- Email: [email protected]
Affinity and efficacy of selective agonists and antagonists for vasopressin and oxytocin receptors: an “easy guide” to receptor pharmacology
I.D. Neumann and R. Landgraf (Eds.) Progress in Brain Research, Vol. 170 ISSN 0079-6123 Copyright r 2008 Elsevier B.V. All rights reserved
CHAPTER 38
Affinity and efficacy of selective agonists and antagonists for vasopressin and oxytocin receptors: an ‘‘easy guide’’ to receptor pharmacology Bice Chini1,, Maurice Manning2 and Gilles Guillon3 1 CNR Institute of Neuroscience, Milan, Italy Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH, USA 3 Institut de Ge´nomique Fonctionnelle, UMR5203-CNRS, INSERM U661, Universite´ Montpellier I, Universite´ Montpellier II, Montpellier, France 2
Abstract: The development of ‘‘selective’’ drugs targeting oxytocin/vasopressin receptors has enormously progressed since the original synthesis of oxytocin more than 50 years ago. However, several factors still hamper the availability of a rich and complete range of selective agonists and antagonists acting at the different oxytocin/vasopressin receptor subtypes, making the use of these drugs still a daunting task. In this paper we will briefly review the major problems encountered when dealing with oxytocin/vasopressin selective ligands, proving few rules for their correct pharmacological use, in order to avoid common pitfalls. Finally, we will glimpse at new challenges, such us the discovery of coupling selective ligands, which foster the search for new classes of selective compounds. Keywords: vasopressin; oxytocin; agonist; antagonist; selectivity; species difference
The design and use of selective agonists and antagonists acting at the oxytocin/vasopressin (OT/AVP) receptors has been and continues to be a difficult task for two principal reasons:
are responsible for important changes in the selectivity profile of some ligands; for this reason, the pharmacological data obtained in one species cannot be extrapolated ‘‘tout court’’ to other species.
The high sequence homology among the AVP and OT V1a/V1b/V2 and OT receptor subtypes and the high degree of chemical similarity among the active ligands often results in overlapping selectivity profiles. The presence of subtle differences among receptor sequences in various animal species
Here we propose a quick guide to the use of ‘‘selected’’ useful drugs together with few key principles to be followed to design and perform pharmacological assays on OT/AVP receptors. We will not consider here the use of selective analogues in in vivo bioassays, as this is the subject of an extensive review that also appears in this issue, to which we refer for a historical and critical evaluation of the different analogues (Manning et al., 2008).
Corresponding author. Tel.: +39 02 50316958; Fax: +39 02 7490574; E-mail: [email protected]
DOI: 10.1016/S0079-6123(08)00438-X
513
514
Let us first discuss the operational definition of a ‘‘selective’’ ligand. In current pharmacological terms, a selective ligand is defined on the basis of either its high affinity or biological activity on a specific receptor when compared to the others (Kenakin, 2003). However, it is to be kept in mind that all these structurally related analogues lack an absolute receptor subtype selectivity; their receptor subtype selectivity is always relative and concentration dependent. As selectivity is defined on the basis of the capability of a single analogue to bind to and/or activate a single OT/AVP receptor subtype, it is of course an absolute requirement for these analogues to have been tested on all the OT/AVP receptor subtypes within the species under investigation. Unfortunately, pharmacological data are often partial and, for several analogues, the full characterization on all the OT/AVP receptor subtypes is
missing, making their use as ‘‘selective’’ analogues impossible. Selectivity may be defined on the basis of binding affinity of the ligand. In this case, because the affinity of the natural ligands, OT or AVP, defined by their affinity constants (Kd), is in the nanomolar range, a good ligand should possess an affinity, again defined by its affinity constant (Kd or Ki), in the same range. To be ‘‘selective’’ on one receptor subtype, its affinity constant on the other three members of the family should be at least two orders of magnitude higher. Using these very tight criteria, the most selective ligands for the human and the rat OT/AVP receptors are listed in Fig. 1 (Ahrabi et al., 2007; Guillon et al., 2004; MacSweeney et al., 2007; Murat et al., 2007; Pena et al., 2007; Serradeil-Le Gal et al., 2004, 2007). A crucial issue concerning the use of these analogues is their availability; as some of these
Fig. 1. Highly selective receptor subtype OT/AVP ligands. To be included in this figure, a selective ligand fulfilled the following pharmacological criteria: (1) to have been tested on all the OT/AVP receptor subtypes (OTR, V1a, V1b, V2) present in a single species, (2) to possess an affinity (defined by its affinity constants Kd or Ki) in the nanomolar range for the receptor subtype it is specific for and (3) to possess an affinity constant at least two orders of magnitude higher for the three other receptor subtypes. The affinity constant of the analogue for its selective receptor subtype, expressed in nanomolar, is reported in round brackets with the appropriate reference in square brackets.
