- Email: [email protected]
Endogenous ligands and inverse agonism
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state for some receptors. For example, the binding of p-aminoclonidine to %-adrenoceptors can be shown to decrease the interaction of anti-G,i serum with the G protein, when compared to spontaneous receptor-G protein complexes without agonist binding 1°. These data suggest that the presence of the agonist on the receptor can be recognized by the G protein. Also, there are selected examples where different agonists 'traffic' receptors to bind to different G proteins in the same membrane, suggesting that the activated complexes with these different agonists are not identical n<3. The postulate of separate receptor conformations for agonists is formally indistinguishable from the idea of allosteric effectors. Specifically, if a ligand (allosteric effector) affects the affinity of another ligand, then the receptor could be considered as another conformation, at least with respect to the allosterically modified ligand. Allosteric ligands for many receptors, including GABAA receptors, muscarinic acetylcholine receptors, %-adrenoceptors, dopamine D2 receptors, and adenosine A 1 receptors, have been described (for review see Ref. 14). The only extension
E required for the idea of different References coupling states would be to postulate 1 Black, J. W. (1981) Postgrad. Med. J. 57, 110-112 that the allosterically modified recep- 2 Costa, T. and Herz, A. (1989) Proc. Natl tor had different coupling behaviour Acad. Sci. USA 86, 7321-7325 with G proteins (which is logical in 3 Wyman, J. (1975) Proc. Natl Acad. Sci. USA 72, 3983-3987 view of thermodynamic reversibil- 4 Chidiac, P., Hebert, T. E., Valiquette, M., ity). Therefore, protean ligands could Dennis, M. and Bouvier, M. (1994) Mol. Pharmacol. 44, 490-499 be allosteric effectors that allosteri5 Burgen, A. S. V. (1981) Fed. Proc. 40, cally modify the behaviour of that 2723-2728 receptor with the G protein (for 6 Samama, P., Cotecchia, S., Costa, T. and Lefkowitz, R. I. (1993) ]. Biol. Chem. 268, example, the adenosine receptor 4625-4636 ligand PD81723 stabilizes receptor--G 7 Senogles, S. E., Spiegel, A. M., Pardrell, E., protein interactionslS). Iyengar, R. and Caron, M. (1990) ]. Biol. Chem. 265, 4507-4514 In general, this is still an open 8 Coast, T. and Herz, A. (1989) Proc. Nat/ question but there may be a unique Acad. Sci. USA 86, 7321-7325 way to answer it with the appropri9 Freissmuth, M., Selzer, E. and Schutz, W. (1991) Biochem. J. 275, 651~56 ate tools. If controlled experiments 10 Okuma, Y. and Reisine, T. (1992) J. Biol. can identify pharmacologically proChem. 267, 14826-14831 tean ligands that behave as agonists 11 Spengler, D. et al. (1993) Nature 365, 170--175 under some conditions and inverse 12 Robb, S. et al. (1994) EMBO J. 13,1325-1330 agonists under others, this would 13 Meller, E., Puza, T., Diamond, J., Lieu, suggest that receptor activation and H-D. and Bohmaker, K. (1992)J. Pharmacol. Exp. Ther. 263, 462-469 G protein activation relate to different 14 Birdsall, N. J. M., Cohen, F., Lazareno, S. receptor conformations. It will be and Matsui, H. (1995) Biochem. Soc. Trans. extremely interesting to see, as these 23, 108-111 receptor systems become more 15 Bhattacharya, S. and Linden, J. (1995) Biochim. Biophys. Acta 1265, 15-21 widely used, if such ligands are discovered. Chemical name Terry Kenakin Department of Cellular Biochemistry, Glaxo Wellcorne plc, Five Moore Drive, PO Box 13358, Reseach Triangle Park, NC 27709, USA.
Endogenous ligands and inverse agonism Terry Kenakin continues a theme in which the absence of an agonist has been an essential prerequisite for the generation of a 'modem' biomolecular hypothesis. While the mechanistic detail remains open to debate, the existence of inverse agonists at G protein-coupled receptors now appears to be widely accepted and there are already suggestions that such ligands may have therapeutic potential 1. It is clear, however, that any realization of this potential requires a greater appreciation of the pharmacological features of this putative ligand class. This will allow, for example, definition of the conditions, under which protean ligand behaviour may be of
2 5 8
TiPS - August 1995 (Vol. 16)
analytical value and may, in turn, allow the development of robust classification criteria to aid selection of the most appropriate molecules. The challenge to the analytical pharmacologist is significant, not least, because the behaviour of inverse agonists is in many ways identical to that expected of more conventional ligands (for example, antagonists) in systems in which the stimulus results not from constitutive activity but from the presence of a contaminating agonist. To address this issue, several investigators have attempted to measure levels of potential contaminating agonist directly2,3. Unfortu-
PD81723: 2-amino-4,5-dimethyl-
3-thienyl- [3-(trifluoromethyl)phenyl]methanone
nately, both the experimental observations and the predictions of contemporary pharmacological theory4, indicate that the levels required for stimulation may be very low, and therefore difficult to assess. Some aspects of ligand behaviour may strongly suggest that a contaminating agonist is not an issue2,3,5-7.For example, how, if so-called inverse agonists are simply antagonists, is it possible to obtain different levels of inverse intrinsic activityZ3,,~7? How can the existence of antagonistsZ3,6, which block the actions of both agonists and inverse agonists alike, be explained? Such observations can be accommodated by modifying established models of G protein-coupled receptor signalling5. However, it is also possible to simulate such behaviour by assuming the existence of a
© 1995, Elsevier Science Ltd
D contaminating agonist and applying existing models, without fundamental modification. Indeed, differing levels of negative intrinsic activity can be simulated using a model originally developed to quantify the interaction between a full and partial agonist 4. If it is assumed that a contaminating agonist is present, then the efficacy, relative to the contaminant, of any added ligand dictates whether the ligand acts as an agonist or inverse agonist and the level of inverse intrinsic activity attained. The premise of this argument is that each of the antagonists, by virtue of an increased efficacy in the recombinant or mutated system, now have the capacity to impart positive stimuli. This transition does not challenge conventional wisdom. Modification of the model to account for the introduction of a third agonist species (using a similar algebraic argument4), permits simulation of antagonists. In this case the
so-called antagonist is a partial agonist whose maximum capacity to stimulate matches that imparted by the contaminant. If it is possible to simulate the behaviour both of inverse agonists and antagonists using a combination of two or more conventional ligands, such behaviour cannot be regarded as robust evidence for their classification. Likewise, the suggestion by Kenakin that a proteanligand may be of some analytical value is also open to question as such behaviour would presumably occur in the face of variable levels of endogenous agonist. In this regard, while it may be considered unlikely that contaminating agonist is a factor in either isolated cells or membranes (although this too may be open to debate), its presence in isolated tissues or in vivo is certainly a realistic proposition. Until appropriate and reliable discriminatory criteria are developed, it
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Molecular mechanisms of agonism Agonists determine receptor conformation and hence G protein affinity In a recent issue of TiPSLeft described the application of a two-state model of receptor activation 1. In this model he describes a G protein-coupled receptor as an entity that can be found in two states, an inactive state (R) and an active state (R'); the receptor can only interact with a G protein when in the R" state. This is a simplification of the ternary complex model 2 that explains how agonists allow a receptor to interact with G proteins. It has been shown that the affinity of the receptor for G protein is dependent upon the agonist boundZ3. This would imply that different agonists cause the receptor to take up different conformations that have different affinities for G proteins, and presumably inverse agonists have the opposite effect. This does not agree with the model being
proposed by Leffi for the action of ligands, where the receptor would appear to have a fixed affinity for the G protein when in the R" state, and ligands affect the proportion of the receptors found in the R* state rather than the affinity of receptor for G protein. If the receptor is considered to be a flexible entity, as suggested by the studies of De Lean and colleagues 2, and Costa and co-workers 3, among others, rather than a rigid entity that exists in two states 1 then the interaction of the receptor with the G protein needs to be visualized in an alternative maimer. Bourne and colleagues 4 proposed that the receptor can be considered as a guanine nucleotide releasing protein that catalyses the exchange of GDP for
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is not only the therapeutic potential, but, in such systems, the existence of the inverse agonist that must remain somewhat speculative. Gordon S. Baxter and Nick S. Tilford Neurology Research Department, SmithKline Beecham, New Frontiers Science Park, Third Avenue, Harlow, UK CM19 SAW. References 1 Milligan, G., Bond, R. A. and Lee, M (1995) Trends Pharmacol. Sci. 16, 10-13 2 Bond, R. A. et al. (1995) Nature 374, 272-276 3 Barker, E. L., Westphal, R. S., Schmidt, D. and Sanders-Bush, E. (1994) J. Biol. Chem. 269, 11687-11690 4 Left, P., Dougall, I. G. and Harper, D. (1993) Br. J. Pharmacol. 110, 239-244 5 Samama, P., Pei, G., Costa, T., Cotecchia, S. and Lefkowitz, R. J. (1994) Mol. Pharmacol. 45, 390-394 6 Costa, T. and Herz, A. (1989) Proc. Natl Acad. Sci. USA 86, 7321-7325 7 Chidiac, P., Hebert, T. E., Valiquette, M., Dennis, M. and Bouvier, M (1993) Mol. Pharmacol. 45, 490-499
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GTP at the interacting G protein. Under this scheme a G proteincoupled receptor can be considered an allosterically regulated enzyme, an agonist would be a positive allosteric modulator, an inverse agonist a negative allosteric modulator, and an antagonist neutral, that is, it has no effect on the GDP-GTP exchange rate. This is consistent with the model of agonist-inverse agonist action proposed by Costa and colleagues 3, which is based on the ternary complex model. The action of an agonist is to increase the affinity of the receptor for the G protein, an inverse agonist would decrease the affinity of the receptor for the G protein, and an antagonist does not affect the affinity of the receptor for the G protein. The essential differences between this model and that proposed by Left for the action of agonists and inverse agonists, is that it assumes a 'flexible' rather than a'rigid' receptor and, that agonists and inverse agonists affect
TiPS - August 1995 (Vol. 161 2 5 9
E
B
A
T
state for some receptors. For example, the binding of p-aminoclonidine to %-adrenoceptors can be shown to decrease the interaction of anti-G,i serum with the G protein, when compared to spontaneous receptor-G protein complexes without agonist binding 1°. These data suggest that the presence of the agonist on the receptor can be recognized by the G protein. Also, there are selected examples where different agonists 'traffic' receptors to bind to different G proteins in the same membrane, suggesting that the activated complexes with these different agonists are not identical n<3. The postulate of separate receptor conformations for agonists is formally indistinguishable from the idea of allosteric effectors. Specifically, if a ligand (allosteric effector) affects the affinity of another ligand, then the receptor could be considered as another conformation, at least with respect to the allosterically modified ligand. Allosteric ligands for many receptors, including GABAA receptors, muscarinic acetylcholine receptors, %-adrenoceptors, dopamine D2 receptors, and adenosine A 1 receptors, have been described (for review see Ref. 14). The only extension
E required for the idea of different References coupling states would be to postulate 1 Black, J. W. (1981) Postgrad. Med. J. 57, 110-112 that the allosterically modified recep- 2 Costa, T. and Herz, A. (1989) Proc. Natl tor had different coupling behaviour Acad. Sci. USA 86, 7321-7325 with G proteins (which is logical in 3 Wyman, J. (1975) Proc. Natl Acad. Sci. USA 72, 3983-3987 view of thermodynamic reversibil- 4 Chidiac, P., Hebert, T. E., Valiquette, M., ity). Therefore, protean ligands could Dennis, M. and Bouvier, M. (1994) Mol. Pharmacol. 44, 490-499 be allosteric effectors that allosteri5 Burgen, A. S. V. (1981) Fed. Proc. 40, cally modify the behaviour of that 2723-2728 receptor with the G protein (for 6 Samama, P., Cotecchia, S., Costa, T. and Lefkowitz, R. I. (1993) ]. Biol. Chem. 268, example, the adenosine receptor 4625-4636 ligand PD81723 stabilizes receptor--G 7 Senogles, S. E., Spiegel, A. M., Pardrell, E., protein interactionslS). Iyengar, R. and Caron, M. (1990) ]. Biol. Chem. 265, 4507-4514 In general, this is still an open 8 Coast, T. and Herz, A. (1989) Proc. Nat/ question but there may be a unique Acad. Sci. USA 86, 7321-7325 way to answer it with the appropri9 Freissmuth, M., Selzer, E. and Schutz, W. (1991) Biochem. J. 275, 651~56 ate tools. If controlled experiments 10 Okuma, Y. and Reisine, T. (1992) J. Biol. can identify pharmacologically proChem. 267, 14826-14831 tean ligands that behave as agonists 11 Spengler, D. et al. (1993) Nature 365, 170--175 under some conditions and inverse 12 Robb, S. et al. (1994) EMBO J. 13,1325-1330 agonists under others, this would 13 Meller, E., Puza, T., Diamond, J., Lieu, suggest that receptor activation and H-D. and Bohmaker, K. (1992)J. Pharmacol. Exp. Ther. 263, 462-469 G protein activation relate to different 14 Birdsall, N. J. M., Cohen, F., Lazareno, S. receptor conformations. It will be and Matsui, H. (1995) Biochem. Soc. Trans. extremely interesting to see, as these 23, 108-111 receptor systems become more 15 Bhattacharya, S. and Linden, J. (1995) Biochim. Biophys. Acta 1265, 15-21 widely used, if such ligands are discovered. Chemical name Terry Kenakin Department of Cellular Biochemistry, Glaxo Wellcorne plc, Five Moore Drive, PO Box 13358, Reseach Triangle Park, NC 27709, USA.
Endogenous ligands and inverse agonism Terry Kenakin continues a theme in which the absence of an agonist has been an essential prerequisite for the generation of a 'modem' biomolecular hypothesis. While the mechanistic detail remains open to debate, the existence of inverse agonists at G protein-coupled receptors now appears to be widely accepted and there are already suggestions that such ligands may have therapeutic potential 1. It is clear, however, that any realization of this potential requires a greater appreciation of the pharmacological features of this putative ligand class. This will allow, for example, definition of the conditions, under which protean ligand behaviour may be of
2 5 8
TiPS - August 1995 (Vol. 16)
analytical value and may, in turn, allow the development of robust classification criteria to aid selection of the most appropriate molecules. The challenge to the analytical pharmacologist is significant, not least, because the behaviour of inverse agonists is in many ways identical to that expected of more conventional ligands (for example, antagonists) in systems in which the stimulus results not from constitutive activity but from the presence of a contaminating agonist. To address this issue, several investigators have attempted to measure levels of potential contaminating agonist directly2,3. Unfortu-
PD81723: 2-amino-4,5-dimethyl-
3-thienyl- [3-(trifluoromethyl)phenyl]methanone
nately, both the experimental observations and the predictions of contemporary pharmacological theory4, indicate that the levels required for stimulation may be very low, and therefore difficult to assess. Some aspects of ligand behaviour may strongly suggest that a contaminating agonist is not an issue2,3,5-7.For example, how, if so-called inverse agonists are simply antagonists, is it possible to obtain different levels of inverse intrinsic activityZ3,,~7? How can the existence of antagonistsZ3,6, which block the actions of both agonists and inverse agonists alike, be explained? Such observations can be accommodated by modifying established models of G protein-coupled receptor signalling5. However, it is also possible to simulate such behaviour by assuming the existence of a
© 1995, Elsevier Science Ltd
D contaminating agonist and applying existing models, without fundamental modification. Indeed, differing levels of negative intrinsic activity can be simulated using a model originally developed to quantify the interaction between a full and partial agonist 4. If it is assumed that a contaminating agonist is present, then the efficacy, relative to the contaminant, of any added ligand dictates whether the ligand acts as an agonist or inverse agonist and the level of inverse intrinsic activity attained. The premise of this argument is that each of the antagonists, by virtue of an increased efficacy in the recombinant or mutated system, now have the capacity to impart positive stimuli. This transition does not challenge conventional wisdom. Modification of the model to account for the introduction of a third agonist species (using a similar algebraic argument4), permits simulation of antagonists. In this case the
so-called antagonist is a partial agonist whose maximum capacity to stimulate matches that imparted by the contaminant. If it is possible to simulate the behaviour both of inverse agonists and antagonists using a combination of two or more conventional ligands, such behaviour cannot be regarded as robust evidence for their classification. Likewise, the suggestion by Kenakin that a proteanligand may be of some analytical value is also open to question as such behaviour would presumably occur in the face of variable levels of endogenous agonist. In this regard, while it may be considered unlikely that contaminating agonist is a factor in either isolated cells or membranes (although this too may be open to debate), its presence in isolated tissues or in vivo is certainly a realistic proposition. Until appropriate and reliable discriminatory criteria are developed, it
L
E
Molecular mechanisms of agonism Agonists determine receptor conformation and hence G protein affinity In a recent issue of TiPSLeft described the application of a two-state model of receptor activation 1. In this model he describes a G protein-coupled receptor as an entity that can be found in two states, an inactive state (R) and an active state (R'); the receptor can only interact with a G protein when in the R" state. This is a simplification of the ternary complex model 2 that explains how agonists allow a receptor to interact with G proteins. It has been shown that the affinity of the receptor for G protein is dependent upon the agonist boundZ3. This would imply that different agonists cause the receptor to take up different conformations that have different affinities for G proteins, and presumably inverse agonists have the opposite effect. This does not agree with the model being
proposed by Leffi for the action of ligands, where the receptor would appear to have a fixed affinity for the G protein when in the R" state, and ligands affect the proportion of the receptors found in the R* state rather than the affinity of receptor for G protein. If the receptor is considered to be a flexible entity, as suggested by the studies of De Lean and colleagues 2, and Costa and co-workers 3, among others, rather than a rigid entity that exists in two states 1 then the interaction of the receptor with the G protein needs to be visualized in an alternative maimer. Bourne and colleagues 4 proposed that the receptor can be considered as a guanine nucleotide releasing protein that catalyses the exchange of GDP for
E
B
A
T
E
is not only the therapeutic potential, but, in such systems, the existence of the inverse agonist that must remain somewhat speculative. Gordon S. Baxter and Nick S. Tilford Neurology Research Department, SmithKline Beecham, New Frontiers Science Park, Third Avenue, Harlow, UK CM19 SAW. References 1 Milligan, G., Bond, R. A. and Lee, M (1995) Trends Pharmacol. Sci. 16, 10-13 2 Bond, R. A. et al. (1995) Nature 374, 272-276 3 Barker, E. L., Westphal, R. S., Schmidt, D. and Sanders-Bush, E. (1994) J. Biol. Chem. 269, 11687-11690 4 Left, P., Dougall, I. G. and Harper, D. (1993) Br. J. Pharmacol. 110, 239-244 5 Samama, P., Pei, G., Costa, T., Cotecchia, S. and Lefkowitz, R. J. (1994) Mol. Pharmacol. 45, 390-394 6 Costa, T. and Herz, A. (1989) Proc. Natl Acad. Sci. USA 86, 7321-7325 7 Chidiac, P., Hebert, T. E., Valiquette, M., Dennis, M. and Bouvier, M (1993) Mol. Pharmacol. 45, 490-499
T
T
E
R
S
GTP at the interacting G protein. Under this scheme a G proteincoupled receptor can be considered an allosterically regulated enzyme, an agonist would be a positive allosteric modulator, an inverse agonist a negative allosteric modulator, and an antagonist neutral, that is, it has no effect on the GDP-GTP exchange rate. This is consistent with the model of agonist-inverse agonist action proposed by Costa and colleagues 3, which is based on the ternary complex model. The action of an agonist is to increase the affinity of the receptor for the G protein, an inverse agonist would decrease the affinity of the receptor for the G protein, and an antagonist does not affect the affinity of the receptor for the G protein. The essential differences between this model and that proposed by Left for the action of agonists and inverse agonists, is that it assumes a 'flexible' rather than a'rigid' receptor and, that agonists and inverse agonists affect
TiPS - August 1995 (Vol. 161 2 5 9