Cholesterol and [³H]cholesterol were from Steraloids Inc (Newport, RI) and American Radiolabeled Chemicals, Inc (St. Louis, MO), respectively. All other chemicals were from Sigma–Aldrich unless otherwise

Effect of CYP46A1 inhibitors on activity of CYP11A1

Only compounds inhibiting CYP46A1 activity by more than 45% in our previous studies (Mast et al., 2012) were tested in this CYP11A1 enzyme assay. More than half of these pharmaceuticals did not significantly affect CYP11A1 activity under the experimental conditions used (Fig. 1, Supplementary Table 1). Yet, ketoconazole, posaconazole, carbenoxolone and selegeline decreased pregnenolone production by >65%, whereas clobenpropit and dexmedetomidine increased CYP11A1-mediated cholesterol metabolism

Discussion

The present work capitalized on our previous structural studies of CYP46A1 and CYP11A1 (Mast et al., 2008, Mast et al., 2010, Mast et al., 2011) and our recent screening of the experimental or approved by the FDA drugs for the inhibitory effect on CYP46A1 in vitro (Mast et al., 2012). We established that the shape of the active site is similar in the two enzymes and hypothesized that some of the compounds that inhibit CYP46A1 in vitro may also inhibit CYP11A1. We also identified pharmaceuticals

Acknowledgements

This study was supported by United Public Health Service Grant GM62882. I.A.P. is a recipient of the Jules and Doris Stein Professorship from the Research to Prevent Blindness.

References (31)

A. Berwanger et al.
Polyamines: naturally occurring small molecule modulators of electrostatic protein–protein interactions
J. Inorg. Biochem.
(2010)
H.V. Bossche
Inhibitors of P450-dependent steroid biosynthesis: from research to medical treatment
J. Steroid Biochem. Molec. Biol.
(1992)
P.L. Gigon et al.
Effect of drug substrates on the reduction of hepatic microsomal cytochrome P-450 by NADPH
Biochem. Biophys. Res. Commun.
(1968)
G.Y. Heo et al.
Features of the retinal environment which affect the activities and product profile of cholesterol-metabolizing cytochromes P450 CYP27A1 and CYP11A1
Arch. Biochem. Biophys.
(2012)
T. Kido et al.
Adrenal cortex mitochondrial cytochrome P-450 specific to cholesterol side chain cleavage reaction. Spectral changes induced by detergents, alcohols, amines, phospholipids, steroid hydroxylase inhibitors, and steroid substrates, and conditions for adrenodoxin binding to the cytochrome
J. Biol. Chem.
(1979)
J.D. Lambeth et al.
Phosphatidylcholine vesicle reconstituted cytochrome P-450scc. Role of the membrane in control of activity and spin state of the cytochrome
J. Biol. Chem.
(1980)
N. Mast et al.
Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1
J. Biol. Chem.
(2011)
N. Mast et al.
Structural basis of drug binding to CYP46A1, an enzyme that controls cholesterol turnover in the brain
J. Biol. Chem.
(2010)
W.L. Miller et al.
Early steps in steroidogenesis: intracellular cholesterol trafficking
J. Lipid Res.
(2011)
I.A. Pikuleva
Cytochrome P450s and cholesterol homeostasis
Pharmacol. Ther.
(2006)

I.A. Pikuleva et al.

Expression, purification, and enzymatic properties of recombinant human cytochrome P450c27 (CYP27)

Arch. Biochem. Biophys.

(1997)

I.A. Pikuleva et al.

Active-site topology of bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) and evidence for interaction of tyrosine 94 with the side chain of cholesterol

Arch. Biochem. Biophys.

(1995)

J. Pitha et al.

Drug solubilizers to aid pharmacologists: amorphous cyclodextrin derivatives

Life Sci.

(1988)

Y. Sagara et al.

Direct expression in Escherichia coli and characterization of bovine adrenodoxins with modified amino-terminal regions

FEBS Lett.

(1992)

D.W. Seybert et al.

Participation of the membrane in the side chain cleavage of cholesterol. Reconstitution of cytochrome P-450scc into phospholipid vesicles

J. Biol. Chem.

(1979)

Cited by (20)

