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Pharmacology and therapeutic actions of drugs which inhibit neuronal transporters
249 References Amara, S. and Kuhar, M.J. (1993) Neurotransmitter transporters: Recent progress. Annu. Rev. Neurosci. 16, 73-93. Cerruti, C., Pilotte, N.S., Uhl, G. and Kuhar, M.J. (1994) Reduction in dopamine transporter mRNA after cessation of repeated cocaine administration. Mol. Brain Res. 22, 132-138. Kitayama, S., Shimada, S., Xu, H., Markham, L., Donovan, D.M. and Uhl, G.R. (1992) Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc. Natl. Acad. Sci. USA 89, 7782-7785. Lew, R., Patel, A., Vaughan, R.A., Wilson, A. and Kuhar, M.J. (1992) Microheterogeneity of dopamine transporters in rat striatum. Brain Res. 584, 266-271.
Pharmacology and therapeutic actions of drugs which inhibit neuronal transporters
S.Z. Langer, M. Agnel, H. Esnaud and D. Graham Synthelabo Recherche, Site de Rueil, BP 248, 92504 Rueil Malmaison, France Key words." Neurotransmitter transporters; 3H-serotonin uptake; 3H-citalopram binding; Serotonin uptake inhibitor Neurotransmitter transport systems play a pivotal role in the re-uptake, recycling and release of neurotransmitters at synapses. The sodium-ion coupled neurotransmitter transport systems localized on the plasma membrane of presynaptic nerve terminals mediate neurotransmitter re-uptake from the synaptic cleft, thereby terminating their physiological action. Upon reuptake, proton-coupled vesicular transporters concentrate and store neurotransmitters in storage vesicles for further release. Agents which modulate these transport systems can produce profound effects upon neurotransmission. Moreover, pharmacological intervention with certain drugs which inhibit the activity of specific transporters has proven therapeutic efficacy, particularly in neuropsychiatric disorder treatment. In recent years, molecular biological techniques have been used to elucidate the structure, function and diversity of these neurotransmitter transport systems. This presentation focuses on these advances and discusses some of the opportunities that this could provide for future drug discovery. Sodium-ion coupled neurotransmitter transporters Molecular cloning techniques have revealed that the sodium-ion coupled neurotransmitter transporters are in fact composed of two structurally distinct gene families. These two families can also be mechanistically distinguished on the basis of their association with other ions. One family, the excitatory amino acid transporters, includes four members that have been cloned to date (GLT1, GLAST1, EAAC1 and ASCT1) and these transporters mediate neurotransmitter influx using the electrochemical energy provided by the co-transport of sodium and the counter-transport of potassium ions. The other family utilizes the co-transport of sodium and chloride ions in neurotransmitter uptake and is composed of transporters for a variety of different neurotransmitters such as GABA, glycine, taurine, proline, noradrenaline, dopamine and serotonin. Studies in our laboratory have particularly focused on the serotonin transporter. Serotonin transporter The serotonin transporter has been cloned from rat (Blakely et al., 1991) and man (Ramamoorthy et al., 1993) and identified in each instance as a 630-amino acid polypeptide, the human sequence showing 92% amino acid identity with its rat homologue (Ramamoorthy et al., 1993). In common with other sodium/chloride-ion coupled neurotransmitter transporters, the serotonin transporter polypeptide sequence predicts secondary structure containing 12 putative transmembrane-spanning zones which would have no signal sequence, thereby suggesting that both the amino and carboxy termini are located cytoplasmically. We have performed a comprehensive study of the properties of the human serotonin transporter polypeptide subsequent to its transient expression in a surrogate HeLa cell line. Using these transfected cells [3H]serotonin uptake was saturable (Kin = I /~M) and potentially inhibited by the selective serotonin uptake inhibitors, indalpine, litoxetine, sertraline and fluoxetine with Ki values in the low nanomolar range. In contrast other compounds, such as maprotiline, GBR12909 and odansetron, had very little effect on [3H]serotonin uptake (Ki values >1 /~M). [3H]Citalopram binding to membrane preparations of these transfected cells occurred to a single class of high-affinity binding sites (Kd = 5.3 nM). Moreover, under equilibrium competition inhibition conditions, [3H]citalopram binding was potently inhibited by selective serotonin uptake
