Human CYP11B1 Stable Cell Line - V79

Osilodrostat (LCI699) is a potent inhibitor targeting human steroidogenic cytochrome P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). LCI699 has been approved by the FDA for the treatment of Cushing disease, a condition characterized by the chronic overproduction of cortisol. Although Phase II and III clinical studies have confirmed the clinical efficacy and tolerability of LCI699 in treating Cushing disease, few studies have attempted to comprehensively evaluate the effects of LCI699 on the process of adrenal steroidogenesis. Here, researchers first conducted a comprehensive analysis of the LCI699-mediated inhibition of steroid synthesis within the NCI-H295R human adrenocortical carcinoma cell line. Subsequently, the inhibitory effects of LCI699 were investigated using HEK-293 or V79 cells stably expressing specific human steroidogenic P450 enzymes. Results from these whole-cell studies confirmed that LCI699 exerts potent inhibitory effects on CYP11B1 and CYP11B2, while demonstrating negligible inhibition of 17α-hydroxylase/17,20-lyase (CYP17A1) and 21-hydroxylase (CYP21A2). Furthermore, LCI699 was found to exert partial inhibitory effects on cholesterol side-chain cleavage enzyme (CYP11A1). To determine the dissociation constants (Kd) between LCI699 and the adrenal mitochondrial P450 enzymes, the P450 enzymes were successfully reconstituted into lipid nanodiscs, and spectrophotometric equilibrium binding and competitive binding assays were performed. The binding assay results confirmed that LCI699 possesses extremely high affinity for CYP11B1 and CYP11B2, whereas its binding affinity for CYP11A1 is significantly weaker. These results not only confirm the selectivity of LCI699 for CYP11B1 and CYP11B2, but also indicate that it exerts partial inhibitory effects on CYP11A1, while exhibiting no inhibitory activity against CYP17A1 and CYP21A2.

Here, researchers established HEK-293 and V79 (Chinese hamster) cell lines that stably express specific steroidogenic P450 enzymes. V79 cells are frequently utilized for studying mitochondrial P450 enzymes because they inherently lack endogenous P450 activity and do not require co-transfection of redox partners (i.e., Adx and its reductase). In contrast, HEK-293 cells are widely employed to investigate drug and steroid metabolism processes mediated by microsomal cytochrome P450 enzymes. In CYP11B1-expressing V79 cells, LCI699 inhibited the conversion of 11-deoxycortisol to cortisol, with an IC50 value of 9.5 ± 0.5 nM (Figure 1). By comparison, in CYP11B2-expressing V79 cells, LCI699 inhibited the conversion of corticosterone to aldosterone, with an IC50 value of 0.28 ± 0.06 nM. In V79 cells expressing CYP11A1, LCI699 demonstrated partial inhibition of pregnenolone production at a concentration of 1000 nM. Conversely, in HEK-293 cells stably expressing CYP17A1 or CYP21A2, the inhibitory effect on enzymatic activity remained negligible, even at LCI699 concentrations as high as 1000 nM. These data confirm that LCI699 potently inhibits CYP11B1 and CYP11B2, exerts virtually no inhibitory effect on CYP17A1 and CYP21A2, but demonstrates partial inhibition of CYP11A1 at high concentrations.

Figure 1. Inhibition of CYP11A1, CYP11B1, and CYP11B2 activity in V79 cells. (Valentín-Goyco J, et al., 2023)