Structure–activity relationship of the allosteric effects of ivermectin at human P2X4 and GABAA receptors

Background and purpose

Positive allosteric modulation of the P2X4 receptor is a potential route to providing cardiovascular benefit through enhancing flow-dependent arterial vasodilation and providing cardioprotection. However, ligands that selectively enhance P2X4 activity are absent. The broad-spectrum antiparasitic ivermectin (MK-933) is a known positive allosteric modulator of P2X4, but not selective for P2X4, acting to enhance the activity of other ion channels including the GABA_A receptor through which its neurotoxic and anti-convulsant properties are mediated. Here, we combine complementary methodology to investigate the structure–activity relationship of ivermectin at human P2X4 and GABA_A receptors.

Experimental approach and key results

Intracellular Ca²⁺ and membrane potential assays in cell lines expressing human P2X4 or human GABA_A α1β3γ2 receptor are used, respectively. A chemical library of ivermectin analogues are pharmacologically characterised at both receptors, and in silico techniques are used to identify ligand binding modes in human P2X4 to interpret pharmacological properties. We identify ivermectin-B1a as a positive allosteric modulation of P2X4, but full agonist at the GABA_A α1β3γ2 receptor. We discover that the large disaccharide moiety of ivermectin-B1a is not required for activity. We identify an intersubunit transmembrane domain binding mode for ivermectin-B1a in P2X4 supported by the structure–activity relationship of ivermectin analogues. A series of novel compounds with selectivity for P2X4 over GABA_A receptor are identified.

Conclusions and implications

Ivermectin-B1a enhances P2X4 and GABA_A α1β3γ2 receptor activity but through differing pharmacological mechanisms. We identify pharmacophore information for the development of positive allosteric modulators selective for human P2X4 over GABA_A receptors.