Mechanistic and Pharmacological Studies of 11 -hydroxylase (P450 11B1) and Aldosterone Synthase (P450 11B2)

Abstract

The cytochrome P450 enzymes, 11β-hydroxylase (P450 11B1), and aldosterone synthase (P450 11B2) carry out the final stages of cortisol and aldosterone synthesis in the adrenal glands. Cortisol, the primary glucocorticoid, plays a crucial role in regulating glucose homeostasis, stress, and immune responses. Excessive production of cortisol, however, leads to Cushing syndrome, which is characterized by a range of debilitating symptoms. These symptoms include hypertension, suppressed immunity, depression, and infertility, among others. Conversely, aldosterone regulates water, sodium, and potassium homeostasis. When aldosterone is produced in excess, it results in primary aldosteronism, the most prevalent form of secondary hypertension. The development of selective inhibitors for P450 11B1 and P450 11B2 has received significant attention due to their roles in endocrine disorders. Unfortunately, the high sequence identity of 93% between both homologous enzymes has hindered the development of such inhibitors, particularly for P450 11B2.

P450 11B2 catalyzes the production of aldosterone in three successive hydroxylation reactions: the 11β-hydroxylation of 11-deoxycorticosterone (DOC) to corticosterone, then the 18-hydroxylation to 18-hydroxycorticosterone, and last 18-oxidation to aldosterone. In contrast, P450 11B1 catalyzes the formation of cortisol from 11-deoxycortisol via a one-step 11β-hydroxylation reaction. Both enzymes can perform 11β-hydroxylation reactions on both 11-deoxycortisol and DOC; however, only P450 11B2 can produce aldosterone from the latter. While it has been shown that P450 11B2 carries out aldosterone synthesis through a processive mechanism, the factors that limit aldosterone production from 11B1 are still not well understood.

To address the disparities in the catalytic activities of P450 11B1 and P450 11B2, we performed two assessments. Firstly, we evaluated their inhibition by osilodrostat (LCI699), a newly approved medication for Cushing syndrome. Secondly, our goal was to identify the distinct biochemical properties of P450 11B2 that limit the 18-oxygenation activities and processivity of P450 11B1. To evaluate LCI699-mediated inhibition, we conducted an analysis of steroid synthesis in the NCI-H295R human adrenocortical cancer cell line. Subsequently, we investigated LCI699 inhibition in cells stably expressing individual human steroidogenic P450 enzymes, utilizing either HEK-293 or V79 cells. Lastly, we assessed the direct binding of LCI699 to purified enzymes incorporated into phospholipid nanodiscs. Our research has demonstrated that osilodrostat is a potent inhibitor of both P450 11B1 and P450 11B2. We also demonstrate partial inhibition of P450 11A1 and no inhibition of P450 17A1 and P450 21A2.

To understand the mechanism that enables and hinders aldosterone synthesis in P450 11B2 and P450 11B1, respectively, we introduced point mutations at residue 320, which partially exchanges the activities of both enzymes. 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 (V320A and A320V, respectively) in phospholipid vesicles and nanodiscs. Altogether, our findings suggest that coupling efficiencies do not directly correlate with an increase in 18-hydroxylase activity. In contrast, slow intermediate dissociation inversely correlates with 18-oxygenation activities in wild-type and mutant enzymes. Therefore, we conclude that slow intermediate dissociation kinetics, rather than high NADPH coupling efficiency, is the major property that enables P450 11B2 – but not P450 11B1 – to synthesize aldosterone via a processive mechanism.