Osilodrostat (LCI699)是人体类固醇合成细胞色素P450 11β-羟化酶（CYP11B1）和醛固酮合酶（CYP11B2）的强效抑制剂。LCI699已被FDA批准用于治疗库欣综合症，该症以皮质醇慢性过度产生为特征。虽然二期和三期临床研究已经证实了LCI699治疗库欣综合症的临床疗效和耐受性，但少有研究尝试全面评估LCI699对肾上腺甾体激素合成的影响。为此，我们首先全面分析了LCI699介导的皮质醇细胞株NCI-H295R的甾体合成抑制。然后，我们使用稳定表达单个人类类固醇生成P450酶的HEK-293或V79细胞研究LCI699抑制。我们使用完整细胞的研究证实了CYP11B1和CYP11B2的强烈抑制作用，对17-羟基化酶/17,20-裂解酶（CYP17A1）和21-羟基化酶（CYP21A2）的抑制微不足道。此外，部分抑制了胆固醇侧链裂解酶（CYP11A1）。为了计算LCI699与肾上腺线粒体P450酶的解离常数（Kd），我们成功地将P450纳入到脂质纳米盘中，并进行了光谱平衡和竞争结合实验。我们的结合实验证实了LCI699与CYP11B1和CYP11B2的高亲和力（Kd ≈ 1nM或更低）以及对CYP11A1的较弱结合（Kd = 18.8μM）。我们的结果证实了LCI699对CYP11B1和CYP11B2的选择性，并证明了对CYP11A1的部分抑制，但不是对CYP17A1和CYP21A2的抑制。版权所有©2023 Elsevier Ltd. 发布。

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 (Kd) 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 (Kd ≈ 1nM or less) and much weaker binding for CYP11A1 (Kd = 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.Copyright © 2023. Published by Elsevier Ltd.