Measuring ligand concentration where it matters: Assessing the “micro pharmacokinetic/pharmacodynamics” of adenosine receptor ligands

The methods used to determine fundamental pharmacological parameters almost exclusively assume that the concentration of drug in the local environment of the target receptor is equal to the concentration of drug that has been added to the system. It has, however, recently been shown that, dependent upon their physiochemical properties, β2-adrenoceptor ligands can interact directly with phospholipids, increasing their local concentration and directly influencing the measured association rate constant at the receptor. This local concentrating effect also been demonstrated directly using a fluorescent β2-ligand with fluorescence correlation spectroscopy (FCS). In this study we expand these early observations by investigating multiple ligands at a different G protein-coupled receptor, the adenosine A2a receptor. In particular, we probe the importance of physicochemical properties on membrane interaction and observed pharmacology by utilising eight fluorescent adenosine receptor ligands with identical pharmacophores (xanthine amine congener (XAC)), but varying fluorophores and linker regions to modulate their properties. These ligands were assessed for kinetic binding profiles, phospholipid affinity, and local concentrations above cell membranes. The binding kinetics of the eight fluorescent ligands was assessed by measuring the time resolved fluorescence energy transfer (TR-FRET) between the terbium-labelled A2a receptor and fluorescent ligand over time. From this series, three ligands with distinct kinetic profiles were chosen for analysis by FCS (XACXBY, kon=24100±6860 min-1mM-1; CA200645, kon=1330±175 min-1mM-1; AV075 kon=791±36.4 min-1mM-1), where their local concentration was measured at distances 2-200µm above live CHO cells. The concentration of all ligands was higher close to the cells, with XAC-X-BY630 having the highest concentration at 2µm above the membrane (1024.6±347.4 nM) compared to CA200645 (62.3±9.5 nM) and AV075 (111.3±30.1 nM). This was consistent XACXBY displaying the fastest association rate and supports previous observations. These studies were then extended to investigate the kinetics and phospholipid interaction of 57 commercially available compounds known to bind at least one adenosine receptor. Binding kinetics were measured at all four adenosine receptors using a competition association assay, and phospholipid affinity (KIAM) was assessed in an Immobilised Artificial Membrane High Performance Liquid Chromatography assay. In this cohort, there was a statistically significant relationship between kon and KIAM (p=0.03), but surprisingly a better correlation with koff (p=0.0012), which may suggest that hydrophobic interactions are important for modulating dissociation rate in this receptor family. In general, the data in this study support the hypothesis that lipophilic ligands have a greater concentration in the local receptor environment close to the cell membrane, which may in turn influence observed pharmacological parameters. This reinforces the importance of considering “micro pharmacokinetics/pharmacodynamics” when determining the pharmacology of novel receptor ligands