Allosteric phenomena at GPCR detection by radioligand binding

Allosteric phenomena at GPCRs can be evidenced using radioligand binding and functional assays. Usually, they are first detected when experimental data deviate from the expectations of simple (competitive) mass-action kinetics. Yet such findings may also reflect experimental artefacts, including inappropriate drug equilibration times, drug solubility problems, exceedingly high receptor concentrations or perturbation of the surrounding lipid bilayer. This latter mechanism allows many types of sufficiently hydrophobic compounds to non-specifically alter receptor conformation so that they could be mistakenly labelled as 'allosteric modulators'. Hence, control experiments need to be performed to check for such potential sources of interference.

Schild plot

Schild plot

Figure 215 AHosteric modulation of [3H]5-HT binding to 5-HT7 receptors by oleamide. Arrow: shift upon increasing the concentration of oleamide. Right: Schild plot of the same data; the dashed line is the predicted behaviour of a competitive antagonist. Reprinted from Biochemistry and Pharmacology, 58, Hedlund, P. B., Carson, M. J., Sutcliffe, J. G. and Thomas, E. A., Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors, 1807-1813. Copyright (1999), with permission from Elsevier.

Figure 215 AHosteric modulation of [3H]5-HT binding to 5-HT7 receptors by oleamide. Arrow: shift upon increasing the concentration of oleamide. Right: Schild plot of the same data; the dashed line is the predicted behaviour of a competitive antagonist. Reprinted from Biochemistry and Pharmacology, 58, Hedlund, P. B., Carson, M. J., Sutcliffe, J. G. and Thomas, E. A., Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors, 1807-1813. Copyright (1999), with permission from Elsevier.

Allosteric phenomena have repercussions on the three standard radioligand binding assays:

• Saturation binding. A negative allosteric modulator may produce a dose- dependent increase in the radioligand's KD in the same way as a competitive antagonist would do. However, whereas this increase has no limit in the case of a competitive antagonist, it will reach a limit in the case of an allosteric modulator (Figure 215). When the saturation binding curve is plotted on a logarithmic scale, the modulator will produce a dose-dependent rightward shift until a maximum is reached. The corresponding Schild plot will be curvilinear and level off at high concentrations of the allosteric modulator. Importantly, allosteric phenomena depend on the nature of the orthosteric ligand, binding assays with different radioligands can yield different results even when the modulator and the receptor are the same.

• Competition binding. A negative allosteric modulator may produce a dose-dependent decrease in the binding of a fixed concentration of radioligand and the competitive ligand will decrease the binding down to non-specific binding levels. The maximal inhibition produced by a negative allosteric modulator will depend upon the magnitude of the co-operativity factor a as well as on the radioligand concentration (Figure 216): i.e. the maximal inhibition is highest at low radioligand concentrations and for modulators with a high degree of negativity (i.e. a »1). In extreme cases, when a negative allosteric modulator decreases the binding to close to the level of non-specific binding, it may be mistaken for a competitive ligand. Hence a 'complete' displacement to non-specific binding levels does not necessarily implicate competitive antagonism.

Log [A'i Log [B]

Figure 216 Inhibition of radioligand (A*) binding by a negative allosteric modulator (B): effect of decreasing the co-operativity factor and increasing the radioligand concentration. Left side of each figure: saturation binding of A* (on a logarithmic scale!) in the absence or presence of a saturating concentration of B. Right side of each figure: 'competition' binding by B with a constant concentration of [A*] indicated by the red dotted lines. Reproduced from Christopoulos, A. and Kenakin, T. (2002) Pharmacological Reviews, 54, 323-374, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 216 Inhibition of radioligand (A*) binding by a negative allosteric modulator (B): effect of decreasing the co-operativity factor and increasing the radioligand concentration. Left side of each figure: saturation binding of A* (on a logarithmic scale!) in the absence or presence of a saturating concentration of B. Right side of each figure: 'competition' binding by B with a constant concentration of [A*] indicated by the red dotted lines. Reproduced from Christopoulos, A. and Kenakin, T. (2002) Pharmacological Reviews, 54, 323-374, with permission from the American Society for Pharmacology and Experimental Therapeutics.

A convenient way to differentiate both types of interactions is to compare the IC50 values of unlabelled compounds at different radioligand concentrations (Figure 217). Whereas the IC50 values increase proportionally with the radioligand concentration for true competitors, the IC50 values will level off for negative allosteric modulators. On the other hand, a positive allosteric modulator may produce a dose-dependent increase in the binding of a fixed concentration of radioligand (Figure 218). Here again, the maximal increase will depend upon the co-operativity factor a, as well as on the radioligand concentration. Importantly, allosteric interactions are unique for each pair of orthosteric and allosteric ligands involved. A positive allosteric modulator of one particular orthosteric ligand is not necessarily a positive modulator of another orthosteric ligand. For example, alcuronium is a positive modulator for a variety of orthosteric ligands at the M2 muscarinic receptor and a negative modulator for others.

