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SCH 57790: A novel M2 receptor selective antagonist
Life Sciences, Vol. 44, Nas. 6/l, pp. 53.5439, 1999 Copyright 0 1999 Ekmier science Inc. Printed in the USA. All rights reserved 00%3205/99 $19.00 + .rm
ELSEVIER
PI1 SOO24-3205(98)00598-O
SCH 57790: A NOVEL M, RECEPTOR SELECTIVE ANTAGONIST
Jean E. Lachowicz, Derek Lowe, Ruth A. Duffy, Vilma Ruperto, Lisa A. Taylor, Henry Guzik, Joan Brown, Joel G. Berger, Melissa Tice, Robert McQuade, Joseph Kozlowski, John Clader, Catherine D. Strader and Nicholas Murgolo Departments of Chemical Research and Central Nervous System Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033
Summary As a decrease in cholinergic neurons has been observed in Alzheimer’s Disease (AD), therapeutic approaches to AD include inhibition of Evidence suggests that acetylcholinesterase to increase acetylcholine levels. acetylcholine release in the CNS is modulated by negative feedback via presynaptic M, receptors, blockade of which should provide another means of acetylcholine release. Structure-activity studies of [4increasing (phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-aThis [4-[[4-methoxyphenyl]sulfinyl]-phenyl]-l-piperazineacetonitle. compound, SCH 57790, binds to cloned human M receptors expressed in CHO cells with an affinity of 2.78 nI@ the affinity at M, receptors is 40-fold lower. SCH 57790 is an antagonist at M, receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M, receptor blockade for enhancement of cognition.
Key Words: SCH 57790, M, receptor antagonist, Alzheimer ’s disease, acetylcholine
The neurotransmitter acetylcholine has been demonstrated to modulate learning and memory processes (1). Thus, the cholinergic system has been of interest in the study of cognitive disorders, including the neurodegenerative disorder known as Alzheimer’s Disease (AD). Cholinergic neurons originating in the nucleus basalis of Meynert project to areas such as the cortex and hippocampus, brain regions which play a role in cognition (2,3). Lesioning these pathways in rodents produces a decrease in acetylcholine release and an impairment of memoryrelated task performance (4). This impairment can be reversed by cholinergic agonists (5). In AD, decreases in acetylcholinesterase levels and choline acetyltransferase activity have been reported (6,7). These observations indicate that the cholinergic system is compromised in AD and that drugs which stimulate cholinergic activity may provide relief from the neurodegenerative symptoms. Currently, AD is treated with inhibitors of acetylcholinesterase, such as donepezil, which reduce the rate at which released acetylcholine is degraded for removal from the synapse (8). Another possible mechanism for augmenting central choline@ activity is to facilitate acetylcholine release. Evidence suggests that in the CNS, presynaptic M, receptors act to inhibit acetylcholine release (9, 10). A selective M, antagonist which blocks this inhibition may therefore prove useful in the treatment of AD. SCH 57790 (4-cyclohexyl-a-[4-[Cmethoxyphenyl]-(S)-sulfinyl]phenyll-1-piperazineacetonitrile) is a selective, high affinity M, antagonist. The interaction of this compound and a number of analogues (Fig. 1) with human muscarinic receptors is described.
536
Vol. 64, Nos. 617, 1999
SCH 57790 as M, Receptor Antagonist
Methods The synthesis of SCH 57790 will be described elsewhere (Lowe, et al., manuscript in preparation). Human muscarinic receptor constructs were transfected into CHO-Kl cells by the calcium phosphate precipitation method. Colonies were selected for G418 resistance and tested for receptor expression by radioligand binding to Z-quinuclidinyl IphenyZ-4-3H]benzilate ( [3H] QNB). Saturation experiments were performed using 5-2500 pM r3H] QNB in 10 mM potassium phosphate buffer, pH 7.4, to determine the Kd of [3H] QNB for each receptor subtype. Competition binding experiments were performed using 180 pM [3H] QNB and lo-50 ug of membrane protein. All binding experiments were performed in the presence of 1% DMSO and 0.4% methylcellulose. Non-specific binding was defined by 0.5 l.tM atropine. After equilibrium was reached (120 minute incubation at room temperature) bound and free radioactivity were separated by filtration using Whatman GF-C filters. Ki values were derived from IC,, values using the Cheng-Prusoff equation (11). Adenylyl cyclase assays were conducted using M, receptor-expressing CHO cells in suspension (approximately 250,000 cells/ 60 l.rIassay volume). To provide a stimulated baseline from which to observe oxotremorine-mediated inhibition, 3 J.&I forskolin was included in the assay. Phosphodiesterase activity was inhibited by 100 uM Ro-02-1724. Cells were pretreated with antagonist for 10 minutes prior to incubation with oxotremorine at 37°C for 5 minutes. A subsequent three minute 100°C incubation terminated the reaction and lysed the cells to release accumulated CAMP. Total CAMP in a 20 l.tl assay sample was quantified by a radioimmunoassay capable of detecting between 0.5 and 100 uM CAMP.
