The hypothesis of synthesizing novel allosteric modulators of purinergic P2X4 receptors was investigated based on click chemistry. A small library of 10 compounds designed from the tritopic pharmacophore structure of ivermectin (IVM), a natural selective allosteric modulator of P2X4 receptor, was efficiently synthesized using a 1,3,5-tris(1,2,3-triazole)benzene scaffold. The efficacy of the compounds, termed MSKs, was screened using voltage patch clamp electrophysiology in microglia cells to assess the specific impact on P2X4 channel kinetics and properties. Since IVM modulates both ion conduction and channel gating of P2X4, MSK compounds were evaluated separately for both parameters following ATP applications. Most compounds induced an increase in the maximal inward current (Imax) indicating enhanced ion conductance. Notably, compounds MSK-7 to MSK-10, bearing two rigid 3-piperidine substituents and a highly hydrophobic menthyl group, also reduced receptor deactivation (τoff). The therapeutic potential of two selected compounds, MSK-5 and MSK-9, each exhibiting distinct mechanisms of interaction with the P2X4 receptor, was evaluated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Compound MSK-5 induced a modest yet significant amelioration of neurological symptoms during the chronic phase of EAE. In contrast, compound MSK-9 delayed EAE onset and showed a higher amelioration of neurological symptoms. We concluded that rigid trivalent click scaffolds constitute a novel class of non-natural platforms enabling the rapid development of positive allosteric modulators of P2X4 receptors that target both ion conductance and receptor deactivation. These results provide a solid foundation for future advances in the discovery of therapeutic lead candidates for multiple sclerosis.
