Fig 1.
Ritonavir-hTAS2R14 complex model (upper (orthosteric) site) predicted by Induced Fit Docking (IFD).
A. The predicted binding site of ritonavir in hTAS2R14 (side view). hTAS2R14 (PDBID:8XQO) is rendered as newcartoon (in orange). Ritonavir is depicted in licorice colored by atom types. B. Predicted ritonavir upper binding site in hTAS2R14. The binding site residues and ritonavir are represented in ball-stick and licorice, respectively, and colored by atom types. C. top (from extracellular side) view of A. D. 2D plot (by Discovery Studio) of the detailed interactions between ritonavir and hTAS2R14 receptor binding site residues.
Fig 2.
Ritonavir-hTAS2R14 complex model (lower (allosteric) site) predicted by Induced Fit Docking (IFD).
A. The predicted binding site of ritonavir in hTAS2R14 (side view). hTAS2R14 (PDBID:8XQO) is rendered as newcartoon (in orange). Ritonavir is depicted in licorice colored by atom types. B. Predicted ritonavir upper binding site in hTAS2R14. The binding site residues and ritonavir are represented in ball-stick and licorice, respectively, and colored by atom types. C. top (from extracellular side) view of A. D. 2D plot (by Discovery Studio) of the detailed interactions between ritonavir and hTAS2R14 receptor binding site residues.
Fig 3.
Point mutations of selected residues in the hTAS2R14 binding pocket (upper (orthosteric) site) affect response to ritonavir.
A. Intracellular calcium signals induced by ritonavir (100 μM) and control compounds (30 μM Aristolochic acid (AA), 30 μM Flufenamic acid (FA) and 100 μM oleuropein aglycone (OA)) in HEK293E cells transfected with hTAS2R14 or mutants. The data presented were from at least six independent measurements (mean ± SEM). ΔF/F (%), percentage of relative fluorescence increase. Mock, without hTAS2R14 receptor transfected cells as a negative control. Asterisks indicate significant differences tested by one-way ANOVA (***p < 0.001, **p < 0.01, *p < 0.05, N ≥ 6). The figure was plotted by GraphPad. B. location of the upper binding site residues tested (Cα atom only in purple) in the hTAS2R14 (side view, cartoon in orange). The TM1 and TM4 were removed for clarity. C. the top view of B.
Fig 4.
Point mutations of selected residues in the hTAS2R14 binding pocket (lower (allosteric) site) affect response to ritonavir.
A. Intracellular calcium signals induced by ritonavir (100 μM) and control compounds (30 μM Aristolochic acid (AA), 30 μM Flufenamic acid (FA) and 100 μM oleuropein aglycone (OA)) in HEK293E cells transfected with hTAS2R14 or mutants. The data presented were from at least six independent measurements (mean ± SEM). ΔF/F (%), percentage of relative fluorescence increase. Mock, without hTAS2R14 receptor transfected cells as a negative control. Asterisks indicate significant differences tested by one-way ANOVA (***p < 0.001, **p < 0.01, *p < 0.05, N ≥ 6). The figure was plotted by GraphPad. B. location of the upper binding site residues tested (Cα atom only in purple) in the hTAS2R14 (side view, cartoon in orange). The TM1 and TM4 were removed for clarity. C. the top view of B.
Fig 5.
Dose-response relationships for hTAS2R14 mutants affect ritonavir activity.
The Results were obtained using a cell-based assay of HEK293E cells co-transfected with hTAS2R14 or its mutants along with Gα16gust44 and GCaMP6s. Mock, without hTAS2R14 receptor transfected cells as a negative control. Each point represents the mean ± SEM from three independent measurements. The figures were plotted by Prism 10. ΔF/F (%), percentage of relative fluorescence increase.
Fig 6.
The upper and lower binding sites of ritonavir in hTAS2R14. hTAS2R14 is shown as cartoon colored in orange, ritonavir and critical interacting residues are shown as licorice colored by atom types.