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Different receptor affinities of the enantiomers of BAY K 8644, a dihydropyridine Ca channel activator
187
European Journal of Pharmacology, 118 (1985) 187-188 Elsevier
Rapid communication DIFFERENT RECEPTOR AFFINITIES OF THE ENANTIOMERS OF BAY K 8644, A DIHYDROPVRIDINE Ca CHANNEL ACTIVATOR PETER BELLEMANN * and G E R H A R D F R A N C K O W I A K
Research Center, Bayer AG, P.O. Box 10 17 09, D-5600 Wuppertal 1, F.R.G. Received 7 October 1985, accepted 7 October 1985
Pharmacologically active compounds exert their biological action on cells predominantly via specific membrane-associated loci that modulate transmembrane fluxes of certain bioactive messenger agents, e.g., Ca 2+. Dihydropyridines (DHP) of the nifedipine type are generally accepted as potent inhibitors of the slow inward current of Ca 2+. A receptor for the DHPs was documented by correlating the binding characteristics of DHPs with their pharmacological response, and by their stereoselectivity (Bellemann et al., 1983). Recent developments in the chemical structure resulted in DHPs, e.g., BAY K 8644, that exhibit the opposite, Ca channel stimulating effects (Schramm et al., 1983). As BAY K 8644 has an asymmetric center, the receptor interaction of the optical isomers was investigated by using the Ca channel antagonistic [ 3H]nimodipine. [3H]Nimodipine (150-180 C i / m m o l ) was synthesized by New England Nuclear (Boston, MA), its purity was assessed by thin-layer radiochromatography and the ligand was then stored in the dark at - 3 0 ° C under nitrogen gas to prevent radiolysis and oxidation. The D H P compounds used were freshly dissolved in dimethyl sulfoxide and stock solutions (10 -2 M) were diluted further with Tris-HC1, pH 7.4. Partially purified membrane fractions from rat brain cortices were prepared as described elsewhere. Binding assays were performed essentially as described by Bellemann et al. (1983) under strict sodium light to prevent breakdown of DHPs. In short, membrane protein (40-60/~g per assay) was incubated (final volume, 0.25 ml) at 37°C in 50 * To whom all correspondenceshould be addressed. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishers B.V.
mM Tris-HC1, pH 7.4, 150 mM NaC1, 1 mM CaCl 2 containing 1.3-1.6 nM of radioligand and the additives, e.g. the optical isomers of BAY K 8644. The reaction (30 min) was terminated by dilution with 3.5 ml of ice-cold Tris-HC1 at pH 7.4. Particle-bound and free [3H]DHP ligand were separated by rapid vacuum filtration through G F / C glass fiber filter (Whatman). The precipitate was washed twice with ice-cold Tris-HC1 and the bound radioactivity was then determined conventionally. Calculation and statistical analysis of the data were done by computer. The Ca channel antagonistic DHPs exhibited a receptor affinity (K i < 1 nM) more than one order of magnitude greater than that of racemic Ca channel activating DHPs. Racemic BAY K 8644 was shown to interact with the D H P receptor when [3H]BAY K 8644 was used (for further details see Bellemann, 1984). Non-identical substituents in the 3- and 5-position of the heterocycle result in chirality of the 1,4-dihydropyridine molecule. Thus, the two enantiomers of BAY K 8644 were separated by preparing their diastereometric derivatives (DOS, 2935 451). These showed distinct differences in affinity to the D H P receptor (fig. 1): the affinity of (-)-(4S)-BAY K 8644 (K i = 25 nM) was double that of its (+)-(4R)counterpart (K i = 65 nM), and the affinity of the racemic BAY K 8644 ranged between 30 and 35 nM. Racemic and ( - ) - B A Y K 8644 showed no, and ( + )-BAY K 8644 showed a moderate positive cooperative tendency. Differences in the functional response of the optical D H P enantiomers in aortic ring and heart preparations have also been observed (Hof et al., 1985; Franckowiak et al., 1985). However, the opposite action of the 1,4-di-
188
[3H] - NIMODIPINE
t~. CF3 O2N~ C O O C H 3
100
H3C
H
CH3
compound. Alterations in the membrane potential, concentration applied, structural and conformational parameters are additional determinants to be taken into account in the design of effective D H P Ca channel blocking or activating drugs.