515
analogues are not commercially available, restrictions in their supply would greatly hamper further scientific advancements in the field. Another important point that clearly emerges from Fig. 1 is the lack of data concerning ligand selectivity in mice. Despite the huge relevance in many areas of basic and preclinical science of genetically modified OT/AVP peptide/receptors in mice, not many analogues have been extensively tested in this species, a gap that certainly needs to be filled. We would also comment on three analogues that have been extensively used in the past as ‘‘selective’’ compounds and that have recently turned out to be less selective than previously thought. First, dDAVP is rather unspecific in humans, where it is a mixed V2/V1b agonist (see Table 4 in Manning et al., 2008). The ‘‘Manning compound’’ is not selective for the V1a receptor as it also binds to the OTR (see Table 10 in Manning et al., 2008). Finally, Atosiban, used as a tocolytic agent in humans, is a mixed V1a/OT receptor antagonist (see Tables 15 and 16 in Manning et al., 2008); furthermore, this analogue has been recently shown to be an antagonist at the OT receptor/Gaq coupling but an agonist at the OTR/Gai coupling, thus belonging to a new class of compounds known as ‘‘biased agonists’’ or ‘‘functional agonists’’ (Reversi et al., 2005). The relevance of couplingselective analogues is due to their capability to selectively activate only one signalling pathway of the many that a single receptor may activate, thanks to its promiscuous coupling to several different G-proteins and to other signalling intermediates (Urban et al., 2007). This is particularly relevant as, in some cases, the signalling pathways activated by a single G-protein-coupled receptor may act synergistically, but they may also give rise to opposite effects on the same cellular function. Developing coupling-selective analogues represents a new challenge in molecular pharmacology, because they will activate, for any single receptor subtype, only one downstream signalling pathway, representing a class of compounds highly selective at the single receptor level. Let us finally discuss a few practical cases in which binding or functional assays are used to demonstrate and characterize OT/AVP receptors in biological samples.
Binding assays The first basic question that often needs to be addressed is whether OT/AVP receptors are present in the sample (cell culture, tissue, organ) under investigation. To answer to this question, we suggest to perform binding assays using the tritiated natural agonists, [3H]AVP and [3H]OT, which are commercially available. In particular, [3H]AVP presents the advantage of binding with similar affinities to V1a/V1b/V2 and OTR in all the mammalian species investigated so far, allowing the simultaneous labelling and identification of all the AVP/OT receptors present in the sample. An incubation time of 60 min at 301C or 371C is usually enough to reach the equilibrium; however, if the assay is performed on intact cells, lower temperatures are necessary to avoid labelled hormone internalization, and thus longer incubation times may be required (up to 4 h at 41C). It may then be necessary to further characterize the receptor subtype(s) by means of binding experiments. To this aim, the best procedure is to perform competition binding experiments, in which the receptors labelled with a fixed concentration of [3H]AVP (usually 1–5 nM) are incubated in the presence of increasing concentrations of unlabelled selective analogues such as those listed in Fig. 1. Because all of the analogues listed in this figure are selective for a given VP/OT receptor isoform and exhibit a nanomolar affinity for the receptor isoform they are specific for, a concentration of 30 nM of any selective analogue should completely inhibit the specific [3H]AVP binding on its specific receptor subtype. In contrast, because they have an affinity at least 100-fold higher for the three other AVP/OT subtypes, the same concentration of 30 nM should only weakly inhibit [3H]AVP binding on the receptor subtypes they are not specific for.
