Concluding remarks

In summary, the investigation of GPCRs first relied on physiological approaches, then on biochemical approaches and nowadays on genetic approaches. The introduction of each new approach has always provided an impulse for the discovery of new receptors or receptor subtypes. These discoveries are very beneficial for the medical treatment of diseases, since it authorizes the use of more and more selective drugs with, hence, fewer possible side effects.

Our understanding of GPCR function has substantially improved during the last decade. It changed the way we look at GPCRs (Figure 232). They are no longer simple 'on/off' switches, but highly dynamic structures that exist in equilibrium between active and inactive conformations. An agonist is recognized as a molecule that can stabilize an active conformation, while an inverse agonist (i.e. an antagonist with negative intrinsic activity) is a molecule that can stabilize an inactive conformation. Thus, it has become clear that not only agonists, but also antagonists, are capable of actively modulating receptor function. Moreover, it has become evident that neither agonists nor antagonists necessarily have to share an overlapping binding site, even if they act at the same receptor.

Moreover, GPCRs no longer exclusively act as monomers, nor do they have to exclusively activate G proteins to produce cell signalling.

Genes coding for GPCR-like proteins have been discovered for some time and the recent sequencing of the human genome unveiled an even greater number of them. Based on the assumption that these proteins function as receptors, much effort is being spent to find their natural messengers as well as other ligands. Traditionally, orphan GPCR ligand identification relies on their expression in an appropriate cell line followed by their exposure to libraries of naturally occuring compounds. This task appears to be notoriously difficult, especially since it does not permit the discovery of antagonist molecules. Constitutively active orphan receptors may be obtained by mutagenesis. They are likely to be more sensitive to agonists, including their natural messenger(s). As they evoke a signal in the absence of agonist, they may respond to inverse agonists as well (Figure 233).

Whereas agonist and antagonist drugs have mainly been discussed in terms of efficacy and potency, there is now a growing tendency to pay attention to their kinetic properties as well. The kinetic properties of the drug-receptor complex, along with pharmacokinetic issues, will determine the drug residence time at its

G Protein- Coupled Receptors: Molecular Pharmacology From Academic Concept to Pharmaceutical Research Georges Vauquelin and Bengt von Mentzer © 2007 John Wiley & Sons, Ltd. ISBN: 978-0-470-51647-8

Figure 232 Molecular cloning, biochemical, immunological and spectroscopic techniques have led to dramatic advancements of our knowledge about G protein-coupled receptor behaviour. Specific issues (a to f) have been outlined in the preceding chapters. Reprinted from Trends in Pharmacological Science, 25, Kenakin, T., Principles: receptor theory in pharmacology, 186-192. Copyright (2004), with permission from Elsevier.

Figure 232 Molecular cloning, biochemical, immunological and spectroscopic techniques have led to dramatic advancements of our knowledge about G protein-coupled receptor behaviour. Specific issues (a to f) have been outlined in the preceding chapters. Reprinted from Trends in Pharmacological Science, 25, Kenakin, T., Principles: receptor theory in pharmacology, 186-192. Copyright (2004), with permission from Elsevier.

target/receptor and this will have profound consequences on its in vivo efficacy and effect duration (Copeland et al., 2006). Whereas drug structure-activity relationship (SAR) studies often merely rely on determinations of their binding affinity and functional effect, these authors recommend an additional screen for the kinetic properties of the drug-receptor interaction. For certain pathologies, such as hypertension, it is now widely accepted that a permanent reduction in blood pressure is beneficial for the patient. Among the drugs which are currently used to this end, the AT receptor antagonist, candesartan, has been shown to dissociate quite slowly from its receptor in in vitro studies (Figure 229) and this process is likely to contribute to its long-lasting clinical effect. Conversely, so-called 'atypical antipsychotics' like clozapine and quetiapine produce fewer extrapyramidal side effects (i.e. symptoms that are similar to those that occur in the disease state of Parkinsonism) as the classical neuroleptics. This is thought to be related to the ability to dissociate rapidly from the D2 receptors so that, in case of a surge of endogenous dopamine in the striatum, physiological dopamine transmission can still take place, to some extent.

