Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology Stuttgart and University of Tübingen, Tübingen, Germany.
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Hamburg, Germany.
Arch Toxicol. 2017 Jun;91(6):2375-2390. doi: 10.1007/s00204-017-1948-3. Epub 2017 Mar 13.
Unintentional activation of xenosensing nuclear receptors pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR) by clinical drug use is known to produce severe side effects in patients, which may be overcome by co-administering antagonists. However, especially antagonizing CAR is hampered by the lack of specific inhibitors, which do not activate PXR. Recently, compounds based on a dibenzazepine carbamate scaffold were identified as potent CAR inhibitors. However, their potential to activate PXR was not thoroughly investigated, even if the lead compound was named "CAR inhibitor not PXR activator 1" (CINPA1). Thus, we performed a comprehensive analysis of the interaction of CINPA1 and four analogs with PXR. Cellular assays were used to investigate intra- and intermolecular interactions and transactivation activity of PXR as a function of the compounds. Modulation of PXR target gene expression was analyzed in primary human hepatocytes. Ligand binding to PXR was investigated by molecular docking and limited proteolytic digestion. We show here that CINPA1 induced the assembly of the PXR ligand-binding domain, released co-repressors from and recruited co-activators to the receptor. CINPA1 and its analogs induced the PXR-dependent activation of a CYP3A4 reporter gene and CINPA1 induced the expression of endogenous cytochrome P450 genes in primary hepatocytes, while not consistently inhibiting CAR-mediated induction. Molecular docking revealed favorable binding of CINPA1 and analogs to the PXR ligand-binding pocket, which was confirmed in vitro. Altogether, our data provide consistent evidence that compounds with a dibenzazepine carbamate scaffold, such as CINPA1 and its four analogs, bind to and activate PXR.
临床药物的使用会导致 xenosensing 核受体孕烷 X 受体 (PXR) 和/或组成型雄烷受体 (CAR) 意外激活，已知这会给患者带来严重的副作用，可通过联合使用拮抗剂来克服。然而，特别是拮抗 CAR 受到缺乏特异性抑制剂的阻碍，这些抑制剂不会激活 PXR。最近，基于二苯并氮杂䓬氨基甲酸酯支架的化合物被鉴定为有效的 CAR 抑制剂。然而，即使先导化合物被命名为“CAR 抑制剂而非 PXR 激活剂 1”（CINPA1），它们对 PXR 的潜在激活作用也没有得到彻底研究。因此，我们对 CINPA1 和四种类似物与 PXR 的相互作用进行了全面分析。细胞测定用于研究化合物作为功能的 PXR 内和分子间相互作用和反式激活活性。在原代人肝细胞中分析 PXR 靶基因表达的调节。通过分子对接和有限的蛋白水解消化研究配体与 PXR 的结合。我们在这里表明，CINPA1 诱导 PXR 配体结合域的组装，从受体上释放共阻遏物并募集共激活物。CINPA1 和其类似物诱导 CYP3A4 报告基因的 PXR 依赖性激活，并且在原代肝细胞中诱导内源性细胞色素 P450 基因的表达，而不持续抑制 CAR 介导的诱导。分子对接显示 CINPA1 和类似物与 PXR 配体结合口袋具有良好的结合，在体外得到了证实。总的来说，我们的数据提供了一致的证据，表明具有二苯并氮杂䓬氨基甲酸酯支架的化合物，如 CINPA1 和其四个类似物，与 PXR 结合并激活 PXR。