Second messengers or functional studies on membranes, cells or tissues sections For functional tests, selective agonists are always preferred (if available) because experiments are easier to perform and to analyse. When
516
experiments are performed on membrane homogenates or on cells in culture, a single dose of the selective agonist at a concentration around its Kd/Ki (generally between 1 nM and 10 nM) may initially be used. If a significant response to this concentration is obtained, dose-response curves must be performed. The Kact obtained for this selective agonist (concentration of agonist leading to half maximal activity) should be around its Kd/Ki. Furthermore, to unambiguously determine the AVP/OT receptor isoform involved, it is necessary to show that other agonists, selective for the other receptor subtypes, when used at their appropriate concentrations (around their Kd/Ki values), are not effective. If working on acute slices of living tissues, this concentration will probably need to be increased to allow the diffusion of the analogue within the tissues. However, special care should be taken in order to keep the selectivity of the peptide, because at concentrations 50–100-fold over its Kd/Ki, the agonist specificity of the agonist may be lost. Alternatively, selective antagonists may be used to abolish the agonist-induced effect. When using a ‘‘selective’’ antagonist to compete for a ‘‘selective’’ agonist, always follow the same rule of not working at a concentration exceeding 10–50-fold its Ki/Kd, as antagonists also usually present a two-log selectivity range in which they are selective. If no selective agonist is available, a selective antagonist can be used in more complex experiments. First, the lowest concentration of AVP/OT leading to a clear but not maximal stimulation should be determined (a nanomolar concentration is generally sufficient for experiments performed on cells or membranes, a 10–30 nM concentration for experiments performed on tissues slices). Then, samples are pre-incubated for 15 min at 371C with increasing amounts of a selective antagonist (at concentrations ranging between 1–100 its Kd/Ki), followed by AVP/OT stimulation at the concentration determined above. If a clear inhibition of AVP/OT-stimulated response is obtained with a concentration of a selective antagonist at a concentration approximately 10-fold its Kd/Ki, the antagonist used identifies the receptor subtype. The rational for choosing the concentration of a selective agonist or antagonist in the experiments
described above is based on the existence of an excellent correlation between binding parameter of a given analogue (Kd) and its ability either to stimulate or inhibit the functional response measured (Cheng et al., 2004). Conclusions Far from being exhaustive, we hope to have provided some general rules to assist with the choice and use of OT/AVP ligands, with the overall aim being to help in avoiding pitfalls when setting up the most common pharmacological assays. Please refer to our paper in this issue (Manning et al., 2008) for an extensive review of the OT/AVP analogues developed through the years, as well as for detailed discussion of their selectivity profiles in in vivo bioassays. Finally, further data on receptor and ligand selectivity can be found at the IUPHAR (The International Union of Basic and Clinical Pharmacology) Database on Receptor Nomenclature and Drug Classification (http:// www.iuphar-db.org).
Acknowledgements This work was supported in part by research grants from the National Institute of General Medical Sciences (No. GM-25280; Maurice Manning), The Italian Association for Cancer Research (AIRC 2006; Bice Chini), Institute de la Sante´ et de la Recherche Me´dicale (INSERM) and Centre National de la Recherche Scientifique (CNRS; Gilles Guillon). References Ahrabi, A.K., Terryn, S., Valenti, G., Caron, N., Serradeil-Le Gal, C., Raufaste, D., Nielsen, S., Horie, S., Verbavatz, J.M. and Devuyst, O. (2007) PKD1 haploinsufficiency causes a syndrome of inappropriate antidiuresis in mice. J. Am. Soc. Nephrol., 18: 1740–1753. Cheng, L.L., Stoev, S., Manning, M., Derick, S., Pena, A., Mimoun, M.B. and Guillon, G. (2004) Design of potent and selective agonists for the human vasopressin V1b receptor based on modifications of [deamino-cys1]arginine vasopressin at position 4. J. Med. Chem., 47: 2375–2388.