H,CO A. Racemic B. (S)-Sulfoxide
(SCH 57790)
H&O C. Racemic D. (S)-Sulfoxide
H3C0 E. Racemic
Fig. 1 Chemical structures of SCH 57790 and analogues. Results
and Discussion
Fig. 2 shows SCH 57790 displacement of [3H] QNB from human M,-M, receptors. The muscarinic receptor binding affinities for SCH 57790 and its analogues are listed in Table 1. The K values derived from these competition experiments provide insights into the structure activity relationships of these compounds and muscarinic receptors. Substitution of a methyl group for the nitrile substituent confers higher affinity binding to muscarinic receptors, illustrated by compounds C and D. However, M, receptor selectivity versus M,, M,, and M4 receptors is decreased by this substitution. The increased affinity for muscarinic receptors overall combined
SCH 57790 as M2 Receptor Antagonist
Vol. 64, Nos. 6/l, 1999
537
with the loss in selectivity suggests that the methyl substituent interacts with amino acids that are common to all muscarinic receptor subtypes. The nitrile substituent may interact with residues that are specific to the M, receptor. The methyl compounds display the same stereoselectivity as the nitrile compounds; the (S)-sulfinyl enantiomer binds to muscarinic receptors with higher affinity than the racemic compound. This Compound E shows the ramifications of replacing the piperazine with a piperidine. It is compound, which lacks the more basic nitrogen, is inactive at M, and M, receptors. postulated that this nitrogen forms an ionic interaction with the Asp 103 in the third transmembrane domain of the M, receptor. The aspartic acid in this position is common to all biogenic amine receptors and has been shown using receptor mutagenesis to be critical for agonist binding in muscarinic and other G-protein-coupled receptors (12, 13). The hypothesis that the piperazine nitrogen interacts with Asp 103 is further supported by results from receptor mutagenesis studies. Substitution of Asp 105 for Ala in the human M, receptor reduced the affinity of SCH 57790 by 14-fold.
loo-
F F ._
80-
m m 605 F 0I 408 20-
?? ml
A v
m2 m3 ?? m4
0
m5
o-
Inhibition
Fig. 2 of r3H] QNB binding by SCH 57790 at the five cloned muscarinic receptors.
TABLE 1 Affinities of SCH 57790 and Analogues at Human Muscarinic Receptors in CHO Cells. M,Ki,nM A : :
114 6.0 112 4.7 >500 NT= Not Tested.
M, Ki, nM 9.1 2.8 A.: ,500
M,Ki,nM ;: 1.0 0.4 NT
M4 Ki, nM 38 2.2 14 0.7 NT
M,Ki,tlM 5; NT NT NT
Vol. 64, Nos. 617, 1999
SCH 57790 as M, Receptor Antagonist
538
Molecular models of SCH 57790 binding to the human MJ receptor suggest that the sulfinyl moiety interacts with Trp 99 in TMID (Fig 3). This interaction, which has been studied for M, receptors (14), is supported by the stereoselectivity observed for SCH 57790 and analogues. Substitution of Ala for Trp 99 in the M, receptor resulted in a lo-fold decrease in the affinity of SCH 57790. In addition, the models suggest that the distance between the piperazine nitrogen and the sulfoxide is 9 A, consistent with the distance between Trp 99 and Aspl03, which represents one helical turn.
TM I, TM II Hydrophobic Pocket
H&O Fig. 3 Model of interactions between the human M, receptor and SCH 57790. The muscarinic agonist oxotremorine-M produces a 30% maximal inhibition of CAMP produced by M,-expressing CHO cells with an EC,, of 10 nM (Fig 4). SCH 57790 (30 r&l) shifts the In the dose-response curve for oxotremorine to the right in a parallel fashion by 130-fold. presence of 100 nM SCH 57790, oxotremorine-M produces no inhibition of adenylyl cyclase.