Figure 217 Unlabelled competitors can be discriminated from allosteric modulators in 'competition' binding studies when their IC50 values are plotted as a function of the radiolabelled orthosteric ligand (A*) concentration. Reproduced from Christopoulos, A. and Kenakin, T. (2002) Pharmacological Reviews, 54, 323-374, with permission from the American Society for Pharmacology and Experimental Therapeutics.

Figure 217 Unlabelled competitors can be discriminated from allosteric modulators in 'competition' binding studies when their IC50 values are plotted as a function of the radiolabelled orthosteric ligand (A*) concentration. Reproduced from Christopoulos, A. and Kenakin, T. (2002) Pharmacological Reviews, 54, 323-374, with permission from the American Society for Pharmacology and Experimental Therapeutics.

• Kinetic studies. A change in receptor conformation induced by an allosteric agent is likely to alter the orthosteric ligand association and/or dissociation rate constants (Figure 219). This alteration is responsible for the effects of allosteric modulators on orthosteric ligand affinity at equilibrium. In practice, the study of the kinetic properties of a radioligand often allows very sensitive detection of allosteric interactions at GPCRs. Positive allosteric modulation can increase the association rate and/or decrease the dissociation rate. Negative allosteric modulators act the opposite way; they may decrease the association rate and/or increase the dissociation rate. Compared to association experiments, dissociation experiments are easier to interpret, as they only reflect the dissociation of a preformed

Figure 218 Increase of radioligand (A*) binding by a positive allosteric modulator (B). Left: saturation binding of A* (on a logarithmic scale!) in the absence or presence of a saturating concentration of B. Right: 'competition' binding by B with constant concentration of [A*] indicated by the red dotted line.

Figure 218 Increase of radioligand (A*) binding by a positive allosteric modulator (B). Left: saturation binding of A* (on a logarithmic scale!) in the absence or presence of a saturating concentration of B. Right: 'competition' binding by B with constant concentration of [A*] indicated by the red dotted line.

Time (min)

Time (min)

Figure 219 Increased dissociation of [3H]yohimbine from the human a2A-adrenergic receptor in the presence of the allosteric modulator 5-(N-ethyl-N-isopropyl)-amiloride (EPA). Reproduced from Leppik, R., Lazareno, S., Mynett, A. and Birdsall, N. (1998) Molecular Pharmacology, 53, 916-925, with permission from the American Society for Pharmacology and Experimental Theraputics.

orthosteric ligand-receptor complex. Hence, they provide the simplest and most reliable means to detect allosterism at GPCRs.

Although dissociation experiments are usually straightforward to interpret, they could also give rise to over-interpretation or even false interpretations:

• Claims of co-operative binding based on dissociation kinetics using high-affinity radioligands and/or highly concentrated receptor preparations need to be viewed with caution due to the increased likelihood of 'rebinding' phenomena (see Figure 46).

• Finally, the dissociation rate of an orthostatic drug can also be affected by drugs that interfere with receptor-G protein coupling (in agonist dissociation experiments) or affect the receptor conformation indirectly by perturbing the surrounding lipid bilayer. For example, the AT receptor antagonist candesartan dissociates much faster from its receptors in cell membrane preparations than from the same receptor in intact cells (Figure 220). The same increase in dissociation rate can be observed by treating the cells with minute amounts of filipin (a cholesterol-binding pore-forming agent) and saponin (a detergent).

Figure 220 Dissociation of the antagonist [3H]candesartan from AT receptor-expressing CHO cells and derived cell membranes. Reprinted from Biochemistry and Pharmacology, 63, Fierens, F., Vanderheyden, P.M.L., Roggeman, C., Vande Gucht, P., De Backer, J.-P. and Vauquelin, G., Distinct binding properties of the ATI receptor antagonist [3H]candesartan to intact cells and membrane preparations, 1273-1279, Copyright (2002), with permission from Elsevier.

Figure 220 Dissociation of the antagonist [3H]candesartan from AT receptor-expressing CHO cells and derived cell membranes. Reprinted from Biochemistry and Pharmacology, 63, Fierens, F., Vanderheyden, P.M.L., Roggeman, C., Vande Gucht, P., De Backer, J.-P. and Vauquelin, G., Distinct binding properties of the ATI receptor antagonist [3H]candesartan to intact cells and membrane preparations, 1273-1279, Copyright (2002), with permission from Elsevier.

Detection of allosteric phenomena at GPCRs by functional assays

The cubic 'allosteric two-state model by Hall (2000) describes the interaction of an allosteric modulator and an orthosteric ligand on a receptor that can adopt active (R*) and inactive (R) conformations (Figure 221). This model allows an allosteric ligand to modulate the orthosteric ligand's affinity as well as its intrinsic efficacy. This model applies to any allosteric modulator. In the case of G proteins and allosteric modulators, the model is formally identical with the 'cubic ternary complex model' by Weiss (Weiss et al, 1996).

Figure 221 The cubic 'allosteric two-state model'. The active receptor conformation (R*) is in red. In practice, allosteric interactions between multiple ligands (usually the orthosteric ligand, (A), the G protein and another allosteric ligand) on the same GPCR may be even more complex. Reproduced from Hall, D. (2000) Molecular Pharmacology, 58, 1412-1423, with permission from the American Society for Pharmacology and Experimental Theraputics.

