Fig. 4: UB-MBX-46 is a potent and selective allosteric antagonist for the hP2X7R.

A 2D chemical structure of UB-MBX-46. B Concentration-dependent inhibition of agonist-induced calcium influx (IC₅₀ curves) for UB-MBX-46 on human, rat, and mouse P2X7Rs. Receptors were recombinantly expressed in 1321N1 astrocytoma cells and activated by an EC₈₀ of the agonist BzATP. C Calcium influx assays were used to measure the potency of UB-MBX-46 at hP2X1Rs, hP2X2Rs, hP2X3Rs, and hP2X4Rs stably expressed in 1321N1 astrocytoma cells. Receptors were activated by an EC₈₀ of ATP at the respective subtype (hP2X1R, 100 nM; hP2X2R, 1000 nM; hP2X3R, 100 nM; hP2X4R, 300 nM). B, C Data represent mean ± SD of at least three independent experiments performed in duplicates. For each trace, the data were normalized to the calcium signal induced by the respective EC₈₀ concentration of agonist (when no antagonist was added). D Ribbon representation of the classical allosteric ligand-binding site in hP2X7R, located at the interface of two protomers (gray and light blue) with one molecule of UB-MBX-46 (tan) shown with its corresponding cryo-EM density (blue mesh) at 2.5 Å. E Residues in the classical allosteric ligand-binding site of the hP2X7R that interact with UB-MBX-46. The polycyclic group of UB-MBX-46 forms hydrophobic interactions with the receptor and the backbone carbonyl from D92, as well as the side chain hydroxyl of Y298 form hydrogen bonding interactions with the hydrazide linker. F Computational values that describe the binding properties of UB-MBX-46 within the classical allosteric pocket of the hP2X7R. The hydrophobic volume reported is the hydrophobic surface of the polycyclic group calculated via Maestro, Schrödinger. Unoccupied space surrounding the polycyclic core was calculated via POVME3.0⁴². LogP values calculated via Maestro, Schrödinger. G Same view as E showing the accessible volume of the classical allosteric pocket in the hP2X7R calculated by Fpocket using the UB-MBX-46-bound inhibited state structure of the hP2X7R with the ligand removed⁷⁷. UB-MBX-46 tightly fits the classical allosteric pocket of the hP2X7R, although unoccupied voids remain above and below the ligand. H Determination of binding kinetics by TEVC recordings. Normalized current responses to 5s-pulses of 300 µM ATP over time during continuous superfusion with the indicated concentrations of UB-MBX-46. All data are represented as mean ± SD with n = 3, 4, 4, and 4 for 3 nM, 10 nM, 30 nM, and 100 nM respectively. I (left) Time course of antagonist dissociation. Normalized responses to 300 µM ATP after a 90–100% block by the antagonists are shown, highlighting virtually irreversible binding of UB-MBX-46 to the hP2X7R. (right) Experimentally determined on-rates (k_obs) were plotted against antagonist concentrations F to obtain an estimate for the off-rate constant k_off (y-intercept) according to the formula k_obs = k_on* F + k_off. All data are represented as mean ± SD with n = 3 for UB-MBX-46 and n = 5 for UB-ALT-P30 for the dissociation (left) and n = 4 for UB-MBX-46 and n = 7 for UB-ALT-P30 for the on-rates (right). Since there was minimal dissociation of UB-MBX-46 after 10 min, its graphically determined off-rate is clearly overestimated, and better values for K_off were obtained based on the measured or extrapolated IC₅₀ values (Supplementary Fig. 10).