517 Guillon, G., Derick, S., Pena, A., Cheng, L.L., Stoev, S., Seyer, R., Morgat, J.L., Barberis, C., Gal, C.S., Wagnon, J. and Manning, M. (2004) The discovery of novel vasopressin V1b receptor ligands for pharmacological, functional and structural investigations. J. Neuroendocrinol., 16: 356–361. Kenakin, T. (2003) Pharmacology Primer: Theory, Applications and Methods. Academic Press Inc., United States. MacSweeney, C.P., Maloumby, R., Neumann, I.D. and Craighead, M. (2007) Effects of the selective V1b antagonist ORG52186 on behaviour and HPA function in male and female HAB and rats. Advances in Vasopressin and Oxytocin — Abstracts from the VII WCNH Regensburg, Germany: 104. Manning, M., Stoev, S., Chini, B., Durroux, T., Mouillac, B. and Guillon, G. (2008) Peptide and nonpeptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents [this issue]. Murat, B., Devost, D., Andres, M., Zingg, H. and Guillon, G. (2007) Heterodimerization of CRHR1 and V1b receptors: potential physiological consequence. Advances in Vasopressin and Oxytocin — Abstracts from the VII WCNH Regensburg, Germany: 104. Pena, A., Murat, B., Trueba, M., Ventura, M.A., Bertrand, G., Cheng, L.L., Stoev, S., Szeto, H.H., Wo, N., Brossard, G., Serradeil-Le Gal, C., Manning, M. and Guillon, G. (2007) Pharmacological and physiological characterization of d[Leu4, Lys8]vasopressin, the first V1b-selective agonist for
rat vasopressin/oxytocin receptors. Endocrinology, 148: 4136–4146. Reversi, A., Rimoldi, V., Marrocco, T., Cassoni, P., Bussolati, G., Parenti, M. and Chini, B. (2005) The oxytocin receptor antagonist atosiban inhibits cell growth via a ‘‘biased agonist’’ mechanism. J. Biol. Chem., 280: 16311–16318. Serradeil-Le Gal, C., Raufaste, D., Derick, S., Blankenstein, J., Allen, J., Pouzet, B., Pascal, M., Wagnon, J. and Ventura, M.A. (2007) Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand. Am. J. Physiol. Regul. Integr. Comp. Physiol., 293: R938–R949. Serradeil-Le Gal, C., Valette, G., Foulon, L., Germain, G., Advenier, C., Naline, E., Bardou, M., Martinolle, J.P., Pouzet, B., Raufaste, D., Garcia, C., Double-Cazanave, E., Pauly, M., Pascal, M., Barbier, A., Scatton, B., Maffrand, J.P. and Le Fur, G. (2004) SSR126768A (4-chloro-3-[(3R)-(+)-5chloro-1-(2,4-dimethoxybenzyl)-3-methyl-2-oxo-2,3- dihydro-1 H-indol-3-yl]-N-ethyl-N-(3-pyridylmethyl)-benzamide, hydrochloride): a new selective and orally active oxytocin receptor antagonist for the prevention of preterm labor. J. Pharmacol. Exp. Ther., 309: 414–424. Urban, J.D., Clarke, W.P., von Zastrow, M., Nichols, D.E., Kobilka, B., Weinstein, H., Javitch, J.A., Roth, B.L., Christopoulos, A., Sexton, P.M., Miller, K.J., Spedding, M. and Mailman, R.B. (2007) Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther., 320: 1–13.