Control 30 nM SCH
404 -12
-11
-10
-9
-8
-7
-6
[Oxotremorine], Log M
Oxotremorine-mediated
Fig. 4 inhibition of forskolin-stimulated
adenylyl cyclase activity.
I
-5
Vol. 64, Nos. 6/l, 1999
SCH 57790 as M, Receptor Antagonist
539
Conclusion Muscarinic receptors are G-protein coupled receptors for which mutagenesis studies have provided insights into structural moieties required for agonist and antagonist binding. The M, selective compound, SCH 57790 and related sulfinyl piperazine analogues provide new tools with which to study muscarinic receptor binding interactions. Among the [4-(phenylsulfonyl) phenyl]methylpiperazines that have been tested for muscarinic receptor affinity and M, receptor selectivity, the highest observed affinity is obtained when a methyl group is located between the phenyl and piperazine rings. Substituting a nitrile for the methyl in this position decreases the affinity at all muscarinic receptors, but the decrease is more pronounced for M, than M, receptors, particularly for the (S)-sulfinyl enantiomer. This enantiomer, SCH 57790, binds to M, receptors with a Ki of 2.8 nM and is forty-fold selective for the M, over the M, receptor. SCH 57790 has a basic nitrogen which appears to interact with Asp 103 in TMIII of the M, receptor. The sulfinyl group of SCH 57790 is postulated to interact with Trp 99. Further site directed mutagenesis studies will be required to elucidate additional interactions with the M, receptor and to determine the key residues which confer selectivity over other muscarinic subtypes. SCH 57790 is an antagonist at M, receptors, demonstrated by the compound’s ability to attenuate oxotremorine-mediated inhibition of adenylyl cyclase activity in CHO cells. This high affinity, M, selective compound may provide a means for increasing synaptic levels of acetylcholine by selectively blocking presynaptic muscarinic autoreceptors in the CNS. Future studies with this compound in animal models of cognition will demonstrate whether this mechanism of increasing acetylcholine release will enhance learning and memory functions.
References 1. 2. 3. 4. 5. 6. 7.
:: 10. 11. 12. 13. 14.
R.T. BARTUS, R.L. DEAN, AND C. FLICKER, Psychopharmacology: the third Generation of Progress, H.Y. Meltzer, (Ed.), 219-232, Raven Press, New York (1987). D.M. ARMSTRONG, C.B. SAPER, A.I. LEVEY, B.H. WAINER, R.D. TERRY RD, J. Comp. Neurol., 216 53-68 (1983). M. MCKJNNEY, J.T. COYLE, J.C. HEDREEN, J. Comp. Neurol., 217 103-121 (1983). C. FLICKER, R.L. Dean, D.L. Watkins, S.K. Fisher, R.T. Bartus, Pharmacol. Biochem. Behav. 18 973-981 (1983). C.L. MURRAY and H.C. FIBIGER, Neuroscience 14 0125-1032 (1985). W. OP DEN VELDE and F.C. STAM, J. Am. Geriatr. Sot. 24 12-16 (1976). E.K. PERRY, P.H. GIBSON, G. BLESSED, R.H. PERRY, B.E. TOMLINSON, J. Neurol. Sci. 34 247-265 (1977). S. NOCHI, N. ASAKAWA, and T. SATO, Biol. Pharm. Bull. 18 1145-l 147 (1995). M.J. STILLMAN, B. SHUKITT-HALE, R.L. GALLI, A. LEVY, H.R. LIEBERMAN, Brain Res. Bull. 41 221-226 (1996). W. BILLARD, H. BINCH, G. CROSBY, R.D. MCQUADE, J. Pharm. Exp. Ther. 273 273-279 (1995). Y-C. CHENG and W.H. PRUSOFF Biochem. Pharmacol. 22 3099 (1973). C.M. FRASER, C.D. WANG, D.A. ROBINSON, J.D. GOCAYNE, J.C. VENTER, Mol. Pharmacol. 36 840-847 (1989). C.D. STRADER, I.S. SIGAL, M.R. CANDELORE, E. RANDS, W.S. HILL, R.A. DIXON, J. Biol. Chem. 263 10267-10271 (1988). H. MATSUI, S. LAZARENO, N.J. BIRDSALL, Mol. Pharmacol. 47 88-98 (1995)
ELSEVIER
PI1 SOO24-3205(98)00598-O
SCH 57790: A NOVEL M, RECEPTOR SELECTIVE ANTAGONIST
Jean E. Lachowicz, Derek Lowe, Ruth A. Duffy, Vilma Ruperto, Lisa A. Taylor, Henry Guzik, Joan Brown, Joel G. Berger, Melissa Tice, Robert McQuade, Joseph Kozlowski, John Clader, Catherine D. Strader and Nicholas Murgolo Departments of Chemical Research and Central Nervous System Research, Schering-Plough Research Institute, Kenilworth, New Jersey 07033