250 inhibitors such as paroxetine, indalpine, sertraline, litoxetine, citalopram and fluoxetine with Ki values in the lower nanomolar range. These uptake and radioligand binding experiments therefore illustrate that the full pharmacological characteristics of the serotonin transport system are conferred by the expression of the 630 amino acid serotonin transporter polypeptide. Neurotransmitter transporters as therapeutic targets
The hypothesis that monoaminergic deficits might be implicated in depression led to the development of selective inhibitors of the serotonin and noradrenaline transport systems. Selective serotonin uptake inhibitors such as fluoxetine, sertraline, paroxetine and citalopram have shown particular promise in that these compounds exhibit considerable therapeutic efficacy as antidepressants and are devoid of the anticholinergic and cardiovascular side effects associated with the tricyclic antidepressants. Moreover, the selective serotonin uptake inhibitors are finding application beyond the clinical management of depression where their efficacy is being studied in disorders such as panic attacks, obsessive compulsive disorders and bulimia. A G A B A uptake inhibitor, tiagabine, has now been advanced to phase llI clinical trials as an anticonvulsant. In this context, identification of G A B A transporter heterogeneity now offers the opportunity to develop GABA transporter subtype selective compounds with the hope of improving therapy by limiting the appearance of unwanted side effects, References
Blakely, R.D., Berson, H.E., Fremeau Jr. R.T., Caron, M.G., Peek, M.M., Prince, H.K. and Bradley, C.C. (1993) Cloning and expression of a functional serotonin transporter from rat brain. Nature 354, 66-70. Ramamoorthy, S., Bauman, A.L., Moore, H.R., Ham H., Yang-Feng, T., Chang, A.S., Ganapathy, V. and Blakely, R.D. (1993) Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc. Natl. Acad. Sci. USA 90, 2542-2546.
Molecular modeling and function of neuronal transporters
O. Edvardsen and S.G. Dahl Department of Pharmacology, Institute of Medical Biology University of Tromso. N-9037 Tromso, Norway Key words'; Carrier protein; Dopamine: 5-HT; Molecular modelling; Molecular structure Knowledge about the detailed molecular mechanisms of neurotransmitter reuptake in presynaptic neurons has therapeutic implications and would be useful in psychotropic drug design. Such knowledge may be inferred from three-dimensional molecular models of the transporter proteins. We have used molecular modelling techniques to build three-dimensional models of the human dopamine transporter (DAT) (Edvardsen and Dahl, 1994) and the human 5-HT transporter (5-HTT). These models were used to study the interaction of neurotransmitters and various reuptake inhibitors with the transporter proteins. The human DAT and 5-HTT models were constructed from the amino acid sequences (Vandenbergh et al., 1992; Lesch et al., 1993), and included all residues in the proteins. The models were based on amino acid sequence analysis and information from site-directed mutagenesis and ligand binding experiments done by others. The DAT model was built by computer graphics, molecular mechanics calculations and molecular dynamics simulations, by a procedure similar to those we previously used to model various neurotransmitter receptors. The membrane spanning segments were identified from the average hydropathy index of 10 different transporter sequences. This procedure identified 12 hydrophobic segments, which were assumed to be transmembrane a-helices (TMHs) with a total length of 25-27 residues. The 12 TMHs were packed in a clockwise order, viewed from the synaptic side, and were arranged in two 7-TMH bundles sharing two TMHs. The loops and terminals were added to form the complete model, which was refined by energy minimization and molecular dynamics simulations using the AMBER 4 united atom force field. A model of the 5-HTT was created using the DAT model as a template. Dopamine, cocaine and cocaine isomers were docked into the DAT model by interactive computer graphics, and the complexes were energy minimized and further refined by molecular dynamics simulations. By a similar procedure, complexes of the 5-HTT and specific 5-HT reuptake inhibitors were constructed. Drug-transporter interaction energies were calculated and interactions between ligands and specific amino acids were examined.
