P2X3-selective mechanism of Gefapixant, a drug candidate for the treatment of refractory chronic cough

Abstract

Gefapixant/AF-219, a selective inhibitor of the P2X3 receptor, is the first new drug other than dextromethorphan to be approved for the treatment of refractory chronic cough (RCC) in nearly 60 years. To date, seven P2X subtypes (P2X1-7) activated by extracellular ATP have been cloned, and subtype selectivity of P2X inhibitors is a prerequisite for reducing side effects. We previously identified the site and mechanism of action of Gefapixant/AF-219 on the P2X3 receptor, which occupies a pocket consisting of the left flipper (LF) and lower body (LB) domains. However, the mechanism by which AF-219 selectively acts on the P2X3 receptor is unknown. Here, we combined mutagenesis, chimera construction, molecular simulations, covalent occupation and chemical synthesis, and find that the negative allosteric site of AF-219 at P2X3 is also present in other P2X subtypes, at least for P2X1, P2X2 and P2X4. By constructing each chimera of AF-219 sensitive P2X3 and insensitive P2X2 subtypes, the insensitive P2X2 subtype was made to acquire the inhibitory properties of AF-219 and AF-353, an analog of AF-219 with higher affinity. Our results suggest that the selectivity of AF-219/AF-353 for P2X3 over the other P2X subtypes is determined by a combination of the accessibility of P2X3 binding site and the internal shape of this pocket, a finding that could provide new perspectives for drug design against P2X3-mediated diseases such as RCC, idiopathic pulmonary fibrosis, hypertension and overactive bladder disorder.

Keywords: Binding sites; DF, dorsal fin; Gefapixant/AF-219; IC50, the concentration yielding half of the maximal inhibition; LB, lower body; LF, left flipper; MD, molecular dynamics; NPM, N-phenylmaleimide; P2X3 receptors; RCC, refractory chronic cough; Refractory chronic cough; Subtype selectivity.

Graphical abstract

Fig. 1

Covalently linking the NPM to residue 238 substituted by cysteine in rP2X3 and to the identical position in other P2X subtypes. (A) View of the pocket formed by the left flipper (LF) and lower body (LB) domains of rP2X3. (B) Illustration of NPM covalently linked to rP2X3^V238C. Equivalent position mutations in hP2X1, rP2X2, rP2X4 and rP2X7 are V252C, V249C, I252C and I251C, respectively. (C-D) Representative current traces (C) and pooled data (D, mean ± S.D., n = 3–7) show the effect of NPM (1 mM, 5 min) on the wild type (WT) and equivalent cysteine substitutions in P2X receptors. ATP concentrations of 10 μM, 100 μM, 10 μM, and 100 μM and 1 mM were applied in hP2X1, rP2X2, rP2X3, and rP2X4, respectively. ^**P < 0.01 versus WT, two-way ANOVA with Bonferroni post-hoc test. (E-F) Representative current traces (E) and pooled data (F, mean ± S.D., n = 3–5) show the effect of NPM (0.5 mM, 2 min) on wild type (WT) and equivalent cysteine substitutions in P2X receptors. **P < 0.01 versus WT, two-way ANOVA with Bonferroni post-hoc test.

Fig. 2

G189 determines the accessibility of small molecules to the negative allosteric site formed by the left flipper (LF) and lower body (LB) domains. (A) Zoom-in view of the pocket fostered by the LF and LB domains of P2X3. Residues forming this pocket are indicated by sticks for emphasis. (B) Zoom-in view of the equivalent pocket in the zfP2X4 receptor. The bulky side chain of R206 (shown on sticks for emphasis) blocks the entrance to this pocket. (C) Amino acid sequence alignment of residues constituting the proposed site in different P2X receptors. (D-G) Typical current traces (D, F) and pooled data (E, G) showing the effect of AF-353 (D, E, 10 μM) and AF-219 (F, G, 30 μM) on the ATP (10 μM)-current of cP2X3^WT and cP2X3^A196G. Data are expressed as mean ± S.D. (n = 4–6). ^**P < 0.01 compared to WT, Student's t-test. (H-K) Typical current traces (H, J) and pooled data (I, K) showing the effect of AF-353 (H, I, 10 μM) and AF-219 (J, K 30 μM) on the ATP (10 μM)-current of rP2X3^WT and rP2X3^G189A. Data are expressed as mean ± S.D. (n = 3–5). ^**P < 0.01 compared to WT, Student's t-test.