Summary As a decrease in cholinergic neurons has been observed in Alzheimer’s Disease (AD), therapeutic approaches to AD include inhibition of Evidence suggests that acetylcholinesterase to increase acetylcholine levels. acetylcholine release in the CNS is modulated by negative feedback via presynaptic M, receptors, blockade of which should provide another means of acetylcholine release. Structure-activity studies of [4increasing (phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-aThis [4-[[4-methoxyphenyl]sulfinyl]-phenyl]-l-piperazineacetonitle. compound, SCH 57790, binds to cloned human M receptors expressed in CHO cells with an affinity of 2.78 nI@ the affinity at M, receptors is 40-fold lower. SCH 57790 is an antagonist at M, receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M, receptor blockade for enhancement of cognition.
Key Words: SCH 57790, M, receptor antagonist, Alzheimer ’s disease, acetylcholine
The neurotransmitter acetylcholine has been demonstrated to modulate learning and memory processes (1). Thus, the cholinergic system has been of interest in the study of cognitive disorders, including the neurodegenerative disorder known as Alzheimer’s Disease (AD). Cholinergic neurons originating in the nucleus basalis of Meynert project to areas such as the cortex and hippocampus, brain regions which play a role in cognition (2,3). Lesioning these pathways in rodents produces a decrease in acetylcholine release and an impairment of memoryrelated task performance (4). This impairment can be reversed by cholinergic agonists (5). In AD, decreases in acetylcholinesterase levels and choline acetyltransferase activity have been reported (6,7). These observations indicate that the cholinergic system is compromised in AD and that drugs which stimulate cholinergic activity may provide relief from the neurodegenerative symptoms. Currently, AD is treated with inhibitors of acetylcholinesterase, such as donepezil, which reduce the rate at which released acetylcholine is degraded for removal from the synapse (8). Another possible mechanism for augmenting central choline@ activity is to facilitate acetylcholine release. Evidence suggests that in the CNS, presynaptic M, receptors act to inhibit acetylcholine release (9, 10). A selective M, antagonist which blocks this inhibition may therefore prove useful in the treatment of AD. SCH 57790 (4-cyclohexyl-a-[4-[Cmethoxyphenyl]-(S)-sulfinyl]phenyll-1-piperazineacetonitrile) is a selective, high affinity M, antagonist. The interaction of this compound and a number of analogues (Fig. 1) with human muscarinic receptors is described.
536
Vol. 64, Nos. 617, 1999
SCH 57790 as M, Receptor Antagonist
Methods The synthesis of SCH 57790 will be described elsewhere (Lowe, et al., manuscript in preparation). Human muscarinic receptor constructs were transfected into CHO-Kl cells by the calcium phosphate precipitation method. Colonies were selected for G418 resistance and tested for receptor expression by radioligand binding to Z-quinuclidinyl IphenyZ-4-3H]benzilate ( [3H] QNB). Saturation experiments were performed using 5-2500 pM r3H] QNB in 10 mM potassium phosphate buffer, pH 7.4, to determine the Kd of [3H] QNB for each receptor subtype. Competition binding experiments were performed using 180 pM [3H] QNB and lo-50 ug of membrane protein. All binding experiments were performed in the presence of 1% DMSO and 0.4% methylcellulose. Non-specific binding was defined by 0.5 l.tM atropine. After equilibrium was reached (120 minute incubation at room temperature) bound and free radioactivity were separated by filtration using Whatman GF-C filters. Ki values were derived from IC,, values using the Cheng-Prusoff equation (11). Adenylyl cyclase assays were conducted using M, receptor-expressing CHO cells in suspension (approximately 250,000 cells/ 60 l.rIassay volume). To provide a stimulated baseline from which to observe oxotremorine-mediated inhibition, 3 J.&I forskolin was included in the assay. Phosphodiesterase activity was inhibited by 100 uM Ro-02-1724. Cells were pretreated with antagonist for 10 minutes prior to incubation with oxotremorine at 37°C for 5 minutes. A subsequent three minute 100°C incubation terminated the reaction and lysed the cells to release accumulated CAMP. Total CAMP in a 20 l.tl assay sample was quantified by a radioimmunoassay capable of detecting between 0.5 and 100 uM CAMP.