CHAPTER 38
Affinity and efficacy of selective agonists and antagonists for vasopressin and oxytocin receptors: an ‘‘easy guide’’ to receptor pharmacology Bice Chini1,, Maurice Manning2 and Gilles Guillon3 1 CNR Institute of Neuroscience, Milan, Italy Biochemistry and Cancer Biology, University of Toledo College of Medicine, Toledo, OH, USA 3 Institut de Ge´nomique Fonctionnelle, UMR5203-CNRS, INSERM U661, Universite´ Montpellier I, Universite´ Montpellier II, Montpellier, France 2
Abstract: The development of ‘‘selective’’ drugs targeting oxytocin/vasopressin receptors has enormously progressed since the original synthesis of oxytocin more than 50 years ago. However, several factors still hamper the availability of a rich and complete range of selective agonists and antagonists acting at the different oxytocin/vasopressin receptor subtypes, making the use of these drugs still a daunting task. In this paper we will briefly review the major problems encountered when dealing with oxytocin/vasopressin selective ligands, proving few rules for their correct pharmacological use, in order to avoid common pitfalls. Finally, we will glimpse at new challenges, such us the discovery of coupling selective ligands, which foster the search for new classes of selective compounds. Keywords: vasopressin; oxytocin; agonist; antagonist; selectivity; species difference
The design and use of selective agonists and antagonists acting at the oxytocin/vasopressin (OT/AVP) receptors has been and continues to be a difficult task for two principal reasons:
are responsible for important changes in the selectivity profile of some ligands; for this reason, the pharmacological data obtained in one species cannot be extrapolated ‘‘tout court’’ to other species.
The high sequence homology among the AVP and OT V1a/V1b/V2 and OT receptor subtypes and the high degree of chemical similarity among the active ligands often results in overlapping selectivity profiles. The presence of subtle differences among receptor sequences in various animal species
Here we propose a quick guide to the use of ‘‘selected’’ useful drugs together with few key principles to be followed to design and perform pharmacological assays on OT/AVP receptors. We will not consider here the use of selective analogues in in vivo bioassays, as this is the subject of an extensive review that also appears in this issue, to which we refer for a historical and critical evaluation of the different analogues (Manning et al., 2008).
Corresponding author. Tel.: +39 02 50316958; Fax: +39 02 7490574; E-mail: [email protected]
DOI: 10.1016/S0079-6123(08)00438-X
513
514
Let us first discuss the operational definition of a ‘‘selective’’ ligand. In current pharmacological terms, a selective ligand is defined on the basis of either its high affinity or biological activity on a specific receptor when compared to the others (Kenakin, 2003). However, it is to be kept in mind that all these structurally related analogues lack an absolute receptor subtype selectivity; their receptor subtype selectivity is always relative and concentration dependent. As selectivity is defined on the basis of the capability of a single analogue to bind to and/or activate a single OT/AVP receptor subtype, it is of course an absolute requirement for these analogues to have been tested on all the OT/AVP receptor subtypes within the species under investigation. Unfortunately, pharmacological data are often partial and, for several analogues, the full characterization on all the OT/AVP receptor subtypes is
missing, making their use as ‘‘selective’’ analogues impossible. Selectivity may be defined on the basis of binding affinity of the ligand. In this case, because the affinity of the natural ligands, OT or AVP, defined by their affinity constants (Kd), is in the nanomolar range, a good ligand should possess an affinity, again defined by its affinity constant (Kd or Ki), in the same range. To be ‘‘selective’’ on one receptor subtype, its affinity constant on the other three members of the family should be at least two orders of magnitude higher. Using these very tight criteria, the most selective ligands for the human and the rat OT/AVP receptors are listed in Fig. 1 (Ahrabi et al., 2007; Guillon et al., 2004; MacSweeney et al., 2007; Murat et al., 2007; Pena et al., 2007; Serradeil-Le Gal et al., 2004, 2007). A crucial issue concerning the use of these analogues is their availability; as some of these
Fig. 1. Highly selective receptor subtype OT/AVP ligands. To be included in this figure, a selective ligand fulfilled the following pharmacological criteria: (1) to have been tested on all the OT/AVP receptor subtypes (OTR, V1a, V1b, V2) present in a single species, (2) to possess an affinity (defined by its affinity constants Kd or Ki) in the nanomolar range for the receptor subtype it is specific for and (3) to possess an affinity constant at least two orders of magnitude higher for the three other receptor subtypes. The affinity constant of the analogue for its selective receptor subtype, expressed in nanomolar, is reported in round brackets with the appropriate reference in square brackets.