Pharmacology and therapeutic actions of drugs which inhibit neuronal transporters
S.Z. Langer, M. Agnel, H. Esnaud and D. Graham Synthelabo Recherche, Site de Rueil, BP 248, 92504 Rueil Malmaison, France Key words." Neurotransmitter transporters; 3H-serotonin uptake; 3H-citalopram binding; Serotonin uptake inhibitor Neurotransmitter transport systems play a pivotal role in the re-uptake, recycling and release of neurotransmitters at synapses. The sodium-ion coupled neurotransmitter transport systems localized on the plasma membrane of presynaptic nerve terminals mediate neurotransmitter re-uptake from the synaptic cleft, thereby terminating their physiological action. Upon reuptake, proton-coupled vesicular transporters concentrate and store neurotransmitters in storage vesicles for further release. Agents which modulate these transport systems can produce profound effects upon neurotransmission. Moreover, pharmacological intervention with certain drugs which inhibit the activity of specific transporters has proven therapeutic efficacy, particularly in neuropsychiatric disorder treatment. In recent years, molecular biological techniques have been used to elucidate the structure, function and diversity of these neurotransmitter transport systems. This presentation focuses on these advances and discusses some of the opportunities that this could provide for future drug discovery. Sodium-ion coupled neurotransmitter transporters Molecular cloning techniques have revealed that the sodium-ion coupled neurotransmitter transporters are in fact composed of two structurally distinct gene families. These two families can also be mechanistically distinguished on the basis of their association with other ions. One family, the excitatory amino acid transporters, includes four members that have been cloned to date (GLT1, GLAST1, EAAC1 and ASCT1) and these transporters mediate neurotransmitter influx using the electrochemical energy provided by the co-transport of sodium and the counter-transport of potassium ions. The other family utilizes the co-transport of sodium and chloride ions in neurotransmitter uptake and is composed of transporters for a variety of different neurotransmitters such as GABA, glycine, taurine, proline, noradrenaline, dopamine and serotonin. Studies in our laboratory have particularly focused on the serotonin transporter. Serotonin transporter The serotonin transporter has been cloned from rat (Blakely et al., 1991) and man (Ramamoorthy et al., 1993) and identified in each instance as a 630-amino acid polypeptide, the human sequence showing 92% amino acid identity with its rat homologue (Ramamoorthy et al., 1993). In common with other sodium/chloride-ion coupled neurotransmitter transporters, the serotonin transporter polypeptide sequence predicts secondary structure containing 12 putative transmembrane-spanning zones which would have no signal sequence, thereby suggesting that both the amino and carboxy termini are located cytoplasmically. We have performed a comprehensive study of the properties of the human serotonin transporter polypeptide subsequent to its transient expression in a surrogate HeLa cell line. Using these transfected cells [3H]serotonin uptake was saturable (Kin = I /~M) and potentially inhibited by the selective serotonin uptake inhibitors, indalpine, litoxetine, sertraline and fluoxetine with Ki values in the low nanomolar range. In contrast other compounds, such as maprotiline, GBR12909 and odansetron, had very little effect on [3H]serotonin uptake (Ki values >1 /~M). [3H]Citalopram binding to membrane preparations of these transfected cells occurred to a single class of high-affinity binding sites (Kd = 5.3 nM). Moreover, under equilibrium competition inhibition conditions, [3H]citalopram binding was potently inhibited by selective serotonin uptake
250 inhibitors such as paroxetine, indalpine, sertraline, litoxetine, citalopram and fluoxetine with Ki values in the lower nanomolar range. These uptake and radioligand binding experiments therefore illustrate that the full pharmacological characteristics of the serotonin transport system are conferred by the expression of the 630 amino acid serotonin transporter polypeptide. Neurotransmitter transporters as therapeutic targets
