Differential regulation of CYP3A4 promoter activity by a new class of natural product derivatives binding to pregnane X receptor

Abstract

The pregnane X receptor (PXR) regulates drug metabolism by regulating the expression of drug-metabolizing enzymes such as cytochrome P450 3A4 (CYP3A4), which is involved in the metabolism of >50% of clinically prescribed drugs. The activity of PXR can be controlled by the binding of small molecule agonists or antagonists. Because of its unique ligand binding pocket, PXR binds promiscuously to structurally diverse chemicals. To study the structure-activity relationship, novel modulators for PXR are needed. Here we report the virtual screening of ∼25,000 natural product derivatives from the ZINC database using the Molecular Operating Environment docking software tool against the PXR-rifampicin complex X-ray crystal structure. Our screening resulted in identification of compounds based on the lowest S score, which measures Gibbs free energy. Interestingly, we found that the compounds that bind directly to PXR, as revealed in an intrinsic tryptophan fluorescence assay, modulate CYP3A4 promoter activity differentially in HepG2 cells. Mutational analysis and docking studies showed that these compounds bind broadly in the ligand binding pocket but interact with different amino acid residues. We further investigated the mechanism of binding by analyzing the functional groups that are important for distinguishing agonists from antagonists. The approach we used to identify novel modulators that bind to PXR can be useful for finding novel modulators of PXR.

Keywords: Drug metabolism; Molecular docking; Natural product derivative; PXR.

Figure 1. Work flow for identifying novel modulators for PXR

Schematic representations of the virtual screening method and SAR.

Figure 2. Compounds selected after virtual screening based on S values, which measure binding energy

Structures of these compounds and corresponding S values are provided.

Figure 3. Compound 1 activates PXR-regulated CYP3A4 promoter

HepG2 transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of rifampicin or compound 1 prior to luciferase assay.

Figure 4. Analogues of compound 1

Compounds 1, 2, 4, and 7 are agonists, whereas compounds 5, 6, and 8 are antagonists.

Figure 5. Compounds 1, 2, 4, and 7 are PXR agonists

HepG2 cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of compounds prior to luciferase assay.

Figure 6. Compounds 5, 6, and 8 are PXR antagonists

HepG2 cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with 5 μM of rifampicin and indicated concentrations of compound 5, 6, or 8 prior to luciferase assay (A–C). HepG2 cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of compounds prior to luciferase assay (D–F).

Figure 7. Cytotoxicity of compounds in HepG2 cells

Cells were treated with increasing concentrations (1.7 nM to 56 μM) of compounds.

Figure 8. Compounds 1 and 6 modulate PXR-regulated CYP3A4 promoter activity in LS 174T cells

(A) LS 174T cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of rifampicin or compound 1 prior to luciferase assay. (B) LS 174T cells transiently transfected with hPXR, CYP3A4-luc, and CMV-Renilla were treated for 24 h with 5 μM of rifampicin and indicated concentrations of compound 6 prior to luciferase assay.

Figure 9. Compound 1 activate PXR-regulated CYP2B6 promoter activity in HepG2 cells

HepG2 cells transiently transfected with hPXR, CYP2B6-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of rifampicin or compound 1 prior to luciferase assay.

Figure 10. Mammalian two-hybrid assay confirms that compounds affect hPXR interaction with SRC-1

Mammalian two-hybrid assays were performed in HepG2 cells transiently cotransfected with plasmids encoding Gal4-SRC-1 and the reporter gene pG5-luc, along with empty vector pACT as indicated. (A) The cells were treated with DMSO, 5 μM rifampicin, or 3 μM compound 1, 2, 3, 4, or 7. * Indicates p < 0.05 (comparisons were made between compound 1, 2, 3, 4, or 7 and DMSO control for samples transfected with pACT-hPXR). (B) Cells were treated with DMSO, 5 μM rifampicin, or 10 μM compound 5, 6, or 8 in the presence of 5 μM rifampicin (Rif) 24 h after transfection. Luciferase assays were performed 24 h after the compound treatment. The relative luminescence for pG5-luc was determined by normalizing firefly luciferase activity with Renilla luciferase activity. The values represent the means of five independent experiments, and the bars denote the S.D. * Indicates p < 0.05 for samples transfected with pACT-hPXR (samples co-treated with rifampicin and either compound 5, 6, or 8 were compared to rifampicin control; the difference between rifampicin and DMSO is also statistically significant with p < 0.05).

Figure 11. Compounds physically binds to PXR LBD

Decrease in the fluorescence intensities of PXR LBD samples upon addition of compounds as indicated. Solid lines represent the fit of the data to the single-binding site equation for each compound.

Figure 12. Binding modes of different compounds to PXR LBD

Docking mode two-dimensional (2D) interaction schemes of predicted binding poses of compound 1 (A), compound 2 (B), compound 7 (C), compound 5 (D), compound 6 (E), and compound 8 (F) at the PXR LBD binding site.

Figure 13. Comparison of the binding modes of compound 1 and compound 3 to PXR LBD

Main interacting side chains identified through docking of compound 1 (green) and compound 3 (cyan) with the PXR LBD. The aryl group of compound 1 can form pi-pi interactions but not the methyl group of compound 3 shown as circle (blue).

Figure 14. Binding modes of different compounds to PXR LBD

Docking mode three-dimensional (3D) interaction schemes of predicted binding orientations of compound 1 (A), compound 2 (B), compound 7 (C), compound 5 (D), compound 6 (E), and compound 8 (F) at the PXR LBD binding site.

Figure 15. Comparison of hPXR and hPXR mutants upon 5 μM compound 1 treatment

(A) HepG2 cells transiently transfected with hPXR or hPXR mutants, CYP3A4-luc, and CMV-Renilla were treated for 24 h with indicated concentrations of compound 1 prior to luciferase assay. The relative luciferase units (a.u.) were determined by normalizing with the Renilla luciferase control. (B) The expression of hPXR or hPXR mutants upon compound 1 treatment. Actin expression level was used to verify equal loading of lysates. The values represent the means of three independent experiments, and the bars denote the S.D. * Indicates p < 0.05 (comparisons were made between PXR mutant and WT hPXR).