H,CO A. Racemic B. (S)-Sulfoxide
(SCH 57790)
H&O C. Racemic D. (S)-Sulfoxide
H3C0 E. Racemic
Fig. 1 Chemical structures of SCH 57790 and analogues. Results
and Discussion
Fig. 2 shows SCH 57790 displacement of [3H] QNB from human M,-M, receptors. The muscarinic receptor binding affinities for SCH 57790 and its analogues are listed in Table 1. The K values derived from these competition experiments provide insights into the structure activity relationships of these compounds and muscarinic receptors. Substitution of a methyl group for the nitrile substituent confers higher affinity binding to muscarinic receptors, illustrated by compounds C and D. However, M, receptor selectivity versus M,, M,, and M4 receptors is decreased by this substitution. The increased affinity for muscarinic receptors overall combined
SCH 57790 as M2 Receptor Antagonist
Vol. 64, Nos. 6/l, 1999
537
with the loss in selectivity suggests that the methyl substituent interacts with amino acids that are common to all muscarinic receptor subtypes. The nitrile substituent may interact with residues that are specific to the M, receptor. The methyl compounds display the same stereoselectivity as the nitrile compounds; the (S)-sulfinyl enantiomer binds to muscarinic receptors with higher affinity than the racemic compound. This Compound E shows the ramifications of replacing the piperazine with a piperidine. It is compound, which lacks the more basic nitrogen, is inactive at M, and M, receptors. postulated that this nitrogen forms an ionic interaction with the Asp 103 in the third transmembrane domain of the M, receptor. The aspartic acid in this position is common to all biogenic amine receptors and has been shown using receptor mutagenesis to be critical for agonist binding in muscarinic and other G-protein-coupled receptors (12, 13). The hypothesis that the piperazine nitrogen interacts with Asp 103 is further supported by results from receptor mutagenesis studies. Substitution of Asp 105 for Ala in the human M, receptor reduced the affinity of SCH 57790 by 14-fold.
loo-
F F ._
80-
m m 605 F 0I 408 20-
?? ml
A v
m2 m3 ?? m4
0
m5
o-
Inhibition
Fig. 2 of r3H] QNB binding by SCH 57790 at the five cloned muscarinic receptors.
TABLE 1 Affinities of SCH 57790 and Analogues at Human Muscarinic Receptors in CHO Cells. M,Ki,nM A : :
114 6.0 112 4.7 >500 NT= Not Tested.
M, Ki, nM 9.1 2.8 A.: ,500
M,Ki,nM ;: 1.0 0.4 NT
M4 Ki, nM 38 2.2 14 0.7 NT
M,Ki,tlM 5; NT NT NT
Vol. 64, Nos. 617, 1999
SCH 57790 as M, Receptor Antagonist
538
Molecular models of SCH 57790 binding to the human MJ receptor suggest that the sulfinyl moiety interacts with Trp 99 in TMID (Fig 3). This interaction, which has been studied for M, receptors (14), is supported by the stereoselectivity observed for SCH 57790 and analogues. Substitution of Ala for Trp 99 in the M, receptor resulted in a lo-fold decrease in the affinity of SCH 57790. In addition, the models suggest that the distance between the piperazine nitrogen and the sulfoxide is 9 A, consistent with the distance between Trp 99 and Aspl03, which represents one helical turn.
TM I, TM II Hydrophobic Pocket
H&O Fig. 3 Model of interactions between the human M, receptor and SCH 57790. The muscarinic agonist oxotremorine-M produces a 30% maximal inhibition of CAMP produced by M,-expressing CHO cells with an EC,, of 10 nM (Fig 4). SCH 57790 (30 r&l) shifts the In the dose-response curve for oxotremorine to the right in a parallel fashion by 130-fold. presence of 100 nM SCH 57790, oxotremorine-M produces no inhibition of adenylyl cyclase.