No constitutive activity Constitutive activity

No constitutive activity Constitutive activity

drug concentration drug concentration

Figure 233 Production of constitutively activated orphan receptors for the discovery of agonist and inverse agonist ligands (D = detectable in functional studies).

drug concentration drug concentration

Figure 233 Production of constitutively activated orphan receptors for the discovery of agonist and inverse agonist ligands (D = detectable in functional studies).

Another emerging issue is receptor polymorphism. The etiology of many diseases remains unknown, but in major psychiatric conditions such as depression, bipolar disorder and schizophrenia, there is a higher concordance rate for the disease in monozy-gotic versus dizygotic twins. This suggests that genetic factors are involved. Among the potential causative genes, GPCRs are likely to play a primordial role. In support of this allegation, pharmacogenomic studies suggest that the 5-HT2A receptor might be involved in the pathophysiology of hallucinations in humans. Interestingly, metaanalysis of several clinical studies (including 373 patients who responded to the treatment and 360 non-responders) revealed that patients with His452Tyr-5-HT2A receptors were less likely to respond satisfactorily to clozapine (Table 27). This suggests that GPCR polymorphism may also play a significant role in determining drug response.

Table 27 Meta-analysis of studies on genetic variation in 5-HT2A receptors and clozapine response. R is responder, NR is non-responder. Reprinted from Schizophrenia Research, 32, Arranz, M. J., Munro, J., Sham, P., Kirov, G., Murray, R. M., Collier, D. A. and Kerwin, R. W., Meta-analysis of studies on genetic variation in 5-HT2A receptors and clozapine response, 93-99. Copyright (1998), with permission from Elsevier.

Table 27 Meta-analysis of studies on genetic variation in 5-HT2A receptors and clozapine response. R is responder, NR is non-responder. Reprinted from Schizophrenia Research, 32, Arranz, M. J., Munro, J., Sham, P., Kirov, G., Murray, R. M., Collier, D. A. and Kerwin, R. W., Meta-analysis of studies on genetic variation in 5-HT2A receptors and clozapine response, 93-99. Copyright (1998), with permission from Elsevier.

His452Tyr

R (%)

NR (%)

Genotype

His452/His452

320 (85)

238 (79)

His452/Tyr452

52 (14)

56 (18)

Tyr452/Tyr452

2 (1)

8 (3)

Total

374

302

Table 28 Pharmacological testing systems. Reproduced from Kenakin, T. (1996) Pharmacological Reviews, 48, 413-463, with permission from the American Society for Pharmacology and Experimental Theraputics.

Evolution in pharmacological research

Pharmacological receptor testing systems

Animal receptors-animal tissues

Animal genetic receptor material-animal surrogate cells

Human genetic receptor material-animal surrogate cells

Human genetic receptor material-human surrogate cells

Human genetic receptor material-human target cells

Human genetic receptor material-human target cells with appropriate pathology

Pharmacological research at the cellular and molecular levels is still likely to be different in five or ten years from now. Some of the developments pharmacologists dream of are:

• Testing of drugs on the human receptor in exactly the correct tissue under the appropriate pathology (Table 28). Currently, the state of the art mainly resides in systems where human receptor material (i.e., cDNA) coding for receptors is introduced into surrogate cells. This only constitutes a step toward the desired total correspondence between drug and disease.

• The development of the ideally fitting drug based on molecular modelling data without needing long and expensive structure-activity relationship studies. For this purpose, much effort is nowadays devoted towards the determination of the exact molecular structure of the receptors and especially of the binding sites of agonists and antagonists.

• The elucidation of the physiological role of orphan receptors and their potential implication into pathophysiological situations. They might constitute targets for new classes of drugs and, hence, allow new avenues in clinical therapy.

We hope that this book has given a foundation for a deeper understanding of current pharmaceutical research.

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