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Photoaffinity labelling of the benzodiazepine receptor cannot be used to predict ligand efficacy
Neuroscience Letters, 35 (1983) 37-40
37
Elsevier Scientific Publishers Ireland Ltd.
P H O T O A F F I N I T Y LABELLING OF THE B E N Z O D I A Z E P I N E RECEPTOR
CANNOT BE USED TO PREDICT LIGAND EFFICACY
C H L O E L. B R O W N and IAN L. M A R T I N
M R C Neurochemical Pharmacology Unit, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH (U.K.) (Received November 24th, 1982; Revised version received and accepted December 2nd, 1982)
Key words: benzodiazepine receptor -
photoaffinity label quinolinone - B-carboline - agonist - antagonist.
flunitrazepam binding -
pyrazolo-
Irreversible attachment of flunitrazepam to brain membranes occurs on exposure to UV light, resulting in a loss of the number of bindig sites detected by saturation binding of [3H]flunitrazepam, but not of [3H]ethyl-B-carboline-3-carboxylate (BCCE). Changes in the affinity of ligands for the benzodiazepine receptor produced by photoaffinity labelling, as indicated by the relative IC50 values for the displacement of [3H]BCCE in photoaffinity labelled versus control membranes, appear to be related to their chemical structure rather than to their pharmacological profile.
The central nervous system contains specific, high affinity benzodiazepine binding sites which are thought to represent the pharmacological receptors through which these drugs produce their effects. Irreversible attachment of flunitrazepam to membranes in vitro by photoactivation reduces the number of these sites as characterized by saturation analysis of the reversible binding of [3H]diazepam or [3H]flunitrazepam [10]. However, it has been shown that photoaffinity labelling benzodiazepine receptors in this way does not preclude the binding of [3H]ethyl-Bcarboline-3-carboxylate (BCCE) [1]. This compound potently displaces benzodiazepines from their CNS binding sites, although in a complex manner [11], and has actions opposite to those of benzodiazepines [2]. It has also been shown that the binding capacity of membranes for the methyl and propyl esters of /3carboline-3-carboxylic acid (/3CCM and ~CCP) and for the benzodiazepine antagonists Ro15-1788 and CGS8216 is little affected by photoaffinity labelling [3, 4, 6, 9]. The ability of benzodiazepine receptor ligands to displace the specific binding of [3H]BCCE [1], [3H]/3CCM [6] and [3H]Ro15-1788 [9] has been used to assess their affinity for benzodiazepine receptors in photoaffinity labelled membranes. It is clear from such experiments that photoaffinity labelling of the benzodiazepine receptor population in this way does not result in a complete occlusion of the binding sites but causes a pronounced reduction in their affinity for certain ligands. It 0304-3940/83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
38 has been suggested that the affinity is reduced only for those ligands which have agonist activity at this receptor [3, 6, 9], though clearly an equally plausible explanation, from currently available data, is that changes in affinity are associated with the chemical structure of the ligand. The novel pyrazoloquinolinones: CGS 9896, CGS 9895 and CGS 8216, which have benzodiazepine agonist, partial agonist and antagonist properties, respectively [12], are a group of compounds which can be used to differentiate between these possibilities. Well-washed rat whole brain or hippocampal membranes [8] were diluted to 1:80 (w/v) with 0.1 M Tris-citrate buffer, pH 7.1, and incubated in the presence (for photoaffinity labelled) or absence (for control) of 5 nM flunitrazepam on ice for 1 h. Ten ml aliquots were irradiated on ice at 10 cm from an UV light of wavelength 366 nm (intensity 1 m W / c m : ) for 10 min in petri dishes of 5 cm diameter. The tissue from each treatment was pooled and washed 3 times with the buffer to remove unincorporated flunitrazepam. These conditions produce a 75°7o reduction in the number of high affinity binding sites for [3H]flunitrazepam without affecting the number available for [3H]~CCE [1]. The ability of compounds to displace the specific binding of 0.5 nM [3H]/3CCE to photoaffinity labelled and control membranes prepared in this way was assessed in parallel experiments. The ratio of IC5o values in the two types of membranes is a measure of the effect of