.J
i , . \ , + , K,,,4
'°
40
,0
K,(nM)
•
(+t BAY K 8644
:!65 -LOG~0 DISPLACER [M]
Fig. 1. Interaction of BAY K 8644 and its enantiomers with the dihydropyridine receptor. Displacement using [3H]nimodipine ( < 1.5 nM), the calcium entry blocker nimodipine (NIM ©) as the reference, the racemic Ca channel stimulating ( + )-BAY K 8644 (O), and its optical isomers ( - ) - B A Y K 8644, (-)-(4S) form (11) and (+)-BAY K 8644, (+)-(4R) form (A). Inset: The compounds' inhibitory characteristics: Ki = IC50/(1 + L C / K d ) , in which LS is radioligand concentration, K d its dissociation constant, and IC50 the concentration of drug causing 50% inhibition of 3H-ligand specific binding. Data are the means of four separate experiments in triplicate with less than 5% standard deviation. At least three different protein preparations from rat brain cortex were used. The optically active center of the BAY K 8644 molecule is marked by an asterisk.
hydropyridine antipodes is not based only on different affinities of the counterparts to yield partial agonists/antagonists as a mixture in the racemic
Acknowledgements Thanks to Mrs. B. Oschmann for excellent technical assistance and Mrs. R. Quabeck for careful preparation of the manuscript.
References Bellemann, P., 1984, Interaction of the calcium channel activating [3H]-BAY K 8644 and inhibiting [3H]-verapamil with specific receptor sites on cultured beating myocardial cells, J. Rec. ges. 4, 571. Bellemann, P., A. Schade and R. Towart, 1983, Dihydropyridine receptor in rat brain labeled with [3H]-nimodipine, Proc. Natl. Acad. Sci. U.S.A. 80, 2356. Franckowiak, G., M. Bechem, M. Schramm and G. Thomas, 1985, The optical isomers of the 1,4-dihydropyridine BAY K 8644 show opposite effects on Ca channels, European J. Pharmacol. 114, 223. Hof, R.P., U.T. Rbegg, A. Hof and A. Vogel, 1985, Stereoselectivity at the calcium channel: opposite action of the enantiomers of a 1,4-dihydropyridine, J. Cardiovasc. Pharmacol. 7, 689. Schramm, M., G. Thomas, R. Towart and G. Franckowiak, 1983, Novel dihydropyridines with positive inotropic action through activation of Ca 2+ channels, Nature 303, 535.
European Journal of Pharmacology, 118 (1985) 187-188 Elsevier
Rapid communication DIFFERENT RECEPTOR AFFINITIES OF THE ENANTIOMERS OF BAY K 8644, A DIHYDROPVRIDINE Ca CHANNEL ACTIVATOR PETER BELLEMANN * and G E R H A R D F R A N C K O W I A K
Research Center, Bayer AG, P.O. Box 10 17 09, D-5600 Wuppertal 1, F.R.G. Received 7 October 1985, accepted 7 October 1985
Pharmacologically active compounds exert their biological action on cells predominantly via specific membrane-associated loci that modulate transmembrane fluxes of certain bioactive messenger agents, e.g., Ca 2+. Dihydropyridines (DHP) of the nifedipine type are generally accepted as potent inhibitors of the slow inward current of Ca 2+. A receptor for the DHPs was documented by correlating the binding characteristics of DHPs with their pharmacological response, and by their stereoselectivity (Bellemann et al., 1983). Recent developments in the chemical structure resulted in DHPs, e.g., BAY K 8644, that exhibit the opposite, Ca channel stimulating effects (Schramm et al., 1983). As BAY K 8644 has an asymmetric center, the receptor interaction of the optical isomers was investigated by using the Ca channel antagonistic [ 3H]nimodipine. [3H]Nimodipine (150-180 C i / m m o l ) was synthesized by New England Nuclear (Boston, MA), its purity was assessed by thin-layer radiochromatography and the ligand was then stored in the dark at - 3 0 ° C under nitrogen gas to prevent radiolysis and oxidation. The D H P compounds used were freshly dissolved in dimethyl sulfoxide and stock solutions (10 -2 M) were diluted further with Tris-HC1, pH 7.4. Partially purified membrane fractions from rat brain cortices were prepared as described elsewhere. Binding assays were performed essentially as described by Bellemann et al. (1983) under strict sodium light to prevent breakdown of DHPs. In short, membrane protein (40-60/~g per assay) was incubated (final volume, 0.25 ml) at 37°C in 50 * To whom all correspondenceshould be addressed. 0014-2999/85/$03.30 © 1985 ElsevierSciencePublishers B.V.
