Development of CYP11B1 and CYP11B2 assays utilizing homogenates of adrenal glands: Utility of monkey as a surrogate for human
Graphical abstract
Introduction
The mineralocorticoid, aldosterone, exerts its actions in part through the intracellular mineralocorticoid receptor (MR) found in the distal tubules of the kidney nephron thereby governing sodium and water retention as well as excretion of potassium. These actions serve to regulate blood volume and pressure. In addition to the MR-mediated (i.e. genomic) effects of aldosterone, other non-MR-mediated, “non-genomic” effects have been described which are also thought to contribute to various disease etiologies [1], [2], [3], [4]. Regardless of the exact mechanism(s) involved, elevated levels of aldosterone are associated with arterial hypertension, congestive heart failure, chronic kidney disease, and obesity.
The therapeutic benefits of blocking aldosterone binding to MR have been well characterized in large clinical trials with the MR antagonists (MRAs), spironolactone and eplerenone [5], [6], [7]. Additionally, modulation of the renin-angiotensin-aldosterone system (RAAS) with angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) also provides therapeutic benefit to patients with hypertension and cardiac failure. However, patients on ACEs or ARBs often experience increases in plasma aldosterone, known as “aldosterone breakthrough” [8], [9]. Patients who experience aldosterone breakthrough have also been reported to have a worse clinical prognosis. In addition to its hypertensive effects, aldosterone has been shown to be involved in both cardiac and renal fibrosis [10], [11]. Therefore, an aldosterone synthase inhibitor may offer benefits over MRAs for the treatment of hypertension, congestive heart failure, and chronic kidney disease [12], [13], [14] when used in combination with ACEs and ARBs via reducing both the genomic and non-genomic effects of aldosterone.
Aldosterone is produced by the CYP11B2, predominantly in the zona glomerulosa of the cortex of the adrenal gland. CYP11B2 is a mitochondrial cytochrome P450 enzyme which utilizes molecular oxygen and electrons obtained from NADPH via adrenodoxin reductase (AdR) [15] and adrenodoxin (Adx) [16] to catalyze a three-step sequence of oxidations converting 11-deoxycorticosterone (11-DOC) to aldosterone via the intermediates corticosterone and 18-hydroxycorticosterone (Scheme 1A) [17].
Selective inhibition of CYP11B2, and the associated cascade of reactions leading to aldosterone, is not without its challenges. Several factors make the development of selective and clinically-relevant CYP11B2 inhibitors quite demanding. Firstly, there is a high degree of amino acid sequence similarity between CYP11B2 and CYP11B1 (93%), the enzyme responsible for production of cortisol from 11-deoxycortisol (11-DC), Scheme 1B [18]. Lack of selectivity against CYP11B1 and the concomitant decrease in the ability to produce cortisol is associated with an impaired stress response. This lack of selectivity is likely to blame for the failure of LCI699 as an aldosterone synthase inhibitor [19]. Secondly, significant species differences exist for members of the CYP11B family [18]. The similarity of gene sequences, the number of isoforms expressed in a given species, and the substrates utilized may differ between species. These differences have provided significant challenges to the selection of in vitro and in vivo systems to assess the potency and selectivity of inhibitors. In addition to selectivity for CYP11B2 versus CYP11B1, aldosterone synthase inhibitors should not inhibit other cytochrome P450 enzymes involved in the metabolism of endogenous substances or xenobiotics. Inhibition of steroid-synthesizing P450 enzymes (e.g. CYP11A1, CYP21A2, CYP17A1, and CYP19A1) may affect biosynthesis of mineralocorticoids, glucocorticoids, androgens, and estrogens thus resulting in unintended pharmacological or toxicological consequences. Inhibition of other drug-metabolizing P450s may lead to undesirable drug–drug interactions.
A variety of inhibitor screening assays for both CYP11B1 and CYP11B2 have been published. These include human adenocarcinoma cell systems [20], S. pombe expressing CYP11B isoenzymes [21], CYP11B transfected V79 cell systems [22], [23], [24], V79 cells triply transfected with AdR, Adx, and either CYP11B1 or CYP11B2 [25], CYP11B transfected G402 cells [26], and tissue-derived systems [27], [28]. Product ratios and enzyme activities previously have been shown to be affected by the P450:AdR:Adx ratio, post-translational modifications (e.g. phosphorylation) of P450 and/or their redox partners, the availability of heme, the phospholipid environment, or the allosteric influence of other proteins (e.g. calmodulin and other P450 enzymes) [29]. For these reasons we turned our attention to the more physiologically-relevant tissue-derived assay systems. Being that these assays utilize adrenal tissue abrogates the above stated concerns. Since the availability of high quality human tissue was anticipated to be challenging, and as monkey CYP11B enzymes have been shown to be highly similar to the human enzymes [18], the feasibility of tissue derived from cynomolgus monkey was explored as a suitable surrogate for human tissue.
Based on the work of Rigel et al. [27], cynomolgus monkey adrenal homogenate (CAH) and human adrenal homogenate (HAH) were prepared, and assays for both CYP11B1 and CYP11B2 were developed. Using CAH and HAH we have characterized the kinetics of the CYP11B1-mediated conversion of 11-DC to cortisol and the CYP11B2-mediated oxidation of corticosterone to aldosterone. Inhibition assays for CYP11B1 and CYP11B2 were subsequently developed.
A comparison of amino acid sequences for the CYP11B enzymes, kinetic data, and inhibition values derived for a set of 16 compounds utilizing the CAH and HAH assays indicate that both CYP11B1 and CYP11B2 exhibit a high degree of similarity between monkey and human. Therefore, inhibition assays for CYP11B1 and CYP11B2 derived from cynomolgus monkey adrenal glands are suitable surrogates for the human enzymes.
Section snippets
Materials
11-DOC, corticosterone, 11-DC, cortisol 18-hydroxycorticosterone, aldosterone, dimethylsulfoxide (DMSO), acetonitrile (ACN), and nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma–Aldrich (St. Louis, MO). All inhibitors used were from the Boehringer Ingelheim compound collection (Ridgefield, CT). Structures for compounds designated Saarland-1 [30], Novartis-1 [31], and Novartis-2 [32] are presented in Supplemental Fig. 1. Buffer salts, HPLC solvents, and all other
AH preparation
Preparation of CAH was similar to that described for the preparation of rat adrenal homogenate by Rigel et al. [27]. Whole adrenal glands were thawed on ice, weighed, minced with scissors, and homogenized in ice-cold homogenization buffer (8.5 mM MgCl2, 3.13 mM KCl, 7.59 mM NaCl, 2.7 mM CaCl2, 50 mM Tris/HCl, pH 7.4, and one Complete EDTA-free Protease Inhibitor Cocktail tablet (Roche Applied Science, Indianapolis, IN) per 50 mL buffer) using 5 mL of homogenization buffer per gram of tissue. The
Results and discussion
Many assay options exist to assess the inhibitory effects of compounds on CYP11B enzyme activities. These assays range from contrived cell assays to more physiologically-relevant human adenocarcinoma cell systems such as H295R cells. The latter system is hampered by slow cell growth [34], greater complexity [34], [35], [36], [37], [38], low steroid production rates [20] and, therefore, long incubation times. Low aldosterone production in H295R cells has been overcome by generating H295R cells
Acknowledgement
Melissa Kramer, Steve Weldon, and Nick Brown are gratefully acknowledged for their thoughtful review of this manuscript.
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