The HIV protease inhibitor nelfinavir is currently being analyzed for repurposing as an anticancer drug for many different cancers because it exerts manifold off-target protein interactions, finally resulting in cancer cell death. Xenosensing pregnane X receptor (PXR), which also participates in the control of cancer cell proliferation and apoptosis, was previously shown to be activated by nelfinavir; however, the exact molecular mechanism is still unknown. The present study addresses the effects of nelfinavir and its major and pharmacologically active metabolite nelfinavir hydroxy-tert-butylamide (M8) on PXR to elucidate the underlying molecular mechanism. Molecular docking suggested direct binding to the PXR ligand-binding domain, which was confirmed experimentally by limited proteolytic digestion and competitive ligand-binding assays. Concentration-response analyses using cellular transactivation assays identified nelfinavir and M8 as partial agonists with EC₅₀ values of 0.9 and 7.3 µM and competitive antagonists of rifampin-dependent induction with IC₅₀ values of 7.5 and 25.3 µM, respectively. Antagonism exclusively resulted from binding into the PXR ligand-binding pocket. Impaired coactivator recruitment by nelfinavir as compared with the full agonist rifampin proved to be the underlying mechanism of both effects on PXR. Physiologic relevance of nelfinavir-dependent modulation of PXR activity was investigated in respectively treated primary human hepatocytes, which showed differential induction of PXR target genes and antagonism of rifampin-induced ABCB1 and CYP3A4 gene expression. In conclusion, we elucidate here the molecular mechanism of nelfinavir interaction with PXR. It is hypothesized that modulation of PXR activity may impact the anticancer effects of nelfinavir.

中文翻译：

---

奈非那韦及其活性代谢物M8是人类妊娠X受体的部分激动剂和竞争性拮抗剂。

目前正在对HIV蛋白酶抑制剂奈芬那韦（Nelfinavir）进行重新定位，使其作为针对多种不同癌症的抗癌药物，因为它会产生多种脱靶蛋白相互作用，最终导致癌细胞死亡。异诺孕烷X受体（PXR）也参与控制癌细胞的增殖和凋亡，先前已被奈非那韦激活。然而，确切的分子机制仍是未知的。本研究探讨了奈非那韦及其主要和药理活性代谢物奈非那韦羟基叔丁的作用-PXR上的叔丁基酰胺（M8）阐明潜在的分子机理。分子对接表明直接结合到PXR配体结合域，这通过有限的蛋白水解消化和竞争性配体结合测定法得到了实验证实。使用细胞反式激活分析的浓度反应分析确定奈非那韦和M8为部分激动剂，EC ₅₀值为0.9和7.3 µM，并且是竞争性的利福平依赖性诱导拮抗剂，IC ₅₀值分别为7.5和25.3 µM。拮抗作用完全是由结合到PXR配体结合袋中引起的。与完全激动剂利福平相比，奈非那韦削弱的共激活剂募集被证明是这两种作用于PXR的潜在机制。在分别处理的原代人肝细胞中研究了奈非那韦依赖性PXR活性调节的生理相关性，显示了PXR靶基因的差异诱导以及利福平诱导的ABCB1和CYP3A4基因表达的拮抗作用。总之，在这里我们阐明了奈非那韦与PXR相互作用的分子机制。假设PXR活性的调节可能影响奈非那韦的抗癌作用。