What is pharmacological ‘affinity’? Relevance to biased agonism and antagonism

•Because receptors are allosteric proteins, affinity estimates are always conditional.•At present, all known methods to estimate bias utilize functional affinity.•Binding estimates of affinity can be very different from functional affinity.•Affinity must be considered when measuring bias.•Concepts around signaling bias can be extended to development of biased antagonists.

The differences between affinity measurements made in binding studies and those relevant to receptor function are described. There are theoretical and practical reasons for not utilizing binding data and, in terms of the quantification of signaling bias, it is unnecessary to do so. Finally, the allosteric control of ligand affinity through receptor–signaling protein interaction is discussed within the context of biased antagonism. In this regard, it is shown that both the bias and relative efficacy of a ligand are essential data for fully predicting biased effects in vivo.