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive
2024, Journal of Biological Chemistry
Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450_scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)₂ cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)₂ cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and k_off rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)₂ cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d₃-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.
CYP11A1 silencing suppresses HMGCR expression via cholesterol accumulation and sensitizes CRPC cell line DU-145 to atorvastatin
2023, Journal of Pharmacological Sciences
Statins, which are cholesterol synthesis inhibitors, are well-known therapeutics for dyslipidemia; however, some studies have anticipated their use as anticancer agents. However, epithelial cancer cells show strong resistance to statins through an increased expression of HMG-CoA reductase (HMGCR), an inhibitory target of statins. Castration-resistant prostate cancer (CRPC) cells synthesize androgens from cholesterol on their own. We performed suppression of CYP11A1, a rate-limiting enzyme in androgen synthesis from cholesterol, using siRNA or inhibitors, to examine the effect of steroidogenesis inhibition on statin sensitivity in CRPC cells. Here, we suggested that CYP11A1 silencing sensitized the statin-resistant CRPC cell line DU-145 to atorvastatin via HMGCR downregulation by an increase in intracellular free cholesterol. We further demonstrated that CYP11A1 silencing induced epithelial-mesenchymal transition, which converted DU-145 cells into a statin-sensitive phenotype. This suggests that concomitant use of CYP11A1 inhibitors could be an effective approach for overcoming statin resistance in CRPC. Moreover, we showed that ketoconazole, a CYP11A1 inhibitor, sensitized DU-145 cells to atorvastatin, although not all the molecular events observed in CYP11A1 silencing were reproducible. Although further studies are necessary to clarify the detailed mechanisms, ketoconazole may be effective as a concomitant drug that potentiates the anticancer effect of atorvastatin.
Characterization of the interferences of systemic azole antifungal drugs with adrenal steroid biosynthesis using H295R cells and enzyme activity assays
2023, Current Research in Toxicology
Azole antifungals, designed to inhibit fungal CYP51, have a liability to inhibit human CYP enzymes. Whilst drug-metabolizing CYPs are covered in preclinical safety assessment, those metabolizing endogenous bioactive molecules are usually not. Posaconazole and itraconazole were recently found to cause pseudohyperaldosteronism with hypokalemia and hypertension by inhibiting CYP11B1-dependent adrenal cortisol biosynthesis. Because this was overlooked in preclinical safety assessment, the present study tested whether applying adrenal carcinoma H295R cells could have predicted this liability and whether other systemic triazole antifungals interfere with adrenal steroidogenesis. Forskolin-stimulated H295R cells were exposed to systemic triazole antifungals that are currently used, and key adrenal steroids were quantified by UHPLC-MS/MS. To support the findings from the H295R model, activity assays for steroidogenic enzymes were performed. The analysis of the steroid profiles and product/substrate ratios predicted the CYP11B1 and CYP11B2 inhibition by posaconazole and itraconazole. Comparison of their steroid profiles allowed distinguishing their effects and suggested inhibition of adrenal androgen synthesis by posaconazole but not itraconazole, which was confirmed by CYP17A1 17,20-lyase activity measurements. In line with clinical observations, there was no evidence from these experiments for an inhibition of either CYP11B1/2 or CYP17A1 by voriconazole, fluconazole or isavuconazole. However, itraconazole and isavuconazole exerted an overall inhibition of steroidogenesis by a mechanism warranting further investigations. In conclusion, analyses of steroid profiles from the H295R assay and product/substrate ratios provide important information on the interference of a chemical with adrenal steroidogenesis and the underlying mechanism. This approach facilitates prioritization of further investigations, including enzyme expression and activity studies.
The neurosteroid pregnenolone is synthesized by a mitochondrial P450 enzyme other than CYP11A1 in human glial cells
2022, Journal of Biological Chemistry
Citation Excerpt :
Our results did support the third possibility that another CYP450 enzyme with desmolase activity in the brain can be used to synthesize pregnenolone. Although CYP46A1 could be a candidate given its high expression in brain, involvement in cholesterol metabolism, and structural similarity to CYP11A1, KC inhibits CYP46A1 activity potentially more effectively than CYP11A1 activity (68). Our data showed that the CYP450(s) responsible for producing pregnenolone is(are) not inhibited by AMG and KC.