Control 30 nM SCH
404 -12
-11
-10
-9
-8
-7
-6
[Oxotremorine], Log M
Oxotremorine-mediated
Fig. 4 inhibition of forskolin-stimulated
adenylyl cyclase activity.
I
-5
Vol. 64, Nos. 6/l, 1999
SCH 57790 as M, Receptor Antagonist
539
Conclusion Muscarinic receptors are G-protein coupled receptors for which mutagenesis studies have provided insights into structural moieties required for agonist and antagonist binding. The M, selective compound, SCH 57790 and related sulfinyl piperazine analogues provide new tools with which to study muscarinic receptor binding interactions. Among the [4-(phenylsulfonyl) phenyl]methylpiperazines that have been tested for muscarinic receptor affinity and M, receptor selectivity, the highest observed affinity is obtained when a methyl group is located between the phenyl and piperazine rings. Substituting a nitrile for the methyl in this position decreases the affinity at all muscarinic receptors, but the decrease is more pronounced for M, than M, receptors, particularly for the (S)-sulfinyl enantiomer. This enantiomer, SCH 57790, binds to M, receptors with a Ki of 2.8 nM and is forty-fold selective for the M, over the M, receptor. SCH 57790 has a basic nitrogen which appears to interact with Asp 103 in TMIII of the M, receptor. The sulfinyl group of SCH 57790 is postulated to interact with Trp 99. Further site directed mutagenesis studies will be required to elucidate additional interactions with the M, receptor and to determine the key residues which confer selectivity over other muscarinic subtypes. SCH 57790 is an antagonist at M, receptors, demonstrated by the compound’s ability to attenuate oxotremorine-mediated inhibition of adenylyl cyclase activity in CHO cells. This high affinity, M, selective compound may provide a means for increasing synaptic levels of acetylcholine by selectively blocking presynaptic muscarinic autoreceptors in the CNS. Future studies with this compound in animal models of cognition will demonstrate whether this mechanism of increasing acetylcholine release will enhance learning and memory functions.
References 1. 2. 3. 4. 5. 6. 7.
:: 10. 11. 12. 13. 14.
R.T. BARTUS, R.L. DEAN, AND C. FLICKER, Psychopharmacology: the third Generation of Progress, H.Y. Meltzer, (Ed.), 219-232, Raven Press, New York (1987). D.M. ARMSTRONG, C.B. SAPER, A.I. LEVEY, B.H. WAINER, R.D. TERRY RD, J. Comp. Neurol., 216 53-68 (1983). M. MCKJNNEY, J.T. COYLE, J.C. HEDREEN, J. Comp. Neurol., 217 103-121 (1983). C. FLICKER, R.L. Dean, D.L. Watkins, S.K. Fisher, R.T. Bartus, Pharmacol. Biochem. Behav. 18 973-981 (1983). C.L. MURRAY and H.C. FIBIGER, Neuroscience 14 0125-1032 (1985). W. OP DEN VELDE and F.C. STAM, J. Am. Geriatr. Sot. 24 12-16 (1976). E.K. PERRY, P.H. GIBSON, G. BLESSED, R.H. PERRY, B.E. TOMLINSON, J. Neurol. Sci. 34 247-265 (1977). S. NOCHI, N. ASAKAWA, and T. SATO, Biol. Pharm. Bull. 18 1145-l 147 (1995). M.J. STILLMAN, B. SHUKITT-HALE, R.L. GALLI, A. LEVY, H.R. LIEBERMAN, Brain Res. Bull. 41 221-226 (1996). W. BILLARD, H. BINCH, G. CROSBY, R.D. MCQUADE, J. Pharm. Exp. Ther. 273 273-279 (1995). Y-C. CHENG and W.H. PRUSOFF Biochem. Pharmacol. 22 3099 (1973). C.M. FRASER, C.D. WANG, D.A. ROBINSON, J.D. GOCAYNE, J.C. VENTER, Mol. Pharmacol. 36 840-847 (1989). C.D. STRADER, I.S. SIGAL, M.R. CANDELORE, E. RANDS, W.S. HILL, R.A. DIXON, J. Biol. Chem. 263 10267-10271 (1988). H. MATSUI, S. LAZARENO, N.J. BIRDSALL, Mol. Pharmacol. 47 88-98 (1995)