Selectivity of osilodrostat as an inhibitor of human steroidogenic cytochromes P450

Abstract

Osilodrostat (LCI699) is a potent inhibitor of the human steroidogenic cytochromes P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). LCI699 is FDA-approved for the treatment of Cushing disease, which is characterized by chronic overproduction of cortisol. While phase II and III clinical studies have proven the clinical efficacy and tolerability of LCI699 for treating Cushing disease, few studies have attempted to fully assess the effects of LCI699 on adrenal steroidogenesis. To this end, we first comprehensively analyzed LCI699-mediated inhibition of steroid synthesis in the NCI-H295R human adrenocortical cancer cell line. We then studied LCI699 inhibition using HEK-293 or V79 cells stably expressing individual human steroidogenic P450 enzymes. Our studies using intact cells confirm the potent inhibition of CYP11B1 and CYP11B2 with negligible inhibition of 17-hydroxylase/17,20-lyase (CYP17A1) and 21-hydroxylase (CYP21A2). Furthermore, partial inhibition of the cholesterol side-chain cleavage enzyme (CYP11A1) was observed. To calculate the dissociation constant (K_d) of LCI699 with the adrenal mitochondrial P450 enzymes, we successfully incorporated P450s into lipid nanodiscs and carried out spectrophotometric equilibrium and competition binding assays. Our binding experiments confirm the high affinity of LCI699 to CYP11B1 and CYP11B2 (K_d ≈ 1 nM or less) and much weaker binding for CYP11A1 (K_d = 18.8 μM). Our results confirm the selectivity of LCI699 for CYP11B1 and CYP11B2 and demonstrate partial inhibition of CYP11A1 but not CYP17A1 and CYP21A2.

Keywords: Cushing syndrome; Cytochrome P450; LCI699; Osilodrostat; Steroidogenesis.

Fig. 1.. Overview of Studied Adrenal Steroidogenic Pathways.

Different colors are used to highlight the involvement of individual enzymes in multiple reactions. Abbreviations: B, corticosterone; 18OHB, 18-hydroxycorticosterone; DHEA, dehydroepiandrosterone; 17Preg, 17-hydroxypregnenolone; 17OHP, 17-hydroxyprogesterone; A4, androstenedione; 3βHSD2, 3β-hydroxysteroid dehydrogenase type 2; other enzyme abbreviations as in the text.

Fig. 2.. Inhibition of steroid production in NCI-H295R cells.

After forskolin stimulation, H295R cells were treated with 0–1000 nM LCI699, 1000 nM ketoconazole (Keto.), 1000 nM metyrapone (Mety.), or vehicle in a fresh serum-free medium. Steroid levels measured by LC-MS/MS are given as the mean ± standard error of the mean (SEM) of three individual experiments.

Fig. 3.. Inhibition of CYP11A1, CYP11B1, and CYP11B2 activity in V79 cells.

Cells were plated and incubated in a medium containing 1 μM 22-R-hydroxycholesterol, 11-deoxycortisol, and corticosterone for CYP11A1(♦), CYP11B1(•), and CYP11B2(■) expressing cells, respectively, and 0–1000 nM LCI699. Data were fit to the dose-response inhibitor (three-parameter) equation to determine the half-maximal inhibitory concentration (IC₅₀) of 9.5 ± 0.5 nM and 0.28 ± 0.06 nM for CYP11B1 and CYP11B2, respectively. Data represent the mean ± standard error of the mean (SEM) of three individual experiments.

Fig. 4.. Equilibrium binding titration of LCI699 to P450-incorporated nanodiscs.

(A) CYP11A1(♦), (B) CYP11B1(●), and (C) CYP11B2(■) nanodiscs at 0.16 μM were equilibrated with increasing concentrations of LCI699 in 50 mM potassium phosphate buffer (pH 7.4). (D) Calculated equilibrium dissociation constants (K_d). The inserted image in each panel shows the difference spectra between samples with and without LCI699. Data represent the mean ± standard deviation of three individual experiments.

Fig. 5.. CYP11B1-nanodiscs competitive binding assay.

(A) UV-visible spectral changes of 0.16μM CYP11B1-nanodiscs in 50 mM potassium phosphate buffer (pH 7.4). The addition of 25 μM DOC causes the absorption maximum of the Soret band to shift from 417 nm (dark purple) to 390 nm (blue). Subsequently, with increasing concentrations of LCI699, the maximum of the Soret band shifts to 419 nm (orange). (B) Dose-response curve of CYP11B1-nanodisc. Increasing concentrations of LCI699 can outcompete the bound DOC. Data were fit to the dose-response inhibitor (four-parameter) equation to determine the half-maximal inhibitory concentration (IC₅₀) of 90 ± 3 nM. Data represent the mean ± standard deviation of three individual experiments.

Fig. 6.. CYP11B2 competition binding assay.

(A) UV-visible spectral changes of 0.16 μM CYP11B2-nanodiscs in 50 mM potassium phosphate buffer (pH 7.4). The addition of 0.5 μM DOC causes the absorption maximum of the Soret band to shift from 417 nm (dark purple) to 390 nm (blue). Subsequently, increasing the concentration of LCI699 up to 10 μM shifts the maximum of the Soret band to 414 nm (cyan). (B) Dose-response curve of CYP11B1-nanodisc with (▲) and without adrenodoxin (■). LCI699 cannot outcompete the bound DOC in either condition. Data represent the mean ± standard deviation of three individual experiments.

Fig. 7.. LCI699-mediated inhibition of CY11B2-nanodiscs.

Corticosterone (A), 18-hydroxycorticosterone (B), and aldosterone (C) were quantified with LC-MS/MS. CYP11B2-nanodiscs (0.16 μM) were pre-incubated with either DOC (0.5 μM) or LCI699 (10 μM) in 50 mM potassium phosphate buffer (pH 7.4) before the addition of either NADPH (first bar), LCI699 or DOC in the order specified, then NADPH (second and third bars), or no NADPH control (fourth bar). Data represent the mean ± standard deviation of three individual experiments.