Oken AC et al. (2024), P2X7 receptors exhibit at least three modes of ...

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Sci Adv 2024 Oct 04;1040:eado5084. doi: 10.1126/sciadv.ado5084.

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P2X7 receptors exhibit at least three modes of allosteric antagonism.

Oken AC , Ditter IA , Lisi NE , Krishnamurthy I , Godsey MH , Mansoor SE .

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P2X receptors are trimeric ion channels activated by adenosine triphosphate (ATP) that contribute to pathophysiological processes ranging from asthma to neuropathic pain and neurodegeneration. A number of small-molecule antagonists have been identified for these important pharmaceutical targets. However, the molecular pharmacology of P2X receptors is poorly understood because of the chemically disparate nature of antagonists and their differential actions on the seven constituent subtypes. Here, we report high-resolution cryo-electron microscopy structures of the homomeric rat P2X7 receptor bound to five previously known small-molecule allosteric antagonists and a sixth antagonist that we identify. Our structural, biophysical, and electrophysiological data define the molecular determinants of allosteric antagonism in this pharmacologically relevant receptor, revealing three distinct classes of antagonists that we call shallow, deep, and starfish. Starfish binders, exemplified by the previously unidentified antagonist methyl blue, represent a unique class of inhibitors with distinct functional properties that could be exploited to develop potent P2X7 ligands with substantial clinical impact.

