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Review
. 2008 Apr;153(7):1353-63.
doi: 10.1038/sj.bjp.0707672. Epub 2008 Jan 28.

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

P G Strange  1
Affiliations
Review

Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors

P G Strange. Br J Pharmacol. 2008 Apr.

Abstract

Measurements of affinity and efficacy are fundamental for work on agonists both in drug discovery and in basic studies on receptors. In this review I wish to consider methods for measuring affinity and efficacy at G protein coupled receptors (GPCRs). Agonist affinity may be estimated in terms of the dissociation constant for agonist binding to a receptor using ligand binding or functional assays. It has, however, been suggested that measurements of affinity are always contaminated by efficacy so that it is impossible to separate the two parameters. 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 (KA). Also in pharmacological functional studies, good estimates of agonist dissociation constants are possible. 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)/EC50), a combined parameter E(max)K(obs)/EC50). Here I show that E(max)K(obs)/EC50 provides the best assessment of efficacy for a range of agonists across the full range of efficacy for full to partial agonists. Considerable evidence now suggests that ligand efficacy may be dependent on the pathway used to assess it. The efficacy of a ligand may, therefore, be multidimensional. It is still, however, necessary to have accurate measures of efficacy in different pathways.

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Figures

Figure 1
Figure 1
The Del Castillo and Katz model for activation of ion channel-linked receptors. In this model, the receptor exists in ground (R) and active (R*) states, with the R* state having a higher affinity for the agonist. KA is the dissociation constant for binding of agonist to the ground state of the receptor and E is the equilibrium constant for the AR/AR* transition (E=[AR*]/[AR]). If ligand-binding studies are applied to this scheme, the experimentally determined dissociation constant of an agonist (Kobs) is given by: Kobs=KA/1+E.
Figure 2
Figure 2
The ternary complex model for agonist/receptor/G protein coupling. In this model, the receptor exists in uncoupled (R) and G protein-coupled (RG) states and the agonist binds with higher affinity to the RG state. Equilibrium association constants (KR, KRG) for agonist binding are shown and J is defined as [RG]/([R][G]). We can define a cooperativity factor α for the effect of G protein coupling on agonist binding so that KRG=αKR. If ligand-binding studies are applied to this scheme with excess G protein over receptor, the experimentally determined dissociation constant of an agonist (Kobs) is given by: Kobs=KA ((1+JG)/(1+αJG)).
Figure 3
Figure 3
Partial activation of a receptor by an agonist. The agonist stabilizes a partially active form of the receptor (R*) so that the measured agonist affinity is not equal to the ground-state affinity. Equilibrium association constants for agonist binding (KR, KR*) are shown and L is defined as [R*]/[R]. We can define a factor β for the effect of the R/R* transition on agonist binding so that KR*=βKR. If ligand-binding studies are applied to this scheme, the experimentally determined dissociation constant of an agonist (Kobs) is given by: Kobs=KA ((1+L)/(1+βL)).
Figure 4
Figure 4
Methods for determination of agonist efficacy. (a) Shows a family of concentration/response curves for a range of agonists with different maximal agonist effects (Emax) in the system used to study their activity. The data were simulated using a ternary complex model (Figure 2) (Alder et al., 2003) with the following parameters: association constant for free receptor KR=104 (M−1); association constant for G protein-coupled receptor KRG (M−1)=109, 108, 3.33 × 107, 107, 5 × 106, 3.33 × 106, 2 × 106, 106, 5 × 105, 3.33 × 105, 105, 3.33 × 104; J=108 M−1; [R]=2 × 10−10 M, [G]=10−10 M. On the basis of these simulations values for Emax and EC50 were determined. Simulations of agonist-binding curves were also performed allowing the dissociation constant of the uncoupled state of the receptor (KA) to be determined (Alder et al., 2003). For agonists that do not give a maximal response, comparison of Emax values provides a measure of relative efficacy. (b) Shows concentration/response curves and binding curves for two agonists, each of which exhibits an Emax value of 100% in the test system. Both agonists have the same association constant for