mM Tris-HC1, pH 7.4, 150 mM NaC1, 1 mM CaCl 2 containing 1.3-1.6 nM of radioligand and the additives, e.g. the optical isomers of BAY K 8644. The reaction (30 min) was terminated by dilution with 3.5 ml of ice-cold Tris-HC1 at pH 7.4. Particle-bound and free [3H]DHP ligand were separated by rapid vacuum filtration through G F / C glass fiber filter (Whatman). The precipitate was washed twice with ice-cold Tris-HC1 and the bound radioactivity was then determined conventionally. Calculation and statistical analysis of the data were done by computer. The Ca channel antagonistic DHPs exhibited a receptor affinity (K i < 1 nM) more than one order of magnitude greater than that of racemic Ca channel activating DHPs. Racemic BAY K 8644 was shown to interact with the D H P receptor when [3H]BAY K 8644 was used (for further details see Bellemann, 1984). Non-identical substituents in the 3- and 5-position of the heterocycle result in chirality of the 1,4-dihydropyridine molecule. Thus, the two enantiomers of BAY K 8644 were separated by preparing their diastereometric derivatives (DOS, 2935 451). These showed distinct differences in affinity to the D H P receptor (fig. 1): the affinity of (-)-(4S)-BAY K 8644 (K i = 25 nM) was double that of its (+)-(4R)counterpart (K i = 65 nM), and the affinity of the racemic BAY K 8644 ranged between 30 and 35 nM. Racemic and ( - ) - B A Y K 8644 showed no, and ( + )-BAY K 8644 showed a moderate positive cooperative tendency. Differences in the functional response of the optical D H P enantiomers in aortic ring and heart preparations have also been observed (Hof et al., 1985; Franckowiak et al., 1985). However, the opposite action of the 1,4-di-
188
[3H] - NIMODIPINE
t~. CF3 O2N~ C O O C H 3
100
H3C
H
CH3
compound. Alterations in the membrane potential, concentration applied, structural and conformational parameters are additional determinants to be taken into account in the design of effective D H P Ca channel blocking or activating drugs.
.J
i , . \ , + , K,,,4
'°
40
,0
K,(nM)
•
(+t BAY K 8644
:!65 -LOG~0 DISPLACER [M]
Fig. 1. Interaction of BAY K 8644 and its enantiomers with the dihydropyridine receptor. Displacement using [3H]nimodipine ( < 1.5 nM), the calcium entry blocker nimodipine (NIM ©) as the reference, the racemic Ca channel stimulating ( + )-BAY K 8644 (O), and its optical isomers ( - ) - B A Y K 8644, (-)-(4S) form (11) and (+)-BAY K 8644, (+)-(4R) form (A). Inset: The compounds' inhibitory characteristics: Ki = IC50/(1 + L C / K d ) , in which LS is radioligand concentration, K d its dissociation constant, and IC50 the concentration of drug causing 50% inhibition of 3H-ligand specific binding. Data are the means of four separate experiments in triplicate with less than 5% standard deviation. At least three different protein preparations from rat brain cortex were used. The optically active center of the BAY K 8644 molecule is marked by an asterisk.
hydropyridine antipodes is not based only on different affinities of the counterparts to yield partial agonists/antagonists as a mixture in the racemic
Acknowledgements Thanks to Mrs. B. Oschmann for excellent technical assistance and Mrs. R. Quabeck for careful preparation of the manuscript.
References Bellemann, P., 1984, Interaction of the calcium channel activating [3H]-BAY K 8644 and inhibiting [3H]-verapamil with specific receptor sites on cultured beating myocardial cells, J. Rec. ges. 4, 571. Bellemann, P., A. Schade and R. Towart, 1983, Dihydropyridine receptor in rat brain labeled with [3H]-nimodipine, Proc. Natl. Acad. Sci. U.S.A. 80, 2356. Franckowiak, G., M. Bechem, M. Schramm and G. Thomas, 1985, The optical isomers of the 1,4-dihydropyridine BAY K 8644 show opposite effects on Ca channels, European J. Pharmacol. 114, 223. Hof, R.P., U.T. Rbegg, A. Hof and A. Vogel, 1985, Stereoselectivity at the calcium channel: opposite action of the enantiomers of a 1,4-dihydropyridine, J. Cardiovasc. Pharmacol. 7, 689. Schramm, M., G. Thomas, R. Towart and G. Franckowiak, 1983, Novel dihydropyridines with positive inotropic action through activation of Ca 2+ channels, Nature 303, 535.