Kinetic analysis of fluorescent ligand binding to cell surface receptors: Insights into conformational changes and allosterism in living cells

Abstract

AbstractEquilibrium binding assays are one of the mainstays of current drug discovery efforts to evaluate the interaction of drugs with receptors in membranes and intact cells. However, in recent years, there has been increased focus on the kinetics of the drug–receptor interaction to gain insight into the lifetime of drug–receptor complexes and the rate of association of a ligand with its receptor. Furthermore, drugs that act on topically distinct sites (allosteric) from those occupied by the endogenous ligand (orthosteric site) can induce conformational changes in the orthosteric binding site leading to changes in the association and/or dissociation rate constants of orthosteric ligands. Conformational changes in the orthosteric ligand binding site can also be induced through interaction with neighbouring accessory proteins and receptor homodimerisation and heterodimerisation. In this review, we provide an overview of the use of fluorescent ligand technologies to interrogate ligand–receptor kinetics in living cells and the novel insights that they can provide into the conformational changes induced by drugs acting on a variety of cell surface receptors including G protein‐coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and cytokine receptors.