Structure-based analysis reveals a hydrophobic pocket governing P2Y12 receptor agonist potency and selectivity.

Agonist of the ADP-activated P2Y12 receptor modulate platelet aggregation and microglial function, yet the structural basis of their potency and selectivity remains unclear. Using recent active-state cryo-EM structures of P2Y12 and P2Y1 receptors, we applied an integrated modelling approach to revisit a structurally uncharacterised agonist series. We identify a previously unrecognised secondary hydrophobic pocket (SHP) adjacent to the orthosteric site as a key determinant of sub-nanomolar potency and receptor selectivity. Hydrophobic C2-alkylthio substituents engage this pocket and enhance potency by >2,000-fold, compensating for β-phosphate removal and otherwise intolerant N6 modifications. Differential SHP exposure explains P2Y12/P2Y1 selectivity, while non-conserved residues at positions 4.53 and 4.56 in SHP of P2Y13R create a steric bottleneck to exclude bulky C2 substituents, enabling subtype discrimination. We further propose a revised P2Y1R agonist-binding mode consistent with SAR and independently validated by cryo-EM. These findings establish a structure-based SAR framework and design principles for selective P2Y12 agonists.