the uncoupled form of the receptor (KR=104 M−1) but different efficacies. The data were simulated using a ternary complex model (Alder et al., 2003) as in panel a with the following parameters: agonist A, KRG=109 M−1; agonist B, KRG=108 M−1. Response curves for agonists A and B are shown together with binding curves for the uncoupled ground-state receptor. Analysis of the simulated data gives KA/EC50 values of 1354 and 131 for agonists A and B, respectively. The ratio of these two values is 10.3 (see panel c). (c) Shows response/occupancy curves for two agonists. The data for responses are taken from the simulations in panel b. Percentage occupancy for each agonist concentration was calculated based on the dissociation constants of the uncoupled states of the receptor. The inverse occupancy ratio at 50% response for the two agonists is 10.2 in good agreement with the ratio of KA/EC50 values. KA, microscopic agonist dissociation constant.
Figure 5
Figure 5
Relation between agonist efficacy and different parameters used experimentally to quantify efficacy. The set of simulated agonist concentration/response curves in Figure 4a was used together with simulated agonist-binding curves (Alder et al., 2003) to derive values for maximal agonist effect (Emax), KA/EC50 and EmaxKA/EC50. The ratio of KRG/KR was defined as α the efficacy in the ternary complex model. Values of KA correspond to the dissociation constant of the uncoupled form of the receptor derived as in Alder et al. (2003). The relationships between α and Emax, KA/EC50 and EmaxKA/EC50 are shown, respectively, in panels a, b and c. In panel a, Emax is expressed as a percentage; in panel c, Emax is expressed as a fraction. KA, microscopic agonist dissociation constant.
Figure 6
Figure 6
Relation between agonist efficacy and different parameters used experimentally to quantify efficacy: analysis of real data. The data for a set of imidazolines for contraction of rat aortic rings via α1D-adrenoceptors (Ruffolo et al., 1979b) have been analysed in terms of the different parameters shown in Figure 5. These functional data are very complete in that a large set of agonists of differing efficacies was used and maximal agonist effect (Emax), Kobs and EC50 were reported. Additionally, functional relative efficacy estimates were obtained using occupancy/response curves. Further data from the same lab examining contraction of aorta from different species (Ruffolo et al., 1979a; Ruffolo and Waddell, 1982, 1983) provide additional support for these results. In each of the studies, there is a good correlation between log EmaxKobs/EC50 and the log functional efficacy (P<0.05). Similar relationships are seen for the set of data for the inhibition of cAMP production via m2 muscarinic receptors (McKinney et al., 1991). If these are analysed in this manner, EmaxKobs/EC50 provides a good estimate of the functional efficacy. Kobs, experimentally determined agonist dissociation constant.
Figure 7
Figure 7
The multidimensional nature of efficacy. Data for the maximal agonist effect (Emax) values for agonists in different assays are plotted as an x/y plot. In (a), data for stimulation of [35S]GTPγS binding by agonists at the D2 dopamine receptor in the presence of Na+ and in the absence of Na+ (with N-methyl-D-glucamine (NMDG) substitution) are given as Emax relative to dopamine (Lin et al., 2006). In (b), data for stimulation of phospholipase C and arachidonic acid (AA) release via agonists at the serotonin 5-HT2C receptor (Moya et al., 2007) are given as Emax relative to 5-hydroxytryptamine.
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References

    1. Alder JT, Hacksell U, Strange PG. Analysis of molecular determinants of affinity and relative efficacy of a series of R- and S-2-(dipropylamino)tetralins at the 5-HT1A serotonin receptor. Br J Pharmacol. 2003;138:1129–1139. - PMC - PubMed
    1. Avalos M, Mak C, Randall PK, Trzeciakowski JP, Abell C, Kwan SW, et al. Nonlinear analysis of partial dopamine agonist effects on cAMP in C6 glioma cells. J Pharmacol Toxicol Methods. 2001;45:17–37. - PubMed
    1. Barlow RB, Scott NC, Stephenson RP. The affinity and efficacy of onium salts on the frog rectus abdominis. Br J Pharmacol Chemother. 1967;31:188–196. - PMC - PubMed
    1. Besse JC, Furchgott RF. Dissociation constants and relative efficacies of agonists acting on alpha adrenergic receptors in rabbit aorta. J Pharmacol Exp Ther. 1976;197:66–78. - PubMed
    1. Black JW, Leff P. Operational models of pharmacological agonism. Proc R Soc London B Biol Sci. 1983;220:141–162. - PubMed