Structure–activity relationships for substrate recognition by the human dopamine transporter (original) (raw)
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European Journal of Pharmacology: Molecular Pharmacology, 1992
The present study addressed the possibility that there are distinct but allosterically interacting populations of binding siles for dopamine/cocaine and BTCP/GBR (N-[l-(2-benzo[b]thiophenyl)cyelohexyl]piperidine/ I-(2-diphenylmethnxy)-ethyl]-4-(3phenylpropyl)piperazine) (selective dopamine uptake blockers) on the dopamine transporter in the rat :.triatum. Dopaminc uptake '.;ite.~ were labeled in vitro with the cocaine analog [3H]CFT (2/3-carbometboxy-3/3-(4-fluorophenyl)-tropane). and the inhibi~b:,n of binding by CFT or cocaine was measured. A graphic method was adopted for studying shifts in inhibilory potency resulting from the addition of a second compound. Under the conditions used, the co-presence of dopamine. GBR 12935, or BTCP decreased the inhibitory potency ~f CFT or cocaine to the cxtcm predicted by a model in which all compounds bind to the same site or the binding of all compounds is mutually exclusive. No evidence for negative allosteric interactions between CFT and BTCP was found in experiments comparing inhibition of [3H]CFT binding by BTCP at a Io. and high concentration of [3HICFT.
Life Sciences, 1990
Structure-activlty relationships for cocaine and analog binding at the depamine, norepinephrine and serotonin transporters were determined. Cocaine inhibition of llgand binding to each of these sites has a stereospecific requirement for the levorotatory isomer. Binding potencies of cocaine derivatives involving N-substltution, C2 and C3 substituent modifications, however, revealed differences in structure-activity relationships for cocaine binding at the transporters. Removal of the N-methyl groups produced little change in binding potency at the dopamlne transporter site hut produced increases in binding potency at norepinephrlne and serotonin transporter sites. Changes in structure at the C2 substituent produced changes in binding potency at the dopamlne transporter which were generally similar in direction, but not necessarily in magnitude at the noreplnephrine and serotonin transporters. Modifications to the C3 substituent, especially substitution of a hydroxyl moiety, produce changes in affinity at norepinephrine and serotonin transporters which are much larger than those observed at dopamine transporters. In general, our results indicate that unique structural requirements exist for each transporter site, but that cocaine binding at norepinephrine and dopamine transporters can be described by more similar structure-actlvity relationships than those found for the serotonin transporter. Reguirements for cocaine binding to the dopamine transporter, which we have previously shown to be associated with the reinforcing effects of cocaine, include levorotatory sterospeclficlty, the benzene ring