Abstract

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an “inward” position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 “inward”, like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

中文翻译：

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孕烷 X 受体单个氨基酸的突变通过改变 AF-2 螺旋定位将拮抗剂转换为激动剂

摘要

孕烷 X 受体 (PXR) 被化学物质激活以转录调节药物分布，并可能降低药物疗效并增加耐药性，表明 PXR 拮抗剂具有治疗价值。我们之前报道了拮抗剂 SPA70 及其类似物 SJB7，它出人意料地是一种激动剂。在这里，我们描述了另一个意想不到的观察结果：突变 PXR 配体结合域内的单个残基 (W299A) ​​将 SPA70 转化为激动剂。在表征野生型和 W299A PXR 活性特征后，我们使用分子动力学模拟揭示在野生型 PXR 中，激动剂将激活功能 2 (AF-2) 螺旋稳定在“向内”位置，但 SPA70 取代了 AF- 2. 然而，在 W299A 中，SPA70 像激动剂一样“向内”稳定 AF-2。我们通过预测拮抗剂 SJC2 是 W299A 激动剂来验证我们的模型，实验证实了这一点。我们的工作将先前未观察到的配体诱导的构象变化与 PXR 细胞活性相关联，并首次揭示了 PXR 拮抗剂的工作原理。