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Genes referenced: p2rx7

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	Fig. 1. The classical allosteric rP2X7 ligand-binding site is hydrophobic.(A) Ribbon representation of the apo closed-state structure of rP2X7 (Protein Data Bank code: 8TR5) (48). One protomer is colored and labeled by domain architecture as previously described, and the other two protomers are colored gray and light blue (11, 16, 65). The classical allosteric ligand-binding site found in P2X7 is boxed in red and the orthosteric ATP-binding site is circled in blue. Because of the structural conservation of P2XRs, the known allosteric sites of human P2X3 (circled in yellow) and zebrafish P2X4 (boxed in red) are mapped to their respective spatial positions on rP2X7 (15, 45). (B and C) Hydrophobic surface renderings of A438079-bound (B) and JNJ47965567-bound (C) rP2X7. Hydrophobic regions are colored brown [positive molecular lipophilicity potential (MLP)], and hydrophilic regions are colored turquoise (negative MLP). Surface renderings for A438079-bound and JNJ47965567-bound rP2X7 were generated at the same resolution as their cryo-EM reconstructions (2.2 and 2.4 Å, respectively). (B) Two symmetry-related molecules of A438079 (light pink and purple) are shown. The third molecule is out of frame due to 13-Å inter-ligand distances (dashed red lines). (C) Three symmetry-related molecules of JNJ47965567 (light pink, purple, and dark purple) are each separated by 4-Å inter-ligand distances (dashed red lines).
	Fig. 2. Antagonists bind to rP2X7 at the classical allosteric ligand-binding site.(A) Ribbon representation of the classical allosteric rP2X7 ligand-binding site at the interface of two protomers (gray and light blue) bound to one molecule (left to right) of A438079 (2.2 Å), A839977 (2.5 Å), AZD9056 (2.2 Å), GSK1482160 (2.4 Å), and JNJ47965567 (2.4 Å) with corresponding electron density for each ligand (blue mesh). (B) Residues in the classical allosteric ligand-binding site that interact with (left to right) A438079, A839977, AZD9056, GSK1482160, and JNJ47965567. F103 only participates in hydrophobic interactions with AZD9056, GSK1482160, and JNJ47965567. Toward the surface of the binding site, W167 forms hydrophobic interactions with GSK1482160 and JNJ47965567. The halogen atoms in A438079, A839977, and GSK1482160 all point toward A312. K110 forms cation-pi interactions with A839977 and AZD9056 and hydrogen bonds with GSK1482160 (distance of 2.9 Å). (C) Inhibition dose-response (IC50) curves measured by two-electrode voltage clamp (TEVC) for (left to right) A438079, A839977, AZD9056, GSK1482160, and JNJ47965567. Data points and error bars represent means and SDs of normalized current across triplicate experiments, respectively.
	Fig. 3. Methyl blue is an allosteric antagonist of P2X7.(A) Two-dimensional chemical structure of methyl blue, a symmetric analog of Brilliant Blue G, comprising three resonance-related diphenylamine-4-sulfonate arms. (B) Inhibition dose-response curves recorded by TEVC for methyl blue antagonism of rP2X7 (IC50 = 4 ± 1 μM) and hP2X7 (IC50 = 4 ± 2 μM). Data points and error bars represent means and SDs of normalized current across triplicate experiments, respectively. (C) Inhibition dose-response curves recorded by TEVC for methyl blue antagonism of hP2X1, hP2X2, hP2X3, hP2X4, and hP2X7. Data points and error bars represent means and SDs of normalized current across triplicate experiments, respectively. (D) Representative BLI sensorgram for a methyl blue dilution series binding to biotinylated rP2X7 immobilized on streptavidin biosensors during a 600-s association time and a 600-s dissociation time. Data were fit with a 2:1 Langmuir model with rate constants for association (ka) of ka1 = 4.5 ± 0.2 × 102 M−1 s−1 and ka2 = 9.3 ± 2.3 × 103 M−1 s−1 and rate constants for dissociation (kd) of kd1 = 1.1 ± 0.5 × 10−4 s−1 and kd2 = 1.3 ± 0.1 × 10−2 s−1, respectively. This resulted in a two-component equilibrium disassociation constant (KD) for methyl blue binding with a high-affinity component (KD1 = 260 ± 130 nM; 67 ± 8% of signal) and a low-affinity component (KD2 = 1.5 ± 0.2 μM; 33 ± 8% of signal). Kinetic values represent mean and SD across triplicate experiments.
	Fig. 4. Methyl blue binds to an extended allosteric ligand-binding site.(A) Ribbon representation of the methyl blue–bound inhibited state structure of rP2X7 at 2.7 Å. Protomers are represented by gray, light blue, and blue. Three molecules of methyl blue bind to the receptor, each at one of the three symmetry-related extended allosteric ligand-binding sites. (B) Orthogonal views (top, side-on; bottom, top-down) highlight the unique binding mode and inter-ligand interactions between different molecules of methyl blue (tan, light pink, and light purple). (C to E) Identical views of methyl blue bound at the extended allosteric ligand-binding site of rP2X7. (C) Extended allosteric rP2X7 ligand-binding site showing only one molecule of methyl blue and its corresponding electron density (blue mesh). (D) Hydrophobicity of the extended allosteric ligand-binding site of rP2X7, represented as a transparent 2.7-Å resolution surface rendering, with only two molecules of methyl blue shown (represented in light pink and tan). Hydrophobic regions are colored brown (positive MLP) and hydrophilic regions are colored turquoise (negative MLP). (E) Methyl blue bound in the extended allosteric ligand-binding site, with the allosteric, proximal, and distal arms of the molecule boxed in green, yellow, and red, respectively. (F) Residues that interact with the allosteric arm of methyl blue (F88, Y93, T94, M105, Y108, K110, F293, Y295, and I310) define the classical allosteric pocket. (G) Residues that interact with the proximal arm (relative to the center of the receptor) of methyl blue (T94, P96, L97, Q98, G99, S101, Y291, F293, and R316) define the proximal pocket. (H) Residues that interact with the distal arm of methyl blue (P96, Y295, and K297) define the distal pocket [view rotated by 90° from (E)]. The distal arm of each methyl blue molecule (tan, light pink, and light purple) forms inter-ligand interactions between symmetry-related molecules.
	Fig. 5. Application of methyl blue results in low recovery of activation following maximal antagonism.Recovery of P2X7 receptor activation expressed as a percentage of baseline current in response to 100 μM ATP following variable IC100 application times of antagonist and a fixed buffer wash. (A) Example TEVC trace for a recovery experiment. The percent recovery is the ratio of inward current evoked from a 100 μM ATP application before and after an IC100 application of antagonist for 40, 120, or 240 s. The experiment evaluates the relative rate of association and dissociation of each ligand to and from the receptor, respectively. (B) Recovery of activation after 40-s applications of the six allosteric antagonists tested. Antagonists A438079, A839977, AZD9056, and GSK1482160 permit strong receptor recovery; JNJ47965567 permits negligible receptor recovery; methyl blue permits moderate receptor recovery. (C) Recovery of activation following progressively longer IC100 applications of antagonists (40, 120, and 240 s). A438079, AZD9056, and GSK1482160 permit strong recovery even after long application of antagonist; JNJ47965567 permits negligible receptor recovery; and A839977 permits strong recovery after 40 s, but negligible recovery after longer applications. (D) Recovery of activation following progressively longer IC100 applications of methyl blue at rP2X7 and hP2X7. Channel recovery decreases with longer exposures to methyl blue until there is essentially no recovery after a 240-s application. Data represent means and SDs across triplicate experiments.
	Fig. 6. Three modes of allosteric antagonism of the P2X7 receptor.(A to C) Schematic representations of the distinct binding modes for three classes of allosteric antagonists bound to rP2X7 created using low-resolution synthetic maps generated from structural models. Top: Top-down view of the relative positions of the three symmetry-related ligands for each class of allosteric P2X7 antagonist. Bottom: Side view (rotated 90° from the top row) for the three classes of allosteric ligands bound to a single rP2X7 protomer. (A) Shallow binder A438079 occupies a shallow position in the classical allosteric ligand-binding site. Each ligand is located far from the other two symmetry-related molecules, preventing inter-ligand interactions. (B) Deep binder JNJ47965567 occupies a deep position in the classical allosteric ligand-binding site. Each ligand participates in edge-to-edge inter-ligand interactions with the other two symmetry-related molecules. (C) Starfish binder methyl blue occupies the extended allosteric ligand-binding site. The three binding arms of methyl blue are named allosteric, distal, and proximal according to the binding pocket for which each arm occupies (classical allosteric pocket, distal pocket, and proximal pocket). The distal arm of each starfish binder participates in extensive edge-to-face inter-ligand interactions with the distal arms of the other two symmetry-related molecules.