Resumen de Design, synthesis and pharmacological evaluation of novel blood-brain barrier-permeable non-nucleotide purine derivatives as P2X7 antagonists for the treatment of neurodegenerative diseases
Neuroinflammation is a condition involved in the pathogenesis of several central nervous system (CNS) diseases. The ATP-gated purinergic P2X7 receptor stands out among the druggable biological targets that participate in neuroinflammation. Indeed, P2X7 activation by high concentrations of ATP, which usually occur under damaging conditions, triggers a pro-inflammatory signal in immune cells. Thus, P2X7 is considered a damage signal receptor, and Big Pharma and Academia have developed many effective P2X7 antagonists to mitigate the damage signal propagation. Nevertheless, only recently, their focus has shifted to target P2X7 in the CNS for the treatment of CNS disorders.
This PhD dissertation aims at finding out novel P2X7 antagonists with proper brain-blood barrier (BBB) permeability as new pharmacological tools to study P2X7 signalling and potential therapeutic agents against neurodegenerative diseases (NDDs). We designed and synthesised 31 novel non-nucleotide purine derivatives and assessed their pharmacological activity as potential P2X7 antagonists by YO-PRO-1 dye uptake and cytosolic calcium concentration ([Ca2+]c) increase in HEK293 cells stably-expressing human P2X7 (hP2X7). We also tested their blocking effect and selectivity by two-electrode voltage clamp (TEVC) in Xenopus laevis hP2X7-expressing oocytes. To prove their physiological relevance, we evaluated whether they could modulate the P2X7-mediated IL-1β release in mouse peritoneal macrophages. In addition, the BBB permeability profile of the compounds was predicted using the parallel artificial membrane permeability assay (PAMPA), and the P-glycoprotein ATPase activity assay. Among all the compounds, we discovered that 2-(6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one (compound 9), named ITH15004, (I) inhibits P2X7 in all the experimental models employed, (II) presents high selectivity compared with other P2X subtypes, (III) is active on human, rat and mouse orthologues, (IV) reduces ATP-induced IL-1β release in peritoneal macrophages, (V) has good permeability through lipid membranes, and (VI) does not affect Pgp activity. All of these data suggest that ITH15004 is able to reach the CNS and modulate neuroinflammation. We have studied the possible interactions involved in the ITH15004 binding at the P2X7 allosteric binding pocket by molecular docking to aid further structural optimisations. Our research also revealed a potential P2X4 positive allosteric modulator (compound 36) that may be employed as novel P2X4 selective tool.
Based on the results obtained, we state that ITH15004 is a novel P2X7 antagonist with moderate potency and a good BBB permeability profile. It is currently employed as pharmacological tool for in vitro and in vivo studies, and as a hit compound for further medicinal chemistry programmes to develop novel therapeutic drugs for the treatment of NDDs
