Article Figures & Data
Figures
- Fig. 1.
5-BDBD displays an allosteric mode of action (A) ATP-evoked Ca2+ responses from 1321N1 astrocytoma cells stably expressing human P2X2 (open circles) or P2X4 (closed circles; N = 6). (B) 5-BDBD inhibition (30-minute incubation) of P2X4 (closed circles) but not P2X2 receptors (open circles; N = 8). Both receptors activated with 1 μM ATP. (C) Effect of varying 5-BDBD concentrations (30-minute incubation) on ATP concentration-response relationship in 1321N1 astrocytoma cells stably expressing P2X4 (N = 6). (D) Sequential mutation of 10-amino-acid stretches in the ectodomain of P2X4 to the equivalent residues in P2X2 identified a region between residues 81 and 90 important for 5-BDBD activity. Substitutions between 71–80, 101–110, 131–140, 201–210, 221–220, 261–270, 281–290, and 291–300 produced nonfunctional chimeras (N = 5; * p < 0.05 ANOVA with Dunnett’s post hoc test). (E) Homology model of P2X4 in the ATP-bound (beige) open state based on zebrafish P2X4 (Protein Data Bank:4DW1). Residues 81–90 can be located toward the top of the body region (red) and extended toward the orthosteric site (blue).
- Fig. 2.
Molecular modeling identified an allosteric binding site for 5-BDBD. (A) Representative 5-BDBD docking pose from molecular dynamics simulations. Relevant residues are shown as sticks, and potential hydrogen bonding is indicated by dashes. (B) Carbonyl-guanidine distances between 5-BDBD and R301 (blue) and R82 (red), respectively, for a representative molecular dynamics simulation. (C) Amide-carboxylate oxygen distances between 5-BDBD and E307. Blue and red represent the two carboxylate oxygens. (D) Distance between the Y300 hydroxyl group oxygen and the amide-NH (red) and carbonyl oxygen (blue). (E) Presence of hydrophobic interactions between 5-BDBD and relevant residues. For comparison residues are color-coded as in Fig. 3B.
- Fig. 3.
Site-directed mutagenesis of the allosteric site identified F81, M109, F178, Y300, R301, and I312 as important residues for 5-BDBD inhibition (A) Alignment of amino acids predicted to interact with 5-BDBD in the binding site between P2X1-7 (P2X4 numbering). Blue and orange depict which subunit the amino acid residues are located on. (B) Inhibitory activity of 5-BDBD against mutant P2X4 receptors (N = 5; * p < 0.05 ANOVA with Dunnett’s post hoc test). (C) A representative image of 5-BDBD binding to the wild-type P2X4 receptor. Residues implicated in 5-BDBD (purple) binding are highlighted in red. (D) Representative images of 5-BDBD binding to the mutant receptors M109H, F178L, Y300A, R301K, and I312G. The mutated residue is labeled, and the distances between the amino acids and potential interactions with 5-BDBD are shown by dashes. Images were generated using Chimera.
- Fig. 4.
5-BDBD analogs identified key structural interactions between 5-BDBD and P2X4. (A) The structure of 5-BDBD and the positions of the halogen substitutions at R1, R2 and R3. Unmodified 5-BDBD has a bromine at position R2. (B and C) The IC50 values (B) and inhibitory activity (C) of the 5-BDBD analogs toward P2X4 stably expressed in 1321N1 astrocytoma cells (600 µM ATP and 30 µM 5-BDBD analog were used to quantify inhibitory activity; N = 3; * p < 0.05 ANOVA with Dunnett’s post hoc test). (D) The distance between the carbonyl group of 5-BDBD/5-BDBD analogs and R301 in the binding pocket as determined through docking studies. (E) The penetration distance of 5-BDBD/5-BDBD analogs into the predicted binding site relative to the opening of the pocket as determined by docking studies. Images were generated using Chimera.
- Fig. 5.
Constriction of the P2X4 allosteric binding site in the open state may prevent the activity of 5-BDBD. (A) Representative image depicting the predicted constriction of the 5-BDBD binding pocket and conformational changes between P2X4 in the closed (blue) and open (red) states. (B) The relative distance between amino acids T76, S77, L79, R82, and W84 on one subunit with the atoms CD1 or CD2 of L107 on the other subunit in the closed and open channel. (C) Representative electrophysiology trace of a cell stimulated with 30 µM ATP in the absence of 5-BDBD (black) or after a 3-minute preincubation with 20 µM 5-BDBD (red). (D) Representative electrophysiology trace of cells stimulated with 30 µM ATP for 2 seconds prior to the addition of 30 µM ATP and 20 µM 5-BDBD (red) or ATP and DMSO (black) during the desensitization stage for 5 seconds. (E) Representative electrophysiology trace of cells stimulated with 30 µM ATP for 2 seconds prior to the addition of 30 µM ATP and 10 µM BX-430 (red), or ATP and DMSO (black), during the desensitization stage for 5 seconds. (F) The time constant values for current decay (τ) for the desensitization stage in the presence of DMSO, 5-BDBD, or BX-430. Traces and data are representative of 17, 7, and 5 cells for the vehicle control, 5-BDBD, and BX-430, respectively. *p < 0.05 vs. vehicle, **p < 0.05 vs. 5-BDBD. Images were generated using Chimera.
Additional Files
Data Supplement
- Data Supplement -
Supplemental Table 1. A summary of the distances between the atoms CD1 and CD2 of L107 on one subunit of P2X4, with the atoms of the indicated amino acid residues on the second subunit in both the closed and open state.
Supplementary Figure 1. An overview of the chemical interactions between 5-BDBD and amino acid residues in the predicted allosteric binding pocket of P2X4.
Supplementary Figure 2. (A-C) Backbone root-mean-square deviation (rmsd) for three replicate molecular dynamics simulations of the extracellular domain of P2X4 with 5-BDBD docked into the allosteric site and (D) for the orthosteric control. (E-G).
Supplementary Figure 3. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at R2.
Supplementary Figure 4. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at R3.
Supplementary Figure 5. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at both R1 and R2.
Supplementary Figure 6. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at R1.
Supplemental information on synthesised compounds.
- Data Supplement -
Data Supplement
- Supplemental Data -
Supplemental Table 1. A summary of the distances between the atoms CD1 and CD2 of L107 on one subunit of P2X4, with the atoms of the inducated amino acid residues on the second subunit in both the closed and open state.
Supplemental lTable 1. A summary of the distances between the atoms CD1 and CD2 of L107 on one subunit of P2X4, with the atoms of the indicated amino acid residues on the second subunit in both the closed and open state.
Supplementary Figure 1. An overview of the chemical interactions between 5-BDBD and amino acid residues in the predicted allosteric binding pocket of P2X4.
Supplementary Figure 2. (A-C) Backbone root-mean-square deviation (rmsd) for three replicate molecular dynamics simulations of the extracellular domain of P2X4 with 5-BDBD docked into the allosteric site and (D) for the orthosteric control. (E-G).
Supplementary Figure 3. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at R2.
Supplementary Figure 4. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at R3.
Supplementary Figure 5. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at both R1 and R2.
Supplementary Figure 6. Representative images of the docking poses obtained for the 5-BDBD analogues with modifications at R1.Supplemental information on synthesised compounds.
- Supplemental Data -
