Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y12 receptor

P2Y12 receptor (P2Y12-R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12-R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12-R antagonists with a better clinical benefit–risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12-R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12-R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12-R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12-R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12-R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12-R constitutive activity should allow the discovery of more selective biased P2Y12-R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.