Pharmacological Evaluation of Cyanoguanidine P2X7 Receptor Antagonists

Abstract

ABSTRACT BACKGROUND AND AIMS: The P2X7 receptor (P2X7R) is an ATP-gated, non-selective cation channel highly expressed on monocytes, macrophages and microglia. Prolonged activation of the P2X7R by ATP leads to cytolytic pore formation and the release of inflammatory mediators including interleukin-1β and prostaglandin E2. Accumulating evidence suggests a role for the P2X7R in neuroinflammation and thus P2X7R antagonists might be useful in diseases including chronic pain, depression and Alzheimer’s disease. Both negative allosteric modulators of the P2X7R, such as the adamantyl benzamides, and orthosteric antagonists, such as the aryl cyanoguanidines, inhibit the ATP-induced release of IL-1β from immune cells. This shared ability to inhibit IL-1β release may explain why no attempts have been made to determine the features which promote binding to the allosteric or orthosteric site. An advantage of targeting the allosteric or orthosteric site might emerge however, if a different agonist of the P2X7R is used. An antimicrobial peptide produced within the human body, LL-37, is also able to activate the P2X7R and yet LL-37 is never used in the characterisation of new series of P2X7R antagonists. The aims of this project were to characterise a novel series of P2X7R antagonists, the adamantyl cyanoguanidines, which have a hybrid structure derived from the adamantyl benzamides and aryl cyanoguanidines. Characterisation of the adamantyl cyanoguanidines should allow determination of the features which promote binding to the orthosteric or allosteric site of the P2X7R, which was one of the primary aims of this project. A second aim was to evaluate the potential of the hybrid series for further development as P2X7R antagonists by considering their potency and physicochemical P a g e | 13 properties. The final aim was to determine if there was any advantage of targeting the orthosteric or allosteric site of the P2X7R particularly with regard to inhibiting LL-37-mediated activation of the P2X7R. METHODS: The potency of adamantyl cyanoguanidines and reference P2X7R antagonists were determined in YO-PRO-1 dye uptake assays and interleukin-1β release assays to develop structure-activity relationships. A potent member of the adamantyl cyanoguanidines and several reference P2X7R antagonists were pharmacologically characterised in Schild assays, washout studies and receptor protection studies. The ability of several negative allosteric modulators and an orthosteric antagonist to inhibit LL-37-induced dye uptake was also examined. RESULTS: More compact adamantyl cyanoguanidines including those with a methylene linker between the adamantyl and cyanoguanidine groups and no linker between the cyanoguanidine group and phenyl ring were more potent than analogues with longer linkers. Ortho-substitution of the phenyl ring or substitution of the ring with 5-quinoline led to increased potency. The potency seen in the dye uptake assay was also seen in the interleukin-1β release assay. A potent member of the series 3-19 was determined to be a slowly reversible negative allosteric modulator as was 1-17 an adamantyl benzamide. The parent aryl cyanoguanidine, A-804598, was confirmed to be an orthosteric antagonist. None of the compounds were able to inhibit LL-37-induced dye uptake. DISCUSSION AND CONCLUSIONS: The determined structure-activity relationships for the adamantyl cyanoguanidines confirm their potential for further development since the series was highly amenable to modification and several potent analogues have favourable physicochemical properties including lower molecular weight. Since 3-19 P a g e | 14 was a negative allosteric modulator despite its structural similarity to A-804598 this suggests the adamantyl group promotes binding to the allosteric site and that cyanoguanidine is a tolerated bioisostere for the acetamide group at the allosteric site. The 5-quinolinyl group, in the appropriate position, facilitates binding to either site. The failure of multiple P2X7R antagonists to inhibit LL-37-induced dye uptake is concerning since LL-37 alone has been shown to induce the release of interleukin-1β from human monocytes. Future research must determine if LL-37 is responsible for cytokine release in vivo and develop small molecule antagonists of the action of LL-37.