The hypothesis that monoaminergic deficits might be implicated in depression led to the development of selective inhibitors of the serotonin and noradrenaline transport systems. Selective serotonin uptake inhibitors such as fluoxetine, sertraline, paroxetine and citalopram have shown particular promise in that these compounds exhibit considerable therapeutic efficacy as antidepressants and are devoid of the anticholinergic and cardiovascular side effects associated with the tricyclic antidepressants. Moreover, the selective serotonin uptake inhibitors are finding application beyond the clinical management of depression where their efficacy is being studied in disorders such as panic attacks, obsessive compulsive disorders and bulimia. A G A B A uptake inhibitor, tiagabine, has now been advanced to phase llI clinical trials as an anticonvulsant. In this context, identification of G A B A transporter heterogeneity now offers the opportunity to develop GABA transporter subtype selective compounds with the hope of improving therapy by limiting the appearance of unwanted side effects, References
Blakely, R.D., Berson, H.E., Fremeau Jr. R.T., Caron, M.G., Peek, M.M., Prince, H.K. and Bradley, C.C. (1993) Cloning and expression of a functional serotonin transporter from rat brain. Nature 354, 66-70. Ramamoorthy, S., Bauman, A.L., Moore, H.R., Ham H., Yang-Feng, T., Chang, A.S., Ganapathy, V. and Blakely, R.D. (1993) Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc. Natl. Acad. Sci. USA 90, 2542-2546.
Molecular modeling and function of neuronal transporters
O. Edvardsen and S.G. Dahl Department of Pharmacology, Institute of Medical Biology University of Tromso. N-9037 Tromso, Norway Key words'; Carrier protein; Dopamine: 5-HT; Molecular modelling; Molecular structure Knowledge about the detailed molecular mechanisms of neurotransmitter reuptake in presynaptic neurons has therapeutic implications and would be useful in psychotropic drug design. Such knowledge may be inferred from three-dimensional molecular models of the transporter proteins. We have used molecular modelling techniques to build three-dimensional models of the human dopamine transporter (DAT) (Edvardsen and Dahl, 1994) and the human 5-HT transporter (5-HTT). These models were used to study the interaction of neurotransmitters and various reuptake inhibitors with the transporter proteins. The human DAT and 5-HTT models were constructed from the amino acid sequences (Vandenbergh et al., 1992; Lesch et al., 1993), and included all residues in the proteins. The models were based on amino acid sequence analysis and information from site-directed mutagenesis and ligand binding experiments done by others. The DAT model was built by computer graphics, molecular mechanics calculations and molecular dynamics simulations, by a procedure similar to those we previously used to model various neurotransmitter receptors. The membrane spanning segments were identified from the average hydropathy index of 10 different transporter sequences. This procedure identified 12 hydrophobic segments, which were assumed to be transmembrane a-helices (TMHs) with a total length of 25-27 residues. The 12 TMHs were packed in a clockwise order, viewed from the synaptic side, and were arranged in two 7-TMH bundles sharing two TMHs. The loops and terminals were added to form the complete model, which was refined by energy minimization and molecular dynamics simulations using the AMBER 4 united atom force field. A model of the 5-HTT was created using the DAT model as a template. Dopamine, cocaine and cocaine isomers were docked into the DAT model by interactive computer graphics, and the complexes were energy minimized and further refined by molecular dynamics simulations. By a similar procedure, complexes of the 5-HTT and specific 5-HT reuptake inhibitors were constructed. Drug-transporter interaction energies were calculated and interactions between ligands and specific amino acids were examined.