![]() ![]() According to this view, affinity and efficacy are intrinsically linked for agonists, so it is impossible to obtain a value for the dissociation constant for an agonist that does not include some component of efficacy. It has, however, been stated that all estimates of agonist affinity are fundamentally flawed ( Colquhoun, 1998). Agonist dissociation constants are used in structure/activity studies for drug design, in providing estimates of agonist efficacy (see below) or in mutagenesis studies to examine how ligands and receptors interact. Alternatively, dissociation constants can be estimated using the receptor inactivation method of Furchgott ( Furchgott, 1966 Furchgott and Bursztyn, 1967) or the comparative method of Barlow et al. Agonist dissociation constants can be estimated using in vitro techniques such as ligand-binding assays, providing the assay conditions are carefully controlled (see below). In principle, it should be possible to obtain estimates of agonist affinity in terms of dissociation constants for the binding of ligands to their receptors. GPCRs are of great importance for drug action constituting more than 30% of current drug targets.ĭefining the affinity of agonists at their receptors I shall consider this problem for agonists at G protein-coupled receptors (GPCRs). If these measurements are inaccurate or inadequate, then this has great importance and, in this review, I wish to consider different ways in which affinity and efficacy can be measured. In basic biology studies of receptor structure and function, measurements of affinity and efficacy underpin analyses of ligand/receptor interaction in both SAR and mutagenesis analyses. In the past, this has mainly been in terms of affinity measurements but, more recently, measurements of ligand efficacy have become important ( Williams and Sewing, 2005). In drug discovery, these measurements guide the efforts of medicinal chemists. Accurate measurement of affinity and efficacy for ligands is very important for drug discovery and for basic biology. Agonists are said to have positive efficacy, inverse agonists are said to have negative efficacy and neutral antagonists have zero efficacy. This second attribute has been termed efficacy. Second, the ligand may have effects on the receptor and its associated signalling systems. First, the ligand must bind to the receptor it is said to have affinity for the receptor. The actions of ligands at receptors depend on two fundamental events. It is still, however, necessary to have accurate measures of efficacy in different pathways. The efficacy of a ligand may, therefore, be multidimensional. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. Here I show that E max K obs/EC 50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Efficacy can be quantitated in several ways based on functional data (maximal effect of the agonist ( E max), ratio of agonist dissociation constant to concentration of agonist giving half maximal effect in functional assay ( K obs/EC 50), a combined parameter E max K obs/EC 50). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. Here I show that for many GPCRs, if receptor/G protein coupling is suppressed, experimental measurements of agonist affinity using ligand binding ( K obs) provide quite accurate measures of the agonist microscopic dissociation constant ( K A). It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. ![]()
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