photoaffinity labelling on ligand affinity (Table I). Although photoaffinity labelling the benzodiazepine receptor with flunitrazepam considerably reduced the affinity of diazepam and flunitrazepam, and resulted in a smaller reduction in the affinity of CL 218872, a triazolopyridazine with certain benzodiazepine agonist properties [7], it did not significantly affect the affinity of any of the ~-carboline-3-carboxylic acid esters or pyrazoloquinolinones. This effect did not appear to vary markedly between brain regions. The finding that the affinity of the pyrazoloquinolinones CGS 9896, CGS 9895 and CGS 8216, ligands which are reported to exhibit agonist, partial agonist and antagonist behavioural profiles, respectively [12], is not modified in photoaffinity labelled compared to control membranes indicates that such changes cannot be taken as predictive of pharmacological activity but are dependent on chemical structure of the ligand. Also consistent with this proposal are the lack of affinity changes in photoaffinity labelled membranes for the 3 esters of ~-carboline-3-carboxylic acid, which have been reported to display different pharmacological effects [5]. The data is consonant with photoaffinity labelling resulting in the insertion of some flunitrazepam fragment in the vicinity of the benzodiazepine binding site such that the site is not occupied, but access to it is reduced for certain ligands, perhaps by steric factors. The results suggest that the recognition sites for ~-carbolines, pyrazoloquinolinones, triazolopyridazines and Ro15-1788 are different from, although possibly overlapping with, those for classical 5-phenyl-l,4-benzodiazepines.
39
TABLE 1 AFFINITY OF B E N Z O D I A Z E P I N E R E C E P T O R L I G A N D S IN P H O T O A F F I N 1 T Y L A B E L L E D R E L A T I V E TO C O N T R O L M E M B R A N E S Membranes were prepared as described in the text and incubated (approximate concentration 75 izg protein/ml), with 0.5 nM [JH]IJCCE (spec. act. 24 C i / m m o l , A m e r s h a m ) on ice for 1 h before addition of 5 ml of buffer and vacuum filtration through W h a t m a n G F / B filters, followed by 2 further 5 ml washes. 1 #m 13CCE was used to define non-specific binding. IC50 values were derived from Hill plots of at least 5 concentrations of displacer, each assayed in triplicate. In each case the cold ligand was able to displace over 90°7o of specifically b o u n d [JH]CJCCE in both control and photoaffinity labelled membranes. Values given are mean + S.E.M. of 3 experiments. * P < 0 . 0 5 , photoaffinity labelled different from control, paired sample t-test. IC5o Photoaffinity labelled 1C5o Control Displacer
Whole brain
CGS 9896 CGS9895 CGS 8216 Diazepam Flunitrazepam Ro 15-1788 /JCCM /JCCE 13CCP CL 218872
1.23 1.23 1.23 23.2
+ 0.14 + 0.17 + 0.11 ___ 3.4*
-
Hippocampus
1.1 30.1 11.9 1.1 1.1 1.1 1.0 2.2
+_ 0.1 _+ 7.1" _+ 2.5* _+ 0.1 _+ 0.02 _+ 0.1 _+ 0.1 ___ 0.03*
CGS 9896 and CGS 9895 were the generous gifts of Dr. N. Yokoyama, CibaGeigy, Ardsley, N . Y . C . L . B . is in receipt of an SERC (CASE) award in conjunction with Glaxo, Greenford. 1 Brown, C.L. and Martin, I.L., Photoaffinity labelling of the benzodiazepine receptor does not occlude the ~JCCE binding site, Brit. J. Pharmacol., 75 (1982) 43P. 2 Cowen, P.J., Green, A.R., Nutt, D.J. and Martin, I.L., Ethyl 13-carboline carboxylate lowers seizure threshold and antagonises flurazepam-induced sedation in rats, Nature (Lond.), 290 (1981) 54-55. 3 Gee, K.W. and Y a m a m u r a , I-I.I., Differentiation of benzodiazepine receptor agonist and antagonist: sparing of [~H]benzodiazepine antagonist binding following the photolabelling of benzodiazepine receptors, Europ. J. Pharmacol., 82 (1982) 239-241. 4 Hirsch, .I.D., Photolabelling of benzodiazepine receptors spares [JH]-propyl 13-carboline binding, Pharmacol. Biochem. Behav., 16 (1982) 245-248. 5 Jones, B.J. and Oakley N.R., The convulsant properties of methyl 13-carboline-3-carboxylate in the mouse, Brit. J. Pharmacol., 74 (1981) 884P. 6 Karobath, M. and Supavilai, P., Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro, Neurosci. Lett., 31 (1982) 65-69. 7 Lippa, A.S., Coupet, J., Greenblatt, E.N., Klepner, C.A. and Beer, B., A synthetic nonbenzodiazepine ligand for the benzodiazepine receptor: a probe for investigating neuronal substrates of anxiety, Pharmacol. Biochem. Behav., 11 (1979)99-106.