515
analogues are not commercially available, restrictions in their supply would greatly hamper further scientific advancements in the field. Another important point that clearly emerges from Fig. 1 is the lack of data concerning ligand selectivity in mice. Despite the huge relevance in many areas of basic and preclinical science of genetically modified OT/AVP peptide/receptors in mice, not many analogues have been extensively tested in this species, a gap that certainly needs to be filled. We would also comment on three analogues that have been extensively used in the past as ‘‘selective’’ compounds and that have recently turned out to be less selective than previously thought. First, dDAVP is rather unspecific in humans, where it is a mixed V2/V1b agonist (see Table 4 in Manning et al., 2008). The ‘‘Manning compound’’ is not selective for the V1a receptor as it also binds to the OTR (see Table 10 in Manning et al., 2008). Finally, Atosiban, used as a tocolytic agent in humans, is a mixed V1a/OT receptor antagonist (see Tables 15 and 16 in Manning et al., 2008); furthermore, this analogue has been recently shown to be an antagonist at the OT receptor/Gaq coupling but an agonist at the OTR/Gai coupling, thus belonging to a new class of compounds known as ‘‘biased agonists’’ or ‘‘functional agonists’’ (Reversi et al., 2005). The relevance of couplingselective analogues is due to their capability to selectively activate only one signalling pathway of the many that a single receptor may activate, thanks to its promiscuous coupling to several different G-proteins and to other signalling intermediates (Urban et al., 2007). This is particularly relevant as, in some cases, the signalling pathways activated by a single G-protein-coupled receptor may act synergistically, but they may also give rise to opposite effects on the same cellular function. Developing coupling-selective analogues represents a new challenge in molecular pharmacology, because they will activate, for any single receptor subtype, only one downstream signalling pathway, representing a class of compounds highly selective at the single receptor level. Let us finally discuss a few practical cases in which binding or functional assays are used to demonstrate and characterize OT/AVP receptors in biological samples.
Binding assays The first basic question that often needs to be addressed is whether OT/AVP receptors are present in the sample (cell culture, tissue, organ) under investigation. To answer to this question, we suggest to perform binding assays using the tritiated natural agonists, [3H]AVP and [3H]OT, which are commercially available. In particular, [3H]AVP presents the advantage of binding with similar affinities to V1a/V1b/V2 and OTR in all the mammalian species investigated so far, allowing the simultaneous labelling and identification of all the AVP/OT receptors present in the sample. An incubation time of 60 min at 301C or 371C is usually enough to reach the equilibrium; however, if the assay is performed on intact cells, lower temperatures are necessary to avoid labelled hormone internalization, and thus longer incubation times may be required (up to 4 h at 41C). It may then be necessary to further characterize the receptor subtype(s) by means of binding experiments. To this aim, the best procedure is to perform competition binding experiments, in which the receptors labelled with a fixed concentration of [3H]AVP (usually 1–5 nM) are incubated in the presence of increasing concentrations of unlabelled selective analogues such as those listed in Fig. 1. Because all of the analogues listed in this figure are selective for a given VP/OT receptor isoform and exhibit a nanomolar affinity for the receptor isoform they are specific for, a concentration of 30 nM of any selective analogue should completely inhibit the specific [3H]AVP binding on its specific receptor subtype. In contrast, because they have an affinity at least 100-fold higher for the three other AVP/OT subtypes, the same concentration of 30 nM should only weakly inhibit [3H]AVP binding on the receptor subtypes they are not specific for.
