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. 2018 Feb 2;8:2305. doi: 10.1038/s41598-018-20663-z

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Identification of OBG as a ligand for LXRs. (A) Chemical structure of OBG and ouabain (OUA). (B) OBG has a unique steroidal ring fusion skeleton. OBG is built around a cis-trans-cis-fused A/B/C/D-ring system (left panel), whereas most normal steroid hormones have an all-trans-fused ring system (right panel). (C and D) OBG displays agonistic activity towards LXRs in luciferase reporter assays. 293 T cells transfected with LXRs/RXR, the DR4-tk-Luc reporter and the Renilla reporter plasmid (as a control) were subjected to luciferase assays at 48 h after treatment with 10⁻⁸ M or 10⁻⁹ M OBG and/or 10⁻⁹ M T0901317. The fold change in transcriptional activity is expressed as the mean ± SD of triplicate experiments. Values followed by different letters are statistically different according to analysis of variance (ANOVA) followed by SNK tests (P < 0.01). (E) Competitive binding assay using the fluorescent LXR ligand by detection of fluorescent anisotropy. To the solution of complex of LXR-LBD (0.5 μM) and the fluorescent LXR ligand (0.5 μM), OBG or T0901317 was added with the concentration of 0 μM, 0.1 μM, 0.3 μM, 1.0 μM, 3.0 μM, 10 μM, 30 μM, or 100 μM. Intact means fluorescent anisotropy of the fluorescent ligand in the buffer without protein. Bar graphs represent means ± SD of dependent three experiments and values followed by different letters are statistically different according to ANOVA followed by SNK tests (P < 0.01). (F) OBG has a strong affinity for LXRs in in silico docking simulations. Using previously reported datasets from crystal structures of the LXR/T0901317 complex, OBG was subjected to in silico docking simulations and the results were compared with other known LXR ligands. After docking simulation, both superimposed structures between OBG (green colored structure) and T0901317 (magenta colored structure) were settled down around the similar pocket of LXRα (gray colored ribbon in left panel) and LXRβ (gray colored ribbon in right panel). Based on the simulations, the stabilisation energy was calculated (right table). T0901317 and GW3965 are synthetic ligands of LXRs, and 24(S), 25-epoxycholesterol is a natural ligand.