40 8 Martin, I.L. and Candy, J.M., Facilitation of benzodiazepine binding by sodium chloride and GABA, Neuropharmacology, 17 (1978) 993-998. 9 M6hler, H., Benzodiazepine receptors: differential interaction of benzodiazepine agonists and antagonists after photoaffinity labelling with fhmitrazepam, Europ. J. Pharmacol., 80 (1982) 435-436. 10 Mohler, H., Battersby, M.K. and Richards, J.G., Benzodiazepine receptor protein identified and visualised in brain tissue by a photoaffinity label, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 1666-1670. 11 Nielsen, M. and Braestrup, C., Ethyl fl-carboline-3-carboxylate shows differential benzodiazepine receptor interaction, Nature (Lond.), 286 (1980) 606-609. 12 Yokoyama, N., Ritter, B. and Neubert, A.D., c~-Arylpyrazolo-[4,3-c]quinolin-3-ones: novel agonist, partial agonist and antagonist of benzodiazepines, J. med. Chem., 25 (1982) 337 339.
37
Elsevier Scientific Publishers Ireland Ltd.
P H O T O A F F I N I T Y LABELLING OF THE B E N Z O D I A Z E P I N E RECEPTOR
CANNOT BE USED TO PREDICT LIGAND EFFICACY
C H L O E L. B R O W N and IAN L. M A R T I N
M R C Neurochemical Pharmacology Unit, Medical Research Council Centre, Hills Road, Cambridge CB2 2QH (U.K.) (Received November 24th, 1982; Revised version received and accepted December 2nd, 1982)
Key words: benzodiazepine receptor -
photoaffinity label quinolinone - B-carboline - agonist - antagonist.
flunitrazepam binding -
pyrazolo-
Irreversible attachment of flunitrazepam to brain membranes occurs on exposure to UV light, resulting in a loss of the number of bindig sites detected by saturation binding of [3H]flunitrazepam, but not of [3H]ethyl-B-carboline-3-carboxylate (BCCE). Changes in the affinity of ligands for the benzodiazepine receptor produced by photoaffinity labelling, as indicated by the relative IC50 values for the displacement of [3H]BCCE in photoaffinity labelled versus control membranes, appear to be related to their chemical structure rather than to their pharmacological profile.
The central nervous system contains specific, high affinity benzodiazepine binding sites which are thought to represent the pharmacological receptors through which these drugs produce their effects. Irreversible attachment of flunitrazepam to membranes in vitro by photoactivation reduces the number of these sites as characterized by saturation analysis of the reversible binding of [3H]diazepam or [3H]flunitrazepam [10]. However, it has been shown that photoaffinity labelling benzodiazepine receptors in this way does not preclude the binding of [3H]ethyl-Bcarboline-3-carboxylate (BCCE) [1]. This compound potently displaces benzodiazepines from their CNS binding sites, although in a complex manner [11], and has actions opposite to those of benzodiazepines [2]. It has also been shown that the binding capacity of membranes for the methyl and propyl esters of /3carboline-3-carboxylic acid (/3CCM and ~CCP) and for the benzodiazepine antagonists Ro15-1788 and CGS8216 is little affected by photoaffinity labelling [3, 4, 6, 9]. The ability of benzodiazepine receptor ligands to displace the specific binding of [3H]BCCE [1], [3H]/3CCM [6] and [3H]Ro15-1788 [9] has been used to assess their affinity for benzodiazepine receptors in photoaffinity labelled membranes. It is clear from such experiments that photoaffinity labelling of the benzodiazepine receptor population in this way does not result in a complete occlusion of the binding sites but causes a pronounced reduction in their affinity for certain ligands. It 0304-3940/83/0000-0000/$ 03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd.
