Functional efficacy of adenosine A_2A receptor agonists is positively correlated to their receptor residence time

Abstract

BACKGROUND AND PURPOSEThe adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACHWe set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTSA simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONSThis study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor. AbbreviationsADA, adenosine deaminase; BCA, bicinchoninic acid; CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CI, cell index; HEK293hA2AR, human embryonic kidney 293 cells stably expressing the hA2A receptor; k3, the association rate constant of the unlabelled ligand; k4, the dissociation rate constant of the unlabelled ligand; RT, residence time; Z, cell-electrode impedance; ZM241385, 4-(2-[7-amino-2-(2-furyl) [1,2,4]

Whole-cell biosensor for label-free detection of GPCR-mediated drug responses in personal cell lines

“…The initial experimental setup was based on previously published protocols for adherent cell lines (Guo et al, 2012;Scandroglio et al, 2010;Stallaert et al, 2012). While label-free assays are often deemed incompatible with suspension cells, some application examples exist for various label-free assays based on optical or impedance detection.…”

Ligand Residence Time at G-protein–Coupled Receptors—Why We Should Take Our Time To Study It

“…Notwithstanding the difficulties mentioned above, the concept of residence time optimization of GPCR ligands has led to the development of antagonist drugs with long residence times such as tiotropium (Tautermann et al, 2013). Even for agonists, a positive correlation between agonist efficacy and residence time has been observed in the case of the M 3 acetylcholine receptor (Sykes et al, 2009) and at the adenosine A 2A receptor (Guo et al, 2012), although no such correlation was observed for the adenosine A 1 receptor (Louvel et al, 2014).…”

Structure-Activity Analysis of Biased Agonism at the Human Adenosine A3 Receptor

“…These time-dependent processes are an important consideration, as signaling assays require different incubations times and therefore may be differentially influenced by A 3 AR desensitization and/or internalization. Future studies will also assess the binding kinetics of structurally distinct A 3 AR agonists, as it is becoming appreciated that the agonist residence time can influence the efficacy and bias profile observed (Sykes et al, 2009;Guo et al, 2012;Klein Herenbrink, 2016).…”