Fig. 3

V61 contributes to the binding of small molecules to the deeper region of the pocket. (A) Zoom-in view of interaction between rP2X2 and AF-219 in homology model of the rP2X2/AF-219 complex. (B) Superposition of key residues of rP2X2 (gray) and hP2X3 (cyan) in contact with AF-219. I67 in P2X2 and V61 in P2X3 were zoomed in to show clearly. (C) Distance between the O atom of AF-219 and the C_α of I67 in rP2X2 (black) or V61 in rP2X3 (cyan) measured over time in 180-ns MD simulations. (D) Superposition of all poses of AF-219 during the 180-ns MD simulation. The initial pose of AF-219 is indicated by blue sticks and the poses during the MD simulation by origin lines. (E) Superposition of the initial (wheat) and optimized poses (blue) after MD simulation of the rP2X2 model in complex with the AF-219. The magenta dashed line indicates the distance between AF-219 and V61. (F) Concentration-response curves for AF-353 for rP2X3^WT, rP2X2^WT and rP2X2^I67V (n = 3–6).

Fig. 4

Dynamics of the allosteric site formed by the LF and LB domains. (A) Zoom-view of the pocket fostered by the LF and LB domains of rP2X2. Residues forming this pocket are indicated by sticks for emphasis. (B) Measured RMSD of C_α atoms of amino acids that constitute the pocket during MD simulations for rP2X2^WT and rP2X2^gain over time in 100-ns MD simulations. (C) Comparison of the initial structure (gray) and the snapshot (cyan) after 100 ns of simulation on rP2X2^WT. The red arrow indicates the movement of the LF domain during simulations. (D) Amino acid sequence alignment of the LF, DF and LB structural domains between rP2X2 and rP2X3. (E) The region of rP2X2 (including residues 67, 188–226, and 275–286, brown) is substituted by the corresponding residues of rP2X3. (F) Zoomed-in view of the pocket fostered by the LF and LB domains of rP2X2^gain in the resting state. (G) Volume measurements during MD simulation for rP2X2^gain. (H) Comparison of the initial structure (gray) and a snapshot (green) after 100-ns MD simulation on rP2X2^gain. The red arrow indicates the motion of the LF domain.

Fig. 5

MD simulations reveal the interaction of rP2X2^gain with AF-219 at atomic level. (A) Homology model of rP2X2^gain in complex with AF-219. (B) Superposition of key residues of rP2X2^gain (cyan) and hP2X3 (salmon) interacting with AF-219. (C) The superposition of the initial (cyan) and MD-optimized (yellow) poses of rP2X2^gain in complex with AF-219. (D-E) P2X-ligand contacts of hP2X3 (D) and rP2X2^gain (E) with the AF-219 were monitored throughout the MD simulations. P2X-ligand contacts were categorized into three types: hydrogen Bonds (green), hydrophobic (light blue), and water bridges (blue). The stacked bar charts were normalized over the course of the trajectory (values over 1.0 are possible as some protein residue may make multiple contacts of the same subtype with the ligand). (F-G) Timeline representation of the interactions and contacts between hP2X3 (F) and rP2X2^gain (G) with AF-219 monitored throughout the MD simulations (some residues made more than one specific contact with the ligand were represented by a darker shade of orange). Only the binding site residues interacting with the ligand are marked with numbers.

Fig. 6

Combination of factors producing subtype selectivity of AF-353/AF-219 generates the AF-353/AF-219-sensitive chimera P2X2^gain. (A) Schematic representation of chimera construction. rP2X2 chimeras were generated by replacing certain regions of rP2X2 with the corresponding sequences of rP2X3. (B) Concentration response curves of AF-353 in different chimeras (mean ± SEM, n = 3–5). (C-D) Representative currents (C) and pooled data (D) of ATP (10 μM) induced currents of AF-353 (10 μM) on rP2X2 WT and mutants (mean ± S.D., n = 3–5). ^**P < 0.01 versus rP2X2^WT, ^##P < 0.01 versus rP2X2^gain, one ANOVA with Bonferroni post-hoc test.

Fig. 7

Effect of AF-219 derivatives on the ATP current of rP2X3^WT and rP2X2^gain. (A) Chemical structures of various AF-219 derivatives. (B-C) Concentration response curves of AF-219, AF-353, AF-219-1 and AF-219-2 to rP2X3^WT (B) and rP2X2^gain (C). The data of AF-353 are the same as shown in Figs. 3F and 6B and are shown here for comparison. The data are shown as mean ± SEM (n = 3–6).