38 has been suggested that the affinity is reduced only for those ligands which have agonist activity at this receptor [3, 6, 9], though clearly an equally plausible explanation, from currently available data, is that changes in affinity are associated with the chemical structure of the ligand. The novel pyrazoloquinolinones: CGS 9896, CGS 9895 and CGS 8216, which have benzodiazepine agonist, partial agonist and antagonist properties, respectively [12], are a group of compounds which can be used to differentiate between these possibilities. Well-washed rat whole brain or hippocampal membranes [8] were diluted to 1:80 (w/v) with 0.1 M Tris-citrate buffer, pH 7.1, and incubated in the presence (for photoaffinity labelled) or absence (for control) of 5 nM flunitrazepam on ice for 1 h. Ten ml aliquots were irradiated on ice at 10 cm from an UV light of wavelength 366 nm (intensity 1 m W / c m : ) for 10 min in petri dishes of 5 cm diameter. The tissue from each treatment was pooled and washed 3 times with the buffer to remove unincorporated flunitrazepam. These conditions produce a 75°7o reduction in the number of high affinity binding sites for [3H]flunitrazepam without affecting the number available for [3H]~CCE [1]. The ability of compounds to displace the specific binding of 0.5 nM [3H]/3CCE to photoaffinity labelled and control membranes prepared in this way was assessed in parallel experiments. The ratio of IC5o values in the two types of membranes is a measure of the effect of photoaffinity labelling on ligand affinity (Table I). Although photoaffinity labelling the benzodiazepine receptor with flunitrazepam considerably reduced the affinity of diazepam and flunitrazepam, and resulted in a smaller reduction in the affinity of CL 218872, a triazolopyridazine with certain benzodiazepine agonist properties [7], it did not significantly affect the affinity of any of the ~-carboline-3-carboxylic acid esters or pyrazoloquinolinones. This effect did not appear to vary markedly between brain regions. The finding that the affinity of the pyrazoloquinolinones CGS 9896, CGS 9895 and CGS 8216, ligands which are reported to exhibit agonist, partial agonist and antagonist behavioural profiles, respectively [12], is not modified in photoaffinity labelled compared to control membranes indicates that such changes cannot be taken as predictive of pharmacological activity but are dependent on chemical structure of the ligand. Also consistent with this proposal are the lack of affinity changes in photoaffinity labelled membranes for the 3 esters of ~-carboline-3-carboxylic acid, which have been reported to display different pharmacological effects [5]. The data is consonant with photoaffinity labelling resulting in the insertion of some flunitrazepam fragment in the vicinity of the benzodiazepine binding site such that the site is not occupied, but access to it is reduced for certain ligands, perhaps by steric factors. The results suggest that the recognition sites for ~-carbolines, pyrazoloquinolinones, triazolopyridazines and Ro15-1788 are different from, although possibly overlapping with, those for classical 5-phenyl-l,4-benzodiazepines.
