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.
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We thank all members of the Chen research laboratory and the High-Throughput Bioscience Center for valuable discussions of the paper, the Hartwell Center at St. Jude Children’s Research Hospital for DNA sequencing, and Drs. Bert W. O’Malley and Sridhar Mani for plasmids. This work was supported in part by ALSAC and by the National Institutes of Health (Grants R35-GM118041 [to TC] and P30-CA21765 [to the St. Jude Cancer Center to support Cancer Center shared resources]). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Conflict of interest
Authors Taosheng Chen and Wenwei Lin have filed the following patent: Chen T, Lin W, Wang Y. PCT/US2017/021949: 1,4,5-Substituted 1,2,3-Triazole Analogues as Antagonists of the Pregnane X Receptor. International Patent Application published September 28, 2017 as WO/2017/165139; US Patent Application published March 14, 2019 as US 2019/0077770 A1.
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Huber, A.D., Wright, W.C., Lin, W. et al. Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning. Cell. Mol. Life Sci. 78, 317–335 (2021). https://doi.org/10.1007/s00018-020-03505-y
- Nuclear receptor
- Drug metabolism
- Cytochrome P450