Second messengers or functional studies on membranes, cells or tissues sections For functional tests, selective agonists are always preferred (if available) because experiments are easier to perform and to analyse. When
516
experiments are performed on membrane homogenates or on cells in culture, a single dose of the selective agonist at a concentration around its Kd/Ki (generally between 1 nM and 10 nM) may initially be used. If a significant response to this concentration is obtained, dose-response curves must be performed. The Kact obtained for this selective agonist (concentration of agonist leading to half maximal activity) should be around its Kd/Ki. Furthermore, to unambiguously determine the AVP/OT receptor isoform involved, it is necessary to show that other agonists, selective for the other receptor subtypes, when used at their appropriate concentrations (around their Kd/Ki values), are not effective. If working on acute slices of living tissues, this concentration will probably need to be increased to allow the diffusion of the analogue within the tissues. However, special care should be taken in order to keep the selectivity of the peptide, because at concentrations 50–100-fold over its Kd/Ki, the agonist specificity of the agonist may be lost. Alternatively, selective antagonists may be used to abolish the agonist-induced effect. When using a ‘‘selective’’ antagonist to compete for a ‘‘selective’’ agonist, always follow the same rule of not working at a concentration exceeding 10–50-fold its Ki/Kd, as antagonists also usually present a two-log selectivity range in which they are selective. If no selective agonist is available, a selective antagonist can be used in more complex experiments. First, the lowest concentration of AVP/OT leading to a clear but not maximal stimulation should be determined (a nanomolar concentration is generally sufficient for experiments performed on cells or membranes, a 10–30 nM concentration for experiments performed on tissues slices). Then, samples are pre-incubated for 15 min at 371C with increasing amounts of a selective antagonist (at concentrations ranging between 1–100 its Kd/Ki), followed by AVP/OT stimulation at the concentration determined above. If a clear inhibition of AVP/OT-stimulated response is obtained with a concentration of a selective antagonist at a concentration approximately 10-fold its Kd/Ki, the antagonist used identifies the receptor subtype. The rational for choosing the concentration of a selective agonist or antagonist in the experiments
described above is based on the existence of an excellent correlation between binding parameter of a given analogue (Kd) and its ability either to stimulate or inhibit the functional response measured (Cheng et al., 2004). Conclusions Far from being exhaustive, we hope to have provided some general rules to assist with the choice and use of OT/AVP ligands, with the overall aim being to help in avoiding pitfalls when setting up the most common pharmacological assays. Please refer to our paper in this issue (Manning et al., 2008) for an extensive review of the OT/AVP analogues developed through the years, as well as for detailed discussion of their selectivity profiles in in vivo bioassays. Finally, further data on receptor and ligand selectivity can be found at the IUPHAR (The International Union of Basic and Clinical Pharmacology) Database on Receptor Nomenclature and Drug Classification (http:// www.iuphar-db.org).
Acknowledgements This work was supported in part by research grants from the National Institute of General Medical Sciences (No. GM-25280; Maurice Manning), The Italian Association for Cancer Research (AIRC 2006; Bice Chini), Institute de la Sante´ et de la Recherche Me´dicale (INSERM) and Centre National de la Recherche Scientifique (CNRS; Gilles Guillon). References Ahrabi, A.K., Terryn, S., Valenti, G., Caron, N., Serradeil-Le Gal, C., Raufaste, D., Nielsen, S., Horie, S., Verbavatz, J.M. and Devuyst, O. (2007) PKD1 haploinsufficiency causes a syndrome of inappropriate antidiuresis in mice. J. Am. Soc. Nephrol., 18: 1740–1753. Cheng, L.L., Stoev, S., Manning, M., Derick, S., Pena, A., Mimoun, M.B. and Guillon, G. (2004) Design of potent and selective agonists for the human vasopressin V1b receptor based on modifications of [deamino-cys1]arginine vasopressin at position 4. J. Med. Chem., 47: 2375–2388.
