Discovery of a pathway-selective platelet P2Y ₁ R inverse agonist that suppresses inflammation while preserving hemostasis

The platelet P2Y ₁ receptor (P2Y ₁ R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y ₁ R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y ₁ R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y ₁ R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y ₁ R towards G _α12/13 -mediated pathways, but not those associated by G _αq activation in P2Y ₁ R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y ₁ R-mediated functions. In vivo , KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the ‘neutral’ P2Y ₁ R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y ₁ Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis.