Pharmacological proteus?

CURRENT

A W A R E N E S S

should greatly facilitate the search for subtype selective prostanoids with novel therapeutic applications. Selected references

Acknowledgments The authors acknowledge the financial support of Allergan, RW Johnson, the National Institute of Health, the Arizona Affiliate of the American Heart Association, and the National Science Foundation.

1 Coleman,R.A.,Smith,W.L.andNarumiya, S. (1994)Pharmacol. Rev.46, 205-229 2 Hirata,M. et al. (1991)Nature 349,617-620 3 Abramovitz,M. et al. (1994)J. BioL Chem. 269,2632-2636 4 Boie, Y. et al. (1994) J. Biol. Chem. 269, 12173-12178 5 Funk,C. D. etal. (1993)J. Biol. Chem. 268, 26767-26772 6 Regan,J. W.et al. (1994)MoL Pharmacol. 46, 213-220 7 Regan,J. W. et al. (1994) Br. ]. Pharmacol. 112,377-385 8 Bastien,L., Sawyer, N., Grygorczyk,R., Metters,K.M. and Adam,M. (1994)J. Biol. Chem. 269,11873-11877

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9 Hirata, M., Kakizuka, A., Aizawa, M., Ushikubi, F. and Narumiya, S. (1994) Proc. Natl Acad. Sci. USA 91, 11192-11196 10 Funk, C. D., Furci, L., Moran, N. and Fitzgerald, G. A. (1993) Mol. Pharmacol. 44 934-939 11 Honda, A. et al. (1993) J. Biol. Chem. 268, 7759-7762 12 Nishigaki, N. et al. (1995) FEBS Lett. 364, 339-341 13 Sugimoto, Y. et al. (1993) J. Biol. Chem. 268, 2712-2718 14 Namba, T. et al. (1993) Nature 365, 166-170 15 An, S., Yang, J., So, S. W., Zeng, L. and Goetzl, E. J. (1994) Biochemistry 33, 14496-14502 16 Schmid, A., Thierauch, K., Schleuning, W. and Dinter, H. (1995) Eur. J. Biochem. 228, 23-30

17 Breyer, R. M. et al. (1994) J. Biol. Chem. 269, 6163-6169

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some sort is needed. However, the simplest model of this kind I cannot account for system-dependent variation between agonism and inverse agonism. This behaviour requires extension of the model in the way described below by Terry Kenakin. However, to accept the need for a more complex model we need to be sure that experimental data demand it. Gordon Baxter and Nick Tilford draw attention to the practical

Pharmacological Proteus? One of the central themes in pharmacology is the nature of efficacy. Another has been the proper taxonomy of drugs, that is, the full characterization of the receptor properties of ligands 1. A theoretical new class of receptor ligand may relate to both of these primary questions. The discovery of constitutively active receptor systems and the observation of inverse agonism has raised the possibility for the detection of a new class of ligand. While it is clear that a ligand that destabilizes receptor-G protein coupling, or selectively binds to the inactivated form of the receptor (or both), will be an inverse agonist, theoretically there

Chemical name

18 Lefkowitz, R. J. (1993) Cell 74, 409-412

Inverse agonism: theory and practice The concepts of constitutive receptor activation and inverse agonism are currently the subject of considerable attention. One of the crucial issues that has arisen is whether a ligand can act as an agonist in one system but as an inverse agonist in another. This question needs to be addressed both from a theoretical and a practical point of view. If inverse agonism is accepted as an experimental reality, a two-state receptor model of

19 Negishi, M., Sugimoto, Y., lrie, A., Narumiya, S. and Ichikawa, A. (1993) J. Biol. Chem. 268, 9517-9521 20 Raychowdhury, M. K. et al. (1994) ]. Biol. Chem. 269, 19256-19261 21 Halushka, P. V., Mais, D. E., Mayeux, P. R. and Morinelli, T. A. (1989) Annu. Rev. Pharmacol. Toxicol. 10, 213-239 22 Toh, H., Ichikawa, A. and Narumiya, S. (1995) FEBS Lett. 361, 17-21 23 Niising, R. M. et al. (1993) J. Biol. Chem. 268, 25253-25259

exists the possibility that there are ligands that produce a receptor state that can activate G proteins, but to a lesser extent than the spontaneous active state. Under these circumstances, a spectrum of activity from partial agonism to inverse agonism could be observed. The response to such a ligand would be protean (likened to the Greek god of mythology Proteus who could assume different forms at will) and dependent upon system parameters, not receptor type. In receptor systems where there is little spontaneous formation of the activated receptor state, the receptor-activating property of the ligand will dominate and positive

difficulty of excluding the presence of contaminating agonists. On this basis, they provide an explanation for such variation in agonist activity which avoids complication of the model. Paul Left Astra Research Loughborough, Bakewell Road, Loughborough, UK LE11ORH. Reference

1 Left, P. (1995) Trends Pharmacol. Sci. 16, 89-97

agonism will be observed. In systems where there is considerable formation of the activated receptor state (or the stimulus-response mechanism is tuned to the observation of the resultant of the activated complexes), conversion to the inactivated receptor form will dominate and inverse agonism will result. There would be a spectum of sytem conditions where this would occur therefore a point would also exist for the observance of antagonism. The factors that dictate when these properties will be observed are : (1) the magnitude of the allosteric constant describing the equilibrium between active and inactive receptors; and (2) the stoichiometric ratio of receptors to G proteins.

© 1994, Elsevier Science Ltd

D Figure 1 shows simulation data with a ternary complex model of a receptor system existing in an active (R*) and inactive (R) state coupling to a single type of G protein (G). It is assumed that the ligand produces a new active state that activates G proteins but has a lower association constant than the equilibrium constant between R* and R*G (#kc). This was affected by reducing the ~ term in the model. The effects of such a ligand with changing kact (allosteric constant for [R]/IR*]) can be simulated (Fig. lb). This can be likened to differences between receptors in intact cells and membrane preparations, or under different ionic conditions ]that is removal of Na ÷ (Ref. 2)]. Analogous results can be simulated with changes in receptor level. There are arguments for and against such an idea. On the supportive side, thermodynamically there is no reason to suppose that a ligandbound activated receptor should be identical to an activated receptor with no ligand bound. In fact, the theory of microscopic reversibility 3 dictates that the ligand-bound receptor must be considered differently. Perhaps, more importantly, there are suggestions from experimental data that such compounds already have been found. For example, the #-adrenoceptor ligand dichlorisoprenaline is known to be a partial agonist in many systems, but in Spodoptera fugiperda (Sf9) cells overexpressing ]32-adrenoceptors, it demonstrates inverse agonism 4. On the negative side, the existence of such a compound supposes a separate activated receptor conformation when bound to some ligands, which is a more complicated idea from a mechanism of conformational selection for efficacys, that is, a single activated state, the preponderance of which is enhanced by selective agonist binding. A parsimonious view would suggest that there is no need to postulate a separate agonistactivated state, and that only increases in the spontaneous activated state is enough to describe ligand agonism. There is evidence to show that receptors that spontaneously

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produce the activated form can cause the production of second messenger (that is, cAMP (Ref. 6), phosphoinositol turnover 7) and directly catalyse guanine nucleotide G protein exchange s,9, all in the absence of agonist. At present, there is little

evidence to demonstrate that an agonist-bound receptor is a different species from a spontaneously activated receptor, with respect to G protein coupling. However, there are data that cannot be explained by a single activated

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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

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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