three-dimensional models of neuropeptide g-protein coupled receptors

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Neuropeptides (1994) 26, l-4 0 Longman Group Ltd 1994 Session 1: The Receptors of Neuropeptides: Ligand-receptor Interactions Cl/l Three-Dimensional Models of Neuropeptide G-Protein Coupled Receptors M. F. Hibert, S. Trumpp-Kallmeyer and J. Hoflack Marion Merrell Dow, 16 Rue d’Ankara, 67080 Strasbourg Cedex, France Graphics computer generated three-dimensional models of all cloned G-protein coupled receptors (GPCR) were built and analysed. These models were defmed using pri- mary sequence comparisons, secondary structure predic- tions and 3D homology building taking bacteriorhodopsin as a template. The transmembrane region of the dopamine D,, serotonin 5-HT,, noradrenaline %, adrenaline Sz, acetylcholine m, and a number of neuropeptide receptors were chosen as prototypes and explored in detail. We have localized the ligand binding sites and identify the residues likely to be responsible for receptors affinity, selectivity, stereospecificity and efficacy. The precise nature of the important physico-chemical interactions between differ- ent residue side chains or between the ligand and the adja- cent amino acids have been studied. The models are in agreement with published and novel data obtained from mutagenesis and labelling studies. They represent impor- tant working hypotheses to direct future mutagenesis stud- ies and enable structure-activity relationship studies as well as more rational drug design. 1. 2. 3. 4. 5. 6. 7. Hibert, M., Trumpp-Kallmeyer, S., Bruinvels, A. and Hoflack, J. Mol. Pharmacol. 1991; 40: 8-15. Trumpp-Kallmeyer, S., Hoflack, J., Bruinvels, A. and Hibert, M. J. Med. Chem. 1992; 35; 3448-3462. Hibert, M., Hoflack, J., Trumpp-Kallmeyer, S. and Bruinvels A. MCdecinelSciences 1993; 9: 3 l-40. Hibert, M., Trumpp-Kahmeyer, S., Hoflack, J. and Bruinvels, A. Trends Phamracol. Sci. 1993; 14: 7-12. Trumpp-Kallmeyer, S., Hoflack, J. and Hibert, M. In: Buck, S. H., ed. The Tachykinin Receptors. New York: Humana Press, 1993. Hoflack, J., Hibert, M., Trumpp-Kallmeyer, S. and Bidart, J. M. Drug Design Discov. 1993; 10: 157-171. Trumpp-Kallmeyer, S. and Hibert, M. In: H. Kubinyi, ed. 3D QSAR and Drug Design. Leiden: ESCOM, 1993: 355-373. Cl/2 The Common Non-peptide Antagonist Binding Domain of tbe NK-1 Receptor U. Gether*, S. ZofYinann*, J. A. Lowe III? and T. W. Schwartz* *Laboratory of Molecular Endocrinology, Rigshospitalet 6321, University of Copenhagen, Denmark and TCentral Research Division, Pfizer Inc., Groton, CT, USA In the tachykinin system, several potent and specific, non- peptide antagonists have recently been described. We have studied the molecular mechanism of action for these compounds by development of chimeric constructs between the tachykinin receptors, NK-1, NK-2 and NK-3, followed by systematic pointmutations within the identified domains. Chime& substitutions around transmembrane segment VI (TM VI) indicated that this part of the receptor was of crucial importance for binding of several non-peptide antagonists despite clear differ- ences in their chemical structure and mode of discovery. Thus, mutational transfer of a discontinuous epitope around TM V and VI from the NK-1 to the previously unresponsive NK-3 receptor conveyed full NK- 1 -like atiity for e.g. both CP 96 345 and SR 140 333. In par- allel, transfer from the NK-2 to the NK- 1 receptor of a dis- continuous epitope around the outer part of TM VI and VII conveyed full affinity for the NK-2 selective com- pound, SR 48 968 (K, increased from approx. 10 000 nM to 1 nM). In contrast, the identified domain was not impor- tant for binding of the natural peptide agonists, substance P and neurokinin A. Smaller chimeric substitutions com- bined with pointmutational analysis indicated that the antagonists interact with both distinct and overlapping epitopes within the apparent common target domain. Specific interactions have been identified by combining our pointmutants with series of modified analogues of the non-peptide antagonists. It is concluded that chemically different, tachykinin non-peptide antagonists block the function of their respective target receptor by interacting in distinct ways with a common site located spatially around the outer part of transmembrane segment VI.