Neurosteroids, modulators of neuronal and glial cell functions, are synthesized in the nervous system from cholesterol. In peripheral steroidogenic tissues, cholesterol is converted to the major steroid precursor pregnenolone by the CYP11A1 enzyme. Although pregnenolone is one of the most abundant neurosteroids in the brain, expression of CYP11A1 is difficult to detect. We found that human glial cells produced pregnenolone, detectable by mass spectrometry and ELISA, despite the absence of observable immunoreactive CYP11A1 protein. Unlike testicular and adrenal cortical cells, pregnenolone production in glial cells was not inhibited by CYP11A1 inhibitors DL-aminoglutethimide and ketoconazole. Furthermore, addition of hydroxycholesterols increased pregnenolone synthesis, suggesting desmolase activity that was not blocked by DL-aminoglutethimide or ketoconazole. We explored three different possibilities for an alternative pathway for glial cell pregnenolone synthesis: (1) regulation by reactive oxygen species, (2) metabolism via a different CYP11A1 isoform, and (3) metabolism via another CYP450 enzyme. First, we found oxidants and antioxidants had no significant effects on pregnenolone synthesis, suggesting it is not regulated by reactive oxygen species. Second, overexpression of CYP11A1 isoform b did not alter synthesis, indicating use of another CYP11A1 isoform is unlikely. Finally, we show nitric oxide and iron chelators deferoxamine and deferiprone significantly inhibited pregnenolone production, indicating involvement of another CYP450 enzyme. Ultimately, knockdown of endoplasmic reticulum cofactor NADPH-cytochrome P450 reductase had no effect, while knockdown of mitochondrial CYP450 cofactor ferredoxin reductase inhibited pregnenolone production. These data suggest that pregnenolone is synthesized by a mitochondrial cytochrome P450 enzyme other than CYP11A1 in human glial cells.
Oxysterols profiles in zebrafish (Danio rerio) embryos exposed to bisphenol A
2022, Food and Chemical Toxicology
Citation Excerpt :
Furthermore, at 8 hpf only slight differences between the tested doses of each chemical of interest were observed. At 24 hpf, KCZ confirmed its inhibitory activity on the total amount of oxysterols, inhibition known to be mediated by CYP11A1 and CYP46A1 (Mast et al., 2013). On the contrary, as expected, the higher doses of CBZ and H2O2 showed an increase in total oxysterols (Hole et al., 2018; Chung et al., 2013).
Oxysterols are cholesterol oxidation products and bioactive lipids involved in developmental signalling pathways, embryonic and postembryonic tissue patterning and homeostasis. The embryonic period is a very sensitive window of exposure to bisphenol A (BPA), hence the role of BPA on the levels of oxysterols in the very early stages of zebrafish embryogenesis is a relevant novel field of investigation.
To compare the role of BPA on oxysterols levels in zebrafish embryos at 8 and 24 h post fertilization (hpf) with cytochromes P450 (CYPs)-modulating chemicals (carbamazepine, ketoconazole, and hydrogen peroxide).
Upon a dose range finding, zebrafish embryos were exposed to environmentally relevant (0.04 μM) and toxicological (17.5 μM) BPA concentrations. Seven oxysterols were profiled by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).
Similarly to the CYPs-modulating chemicals, BPA caused: i) no significant changes at 8 hpf and ii) a dose-dependent increase of total oxysterols at 24 hpf, with 27-hydroxycholesterol as the most regulated oxysterol.
In the first day post-fertilization of the zebrafish embryos, the role of BPA alike a CYPs-modulating chemical was confirmed by the similar oxysterol changes observed with the already known CYPs-modulating chemicals.
Neurosteroidogenic enzymes: CYP11A1 in the central nervous system
2021, Frontiers in Neuroendocrinology
Citation Excerpt :
The second hypothesis is that another cytochrome P450 other than CYP11A1 could be responsible for synthesizing pregnenolone in the CNS, as cytochrome P450 enzymes are known to metabolize a wide range of substrates. One potential enzyme is CYP46A1, which is highly expressed in the brain, uses cholesterol as a substrate, and has similar active site structure to CYP11A1 (Mast et al., 2013). The third hypothesis is that the second CYP11A1 isoform may be the main enzyme metabolizing cholesterol to pregnenolone in the CNS rather than the classical isoform.
Neurosteroids, steroid hormones synthesized locally in the nervous system, have important neuromodulatory and neuroprotective effects in the central nervous system. Progress in neurosteroid research has led to the successful translation of allopregnanolone into an approved therapy for postpartum depression. However, there is insufficient evidence to support the assumption that steroidogenesis is exactly the same between the nervous system and the periphery. This review focuses on CYP11A1, the only enzyme currently known to catalyze the first reaction in steroidogenesis to produce pregnenolone, the precursor to all other steroids. Although CYP11A1 mRNA has been found in brain of many mammals, the presence of CYP11A1 protein has been difficult to detect, particularly in humans. Here, we highlight the discrepancies in the current evidence for CYP11A1 in the central nervous system and propose new directions for understanding neurosteroidogenesis, which will be crucial for developing neurosteroid-based therapies for the future.

View all citing articles on Scopus

View full text

Inhibition and stimulation of activity of purified recombinant CYP11A1 by therapeutic agents

Abstract

Highlights

Introduction

Section snippets

Materials

Effect of CYP46A1 inhibitors on activity of CYP11A1

Discussion

Acknowledgements

J. Inorg. Biochem.

J. Steroid Biochem. Molec. Biol.

Biochem. Biophys. Res. Commun.

Arch. Biochem. Biophys.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Lipid Res.

Pharmacol. Ther.

Arch. Biochem. Biophys.

Arch. Biochem. Biophys.

Life Sci.

FEBS Lett.

J. Biol. Chem.