Analgesic effect of cinobufagin is mediated by human-selective P2X7R antagonism governed by distinct extracellular...

Chronic pain management remains a major clinical challenge, limited by the efficacy and safety profile of existing analgesics. The P2X7 receptor (P2X7R), a key driver of neuroinflammatory signaling, represents a promising therapeutic target; however, the clinical advancement of P2X7R antagonists has been hindered by limited efficacy and marked species differences in pharmacological activity. Here, we identify cinobufagin (CBG) as a novel, human-selective allosteric antagonist of P2X7R. CBG potently inhibited human P2X7R in functional assays measuring YO-PRO-1 uptake, calcium flux, and electrophysiological responses, but exhibited minimal activity against rat P2X7R or other P2X subtypes. Structural modeling and mutagenesis confirmed CBG's engagement with the canonical allosteric pocket, with residues F103 and M105 being critical for binding. Notably, species selectivity was determined not by variations within the binding pocket itself, but by distal extracellular domains. We further identified key discriminatory residues (R126, S165, I170, R270, Y288, N303) as novel molecular determinants of this selectivity. In a murine model of complete Freund's adjuvant (CFA)-induced inflammatory pain, CBG demonstrated significant analgesic efficacy. Comprehensive electrophysiological profiling also confirmed its selectivity against other major pain-related ion channels. Collectively, our work elucidates CBG's mechanism of action and reveals a paradigm in which allosteric modulation of P2X7R can be governed by long-range conformational influences from distal extracellular domains, rather than solely by direct ligand-pocket interactions.