517 Guillon, G., Derick, S., Pena, A., Cheng, L.L., Stoev, S., Seyer, R., Morgat, J.L., Barberis, C., Gal, C.S., Wagnon, J. and Manning, M. (2004) The discovery of novel vasopressin V1b receptor ligands for pharmacological, functional and structural investigations. J. Neuroendocrinol., 16: 356–361. Kenakin, T. (2003) Pharmacology Primer: Theory, Applications and Methods. Academic Press Inc., United States. MacSweeney, C.P., Maloumby, R., Neumann, I.D. and Craighead, M. (2007) Effects of the selective V1b antagonist ORG52186 on behaviour and HPA function in male and female HAB and rats. Advances in Vasopressin and Oxytocin — Abstracts from the VII WCNH Regensburg, Germany: 104. Manning, M., Stoev, S., Chini, B., Durroux, T., Mouillac, B. and Guillon, G. (2008) Peptide and nonpeptide agonists and antagonists for the vasopressin and oxytocin V1a, V1b, V2 and OT receptors: research tools and potential therapeutic agents [this issue]. Murat, B., Devost, D., Andres, M., Zingg, H. and Guillon, G. (2007) Heterodimerization of CRHR1 and V1b receptors: potential physiological consequence. Advances in Vasopressin and Oxytocin — Abstracts from the VII WCNH Regensburg, Germany: 104. Pena, A., Murat, B., Trueba, M., Ventura, M.A., Bertrand, G., Cheng, L.L., Stoev, S., Szeto, H.H., Wo, N., Brossard, G., Serradeil-Le Gal, C., Manning, M. and Guillon, G. (2007) Pharmacological and physiological characterization of d[Leu4, Lys8]vasopressin, the first V1b-selective agonist for
rat vasopressin/oxytocin receptors. Endocrinology, 148: 4136–4146. Reversi, A., Rimoldi, V., Marrocco, T., Cassoni, P., Bussolati, G., Parenti, M. and Chini, B. (2005) The oxytocin receptor antagonist atosiban inhibits cell growth via a ‘‘biased agonist’’ mechanism. J. Biol. Chem., 280: 16311–16318. Serradeil-Le Gal, C., Raufaste, D., Derick, S., Blankenstein, J., Allen, J., Pouzet, B., Pascal, M., Wagnon, J. and Ventura, M.A. (2007) Biological characterization of rodent and human vasopressin V1b receptors using SSR-149415, a nonpeptide V1b receptor ligand. Am. J. Physiol. Regul. Integr. Comp. Physiol., 293: R938–R949. Serradeil-Le Gal, C., Valette, G., Foulon, L., Germain, G., Advenier, C., Naline, E., Bardou, M., Martinolle, J.P., Pouzet, B., Raufaste, D., Garcia, C., Double-Cazanave, E., Pauly, M., Pascal, M., Barbier, A., Scatton, B., Maffrand, J.P. and Le Fur, G. (2004) SSR126768A (4-chloro-3-[(3R)-(+)-5chloro-1-(2,4-dimethoxybenzyl)-3-methyl-2-oxo-2,3- dihydro-1 H-indol-3-yl]-N-ethyl-N-(3-pyridylmethyl)-benzamide, hydrochloride): a new selective and orally active oxytocin receptor antagonist for the prevention of preterm labor. J. Pharmacol. Exp. Ther., 309: 414–424. Urban, J.D., Clarke, W.P., von Zastrow, M., Nichols, D.E., Kobilka, B., Weinstein, H., Javitch, J.A., Roth, B.L., Christopoulos, A., Sexton, P.M., Miller, K.J., Spedding, M. and Mailman, R.B. (2007) Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther., 320: 1–13.