Pharmacological modulation of P2X receptors by approved drugs and natural compounds
Final Report Abstract
ATP-sensing P2X receptors of the P2X7 isotype are widely expressed and act as non-selective cation channels, mediating Ca2+ and Na+ entry, K+ efflux and, upon pore dilation, permeability for large organic cations. Within an academic-scale medium throughput screen, we identified a number of approved drugs or natural compounds that modulate human P2X7 in a positive or inhibitory allosteric fashion. To our surprise, most but not all of these modulators display a marked species-selective effect on human P2X7 with lower potency or effectiveness on rat or mouse P2X7. Among the positive modulators, we found that clemastine, ivermectin, agelasine and garinolic acid enhance Ca2+ signals and ionic currents through the channel. The modes of action included a sensitization of the receptor to lower ATP concentrations and stabilization of the open state of the channel (clemastine), or an increase in the channel availability with no changes in the apparent binding affinity of ATP (ivermectin). Within the inhibitory modulators, perazine-type antipsychotic drugs and tanshinone II A sulfonate and teniposide exhibited the highest potencies. Again, the mode of action was dissimilar between the compounds. Prochlorperazine acts via delaying the ATP-induced opening and closure, respectively. Tanshinone II A sulfonate has a slow onset rate of channel inhibition and acts more efficiently in a depolarized setting, indicating a voltage-sensitive access to an intracellularly located binding site. After characterizing the modulators in a heterologous expression system, native P2X7 channel complexes were tested in rat astroglial cells and in human or mouse macrophages, including the formation of a pro-inflammatory cytokine (IL-1β) as a pathophysiologically relevant output. Owing to a marked species selectivity of the effects of P2X7 modulators on human versus murine orthologs, a phylogenetically guided search for conserved modulatory mechanisms turned out to be not feasible. Other findings within the funding period raised concerns about the plethora of P2X7-ascribable biological effects and conflicting reports pertaining to cellular outcomes. Specifically, control of glioblastoma cell proliferation and apoptosis by P2X7 seemed to be inconsistent. After observing spurious background currents in macrophages and in parental HEK cells, which are not considered to express P2X7, we sought to identify the molecular correlate of these conductances. Ion exchange experiments and biophysical characterization led to the identification of TRPM7 as an ion channel, which can be indirectly activated by ATP under conditions that are typically applied in electrophysiological experiments with P2X7. Quantitative PCR and Western blot analysis confirmed the expression of TRPM7 in glioblastoma cell lines. Finally, we tested the controversially discussed pathophysiological role of P2X7 in cerebral ischemic infarction, applying the permanent and transient MCAO model in P2X7-/- mice that have been backcrossed to a BL6 background for 8 generations. Surprisingly, P2X7 deficiency was associated with a trend towards larger infarct volumes, a worse neurological outcome, and a significantly pronounced edema formation at 24 h after transient MCAO. At later time points, neurological scores tended to improve, and histopathological findings revealed an attenuated microglial response with no significant differences of astrogliosis between wild type and P2X7-/- animals. These findings point to a dual role of P2X7 in the ischemic stroke/reperfusion setting: One protective role pertaining to the regulation of fluid exchange and a second role in enhancing microglia activation at the infarct border. Thus, a pharmacological modulation of P2X7 may interfere with edema formation under pathophysiological conditions that lead to a massive release of ATP.
Publications
- Clemastine potentiates the human P2X7 receptor by sensitizing it to lower ATP concentrations. J Biol Chem. 2011; 286: 11067–81
Nörenberg W, Hempel C, Urban N, Sobottka H, Illes P, Schaefer M
(See online at https://doi.org/10.1074/jbc.M110.198879) - Positive allosteric modulation by ivermectin of human but not murine P2X7 receptors. Br J Pharmacol. 2012; 167: 48–66
Nörenberg W, Sobottka H, Hempel C, Plötz T, Fischer W, Schmalzing G, Schaefer M
(See online at https://doi.org/10.1111/j.1476-5381.2012.01987.x) - P2X7 receptors at adult neural progenitor cells of the mouse subventricular zone. Neuropharmacology. 2013; 73C: 122–37
Messemer N, Kunert C, Grohmann M, Sobottka H, Nieber K, Zimmermann H, Franke H, Nörenberg W, Straub I, Schaefer M, Riedel T, Illes P, Rubini P
(See online at https://doi.org/10.1016/j.neuropharm.2013.05.017) - The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor. Neuropharmacology. 2013; 75C: 365–79
Hempel C, Nörenberg W, Sobottka H, Urban N, Nicke A, Fischer W, Schaefer M
(See online at https://doi.org/10.1016/j.neuropharm.2013.07.027) - Natural compounds with P2X7 receptormodulating properties. Purinergic Signal. 2014; 10: 313–26
Fischer W, Urban N, Immig K, Franke H, Schaefer M
(See online at https://doi.org/10.1007/s11302-013-9392-1) - Tanshinone II A sulfonate but not tanshinone II A acts as potent negative allosteric modulator of the human purinergic receptor P2X7. J Pharmacol Exp Ther. 2014; 350: 531–42
Kaiser M, Sobottka H, Fischer W, Schaefer M, Nörenberg W
(See online at https://doi.org/10.1124/jpet.114.214569)