39
TABLE 1 AFFINITY OF B E N Z O D I A Z E P I N E R E C E P T O R L I G A N D S IN P H O T O A F F I N 1 T Y L A B E L L E D R E L A T I V E TO C O N T R O L M E M B R A N E S Membranes were prepared as described in the text and incubated (approximate concentration 75 izg protein/ml), with 0.5 nM [JH]IJCCE (spec. act. 24 C i / m m o l , A m e r s h a m ) on ice for 1 h before addition of 5 ml of buffer and vacuum filtration through W h a t m a n G F / B filters, followed by 2 further 5 ml washes. 1 #m 13CCE was used to define non-specific binding. IC50 values were derived from Hill plots of at least 5 concentrations of displacer, each assayed in triplicate. In each case the cold ligand was able to displace over 90°7o of specifically b o u n d [JH]CJCCE in both control and photoaffinity labelled membranes. Values given are mean + S.E.M. of 3 experiments. * P < 0 . 0 5 , photoaffinity labelled different from control, paired sample t-test. IC5o Photoaffinity labelled 1C5o Control Displacer
Whole brain
CGS 9896 CGS9895 CGS 8216 Diazepam Flunitrazepam Ro 15-1788 /JCCM /JCCE 13CCP CL 218872
1.23 1.23 1.23 23.2
+ 0.14 + 0.17 + 0.11 ___ 3.4*
-
Hippocampus
1.1 30.1 11.9 1.1 1.1 1.1 1.0 2.2
+_ 0.1 _+ 7.1" _+ 2.5* _+ 0.1 _+ 0.02 _+ 0.1 _+ 0.1 ___ 0.03*
CGS 9896 and CGS 9895 were the generous gifts of Dr. N. Yokoyama, CibaGeigy, Ardsley, N . Y . C . L . B . is in receipt of an SERC (CASE) award in conjunction with Glaxo, Greenford. 1 Brown, C.L. and Martin, I.L., Photoaffinity labelling of the benzodiazepine receptor does not occlude the ~JCCE binding site, Brit. J. Pharmacol., 75 (1982) 43P. 2 Cowen, P.J., Green, A.R., Nutt, D.J. and Martin, I.L., Ethyl 13-carboline carboxylate lowers seizure threshold and antagonises flurazepam-induced sedation in rats, Nature (Lond.), 290 (1981) 54-55. 3 Gee, K.W. and Y a m a m u r a , I-I.I., Differentiation of benzodiazepine receptor agonist and antagonist: sparing of [~H]benzodiazepine antagonist binding following the photolabelling of benzodiazepine receptors, Europ. J. Pharmacol., 82 (1982) 239-241. 4 Hirsch, .I.D., Photolabelling of benzodiazepine receptors spares [JH]-propyl 13-carboline binding, Pharmacol. Biochem. Behav., 16 (1982) 245-248. 5 Jones, B.J. and Oakley N.R., The convulsant properties of methyl 13-carboline-3-carboxylate in the mouse, Brit. J. Pharmacol., 74 (1981) 884P. 6 Karobath, M. and Supavilai, P., Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro, Neurosci. Lett., 31 (1982) 65-69. 7 Lippa, A.S., Coupet, J., Greenblatt, E.N., Klepner, C.A. and Beer, B., A synthetic nonbenzodiazepine ligand for the benzodiazepine receptor: a probe for investigating neuronal substrates of anxiety, Pharmacol. Biochem. Behav., 11 (1979)99-106.
40 8 Martin, I.L. and Candy, J.M., Facilitation of benzodiazepine binding by sodium chloride and GABA, Neuropharmacology, 17 (1978) 993-998. 9 M6hler, H., Benzodiazepine receptors: differential interaction of benzodiazepine agonists and antagonists after photoaffinity labelling with fhmitrazepam, Europ. J. Pharmacol., 80 (1982) 435-436. 10 Mohler, H., Battersby, M.K. and Richards, J.G., Benzodiazepine receptor protein identified and visualised in brain tissue by a photoaffinity label, Proc. nat. Acad. Sci. U.S.A., 77 (1980) 1666-1670. 11 Nielsen, M. and Braestrup, C., Ethyl fl-carboline-3-carboxylate shows differential benzodiazepine receptor interaction, Nature (Lond.), 286 (1980) 606-609. 12 Yokoyama, N., Ritter, B. and Neubert, A.D., c~-Arylpyrazolo-[4,3-c]quinolin-3-ones: novel agonist, partial agonist and antagonist of benzodiazepines, J. med. Chem., 25 (1982) 337 339.