Defining the Molecular Mechanisms of Constitutive Activity and Inverse Agonism at the P2Y12 G-protein Coupled Receptor

Abstract

Cardiovascular disease (CVD) is the leading cause of global mortality. Following the build-up of fatty atherosclerotic plaques and their subsequent rupture in the coronary arteries, platelets play a causal role in acute coronary syndromes (ACS). Therefore, anti-platelet drugs, and more specifically purinergic P2Y12 G-protein coupled receptor (P2Y12R) targeting drugs, are a mainstay therapy for treatment of ACS and CVD. Despite its tractable clinical importance, the molecular pharmacology of the P2Y12R remains poorly defined. Although originally classed in the clinic as P2Y12R ‘antagonists’, the true inverse agonist nature of ticagrelor and cangrelor has only recently been fully recognised, alongside the constitutively active nature of the P2Y12R. This thesis aimed to develop our molecular level understanding of this therapeutically significant receptor.
Taking a multidisciplinary approach using in-silico molecular dynamic (MD) simulations, alongside in-vitro bioluminescence resonance energy transfer (BRET) assays, the molecular features of constitutive activity and inverse agonism at the P2Y12R were assessed. Ligands with a spectrum of efficacies, ranging from full agonist to full inverse agonist were docked and subject to MD simulations within the receptor. Correlations between the efficacy of the ligand and interactions with either transmembrane domain (TM) 3, or TM6 were observed. Furthermore, ligand binding was shown to induce significant conformational movements in the intracellular end of TM5. Agonist binding resulted in an enlargement of the intracellular spacing of TM3-TM5, whereas inverse agonists resulted in a decrease in the intracellular spacing of TM3-TM5. Intriguingly, the intracellular spacing between TM3-TM5 is the location of G-protein binding. Further in-vitro mutagenesis studies revealed the significant importance of TM2, 3 and 4 but not TM6 and 7, for the constitutive activity of the receptor. Further in-vitro investigations looking at the function of the highly conserved PIF and NPxxY motifs within the P2Y12R, revealed their importance for receptor constitutive activity.
The works presented here provide a crude ‘molecular definition’ of ligand efficacy at the P2Y12R. These definitions could be applied to currently employed P2Y12R drugs, and the future design of P2Y12R targeting drugs, ensuring effective molecular level characterisation. Given the clinical importance of inverse agonists for anti-platelet treatments, such molecular understanding is necessary if we are to effectively target P2Y12R activity, and treat CVD.

Date of Award	3 Oct 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Stuart J Mundell (Supervisor)