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Neuropeptides (1994) 26, l-4 0 Longman Group Ltd 1994

Session 1: The Receptors of Neuropeptides: Ligand-receptor Interactions

Cl/l Three-Dimensional Models of Neuropeptide G-Protein Coupled Receptors M. F. Hibert, S. Trumpp-Kallmeyer and J. Hoflack Marion Merrell Dow, 16 Rue d’Ankara, 67080 Strasbourg Cedex, France

Graphics computer generated three-dimensional models of all cloned G-protein coupled receptors (GPCR) were built and analysed. These models were defmed using pri- mary sequence comparisons, secondary structure predic- tions and 3D homology building taking bacteriorhodopsin as a template. The transmembrane region of the dopamine D,, serotonin 5-HT,, noradrenaline %, adrenaline Sz, acetylcholine m, and a number of neuropeptide receptors were chosen as prototypes and explored in detail. We have localized the ligand binding sites and identify the residues likely to be responsible for receptors affinity, selectivity, stereospecificity and efficacy. The precise nature of the important physico-chemical interactions between differ- ent residue side chains or between the ligand and the adja- cent amino acids have been studied. The models are in agreement with published and novel data obtained from mutagenesis and labelling studies. They represent impor- tant working hypotheses to direct future mutagenesis stud- ies and enable structure-activity relationship studies as well as more rational drug design.

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Hibert, M., Trumpp-Kallmeyer, S., Bruinvels, A. and Hoflack, J. Mol. Pharmacol. 1991; 40: 8-15. Trumpp-Kallmeyer, S., Hoflack, J., Bruinvels, A. and Hibert, M. J. Med. Chem. 1992; 35; 3448-3462. Hibert, M., Hoflack, J., Trumpp-Kallmeyer, S. and Bruinvels A. MCdecinelSciences 1993; 9: 3 l-40. Hibert, M., Trumpp-Kahmeyer, S., Hoflack, J. and Bruinvels, A. Trends Phamracol. Sci. 1993; 14: 7-12. Trumpp-Kallmeyer, S., Hoflack, J. and Hibert, M. In: Buck, S. H., ed. The Tachykinin Receptors. New York: Humana Press, 1993. Hoflack, J., Hibert, M., Trumpp-Kallmeyer, S. and Bidart, J. M. Drug Design Discov. 1993; 10: 157-171. Trumpp-Kallmeyer, S. and Hibert, M. In: H. Kubinyi, ed. 3D QSAR and Drug Design. Leiden: ESCOM, 1993: 355-373.

Cl/2 The Common Non-peptide Antagonist Binding Domain of tbe NK-1 Receptor U. Gether*, S. ZofYinann*, J. A. Lowe III? and T. W. Schwartz* *Laboratory of Molecular Endocrinology, Rigshospitalet 6321, University of Copenhagen, Denmark and TCentral Research Division, Pfizer Inc., Groton, CT, USA

In the tachykinin system, several potent and specific, non- peptide antagonists have recently been described. We have studied the molecular mechanism of action for these compounds by development of chimeric constructs between the tachykinin receptors, NK-1, NK-2 and NK-3, followed by systematic pointmutations within the identified domains. Chime& substitutions around transmembrane segment VI (TM VI) indicated that this part of the receptor was of crucial importance for binding of several non-peptide antagonists despite clear differ- ences in their chemical structure and mode of discovery. Thus, mutational transfer of a discontinuous epitope around TM V and VI from the NK-1 to the previously unresponsive NK-3 receptor conveyed full NK- 1 -like atiity for e.g. both CP 96 345 and SR 140 333. In par- allel, transfer from the NK-2 to the NK- 1 receptor of a dis- continuous epitope around the outer part of TM VI and VII conveyed full affinity for the NK-2 selective com- pound, SR 48 968 (K, increased from approx. 10 000 nM to 1 nM). In contrast, the identified domain was not impor- tant for binding of the natural peptide agonists, substance P and neurokinin A. Smaller chimeric substitutions com- bined with pointmutational analysis indicated that the antagonists interact with both distinct and overlapping epitopes within the apparent common target domain. Specific interactions have been identified by combining our pointmutants with series of modified analogues of the non-peptide antagonists. It is concluded that chemically different, tachykinin non-peptide antagonists block the function of their respective target receptor by interacting in distinct ways with a common site located spatially around the outer part of transmembrane segment VI.