Figure 221 The cubic 'allosteric two-state model'. The active receptor conformation (R*) is in red. In practice, allosteric interactions between multiple ligands (usually the orthosteric ligand, (A), the G protein and another allosteric ligand) on the same GPCR may be even more complex. Reproduced from Hall, D. (2000) Molecular Pharmacology, 58, 1412-1423, with permission from the American Society for Pharmacology and Experimental Theraputics.

Functional assays may allow the detection of specific receptor conformations promoted by allosteric modulators that may have escaped detection in radioligand binding assays. Indeed the affinity and efficacy of an agonist are independent parameters and an allosteric modulator may differentially affect them. Here again it is important to notice that allosterism may differ from one orthosteric ligand to another. Some specific situations are presented below:

• Unchanged efficacy. The allosteric modulator will produce parallel shifts of the concentration-response curves of an orthosteric agonist with no change in basal and maximal responses. The shift is to the left in the case of positive co-operativity and to the right in the case of negative co-operativity. Similar to radioligand binding assays, the shift will attain a limit (defined by a) as the concentration of allosteric modulator increases. However, modulators with high degrees of negative co-operativity (a >> 1) may be mistaken for competitive antagonists. Detection and quantification of negative allosteric modulation is, here again, improved by investigating the effects of as large a range of modulator concentrations as possible. Indeed, it may only be at high concentrations of modulator that the Schild plot deviates for linearity.

• Unchanged potency. Some allosteric modulators will either increase or decrease the maximal response of the orthosteric agonist without changing its potency (Figure 222). The change in maximal response will increase upon increasing the concentration of the allosteric modulator until a limit is attained. It is likely that such modulators also fail to perturb the binding of the radiolabelled agonist to membrane preparations.

Figure 222 Glutamate-mediated inositol phosphate production in CHO cells expressing the me-tabotropic glutamate receptor 1. Effect of increasing concentrations of the allosteric modulator CPCCOEt. Reproduced from Litschig, S., Gasparini, F., Rueegg, D., Stoehr, N., Flor, P. J., Vranesic, I., Prezeau, L., Pin, J.-P., Thomsen, C. and Kuhn, R. (1999) Molecular Pharmacology, 55, 453-461, with permission from the American Society for Pharmacology and Experimental Theraputics.

Figure 222 Glutamate-mediated inositol phosphate production in CHO cells expressing the me-tabotropic glutamate receptor 1. Effect of increasing concentrations of the allosteric modulator CPCCOEt. Reproduced from Litschig, S., Gasparini, F., Rueegg, D., Stoehr, N., Flor, P. J., Vranesic, I., Prezeau, L., Pin, J.-P., Thomsen, C. and Kuhn, R. (1999) Molecular Pharmacology, 55, 453-461, with permission from the American Society for Pharmacology and Experimental Theraputics.

7500

7500

Figure 223 GABA-mediated [35S]GTPyS binding in membranes from human GABAB-receptor -expressing CHO cells. Arrow: effect of increasing concentrations of CGP7930. Reproduced from Urwyler, S., Mosbacher, J., Lingenhoehl, K., Heid, J., Hofstetter, K., Froestl, W., Bettler, B. and Kaupmann, K. (2001) Molecular Pharmacology, 60, 963-971, with permission from the American Society for Pharmacology and Experimental Theraputics.

Figure 223 GABA-mediated [35S]GTPyS binding in membranes from human GABAB-receptor -expressing CHO cells. Arrow: effect of increasing concentrations of CGP7930. Reproduced from Urwyler, S., Mosbacher, J., Lingenhoehl, K., Heid, J., Hofstetter, K., Froestl, W., Bettler, B. and Kaupmann, K. (2001) Molecular Pharmacology, 60, 963-971, with permission from the American Society for Pharmacology and Experimental Theraputics.

• Changed potency and efficacy. For example, CGP7930 increases the potency of the endogenous agonist, GABA, as well as its maximal response (Figure 223).

• Receptor activation by the allosteric modulator (Figure 224). The cubic allosteric two-state model allows the receptor to be activated by an allosteric modulator, even in the absence of an agonist. This may go along with negative, positive or no co-operativity with respect to agonist binding.

Figure 224 Alcuronium is an allosteric modulator of M1 muscarinic receptors. It elicits significant receptor stimulation in the absence of orthosteric agonists. Reprinted from Proceedings of the National Academy of Science USA, 97, Parnot, C., Bardin, S., Miserey-Lenkei, S., Guedin, D., Corvol, P. and Clauser, E., Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay, 7615-7620. Copyright (2000) National Academy of Sciences, USA.

Figure 224 Alcuronium is an allosteric modulator of M1 muscarinic receptors. It elicits significant receptor stimulation in the absence of orthosteric agonists. Reprinted from Proceedings of the National Academy of Science USA, 97, Parnot, C., Bardin, S., Miserey-Lenkei, S., Guedin, D., Corvol, P. and Clauser, E., Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay, 7615-7620. Copyright (2000) National Academy of Sciences, USA.

0 0

Post a comment