Mapping the binding site of P2X7 receptor antagonists

Abstract

The P2X7 receptor (P2X7R) is a ligand gated ion channel activated by high concentrations of ATP that are usually found in conditions of stress, cellular damage and disease. Selective P2X7R antagonists therefore may have therapeutic potential for treatment of several pathological conditions. Understanding how P2X7R antagonists act on the receptor is useful to facilitate drug design. In this thesis the molecular basis for several selective P2X7R antagonists has been investigated. This was done by using a range of chimeras and mutations between antagonist sensitive hP2X7R and antagonist insensitive hP2X1R. This work showed that the selective P2X7R antagonist AZ11645373 binds at the allosteric pocket between two adjacent subunits away from the orthosteric ATP binding pocket. Molecular docking of AZ11645373 is in line with effects of mutations on the sensitivity to the antagonist. The allosteric binding mode of AZ11645373 explains variation in antagonist sensitivity between human and rat P2X7Rs. Comparing the effects of allosteric point mutations of AZ11645373 with other previously characterized allosteric inhibitors demonstrated that there is a similar reduction in sensitivity with the F88A, T90V, D92A, F103A and V312A mutations. These allosteric mutations were used as a signature to identify the site of action for P2X7R antagonists ZINC58368839, KN-62, Brilliant Blue G and Calmidazolium. The combination of signature allosteric point mutations and molecular docking propose that ZINC58368839 has a mode of binding similar to AZ11645373. However, the binding site for the large molecule P2X7R antagonists KN-62, Brilliant Blue G and Calmidazolium appear to be more complex, because they not only bind at the allosteric site but also extend into the central cavity. In conclusion, this thesis has enhanced our knowledge of selective P2X7R antagonists and advanced a template to develop allosteric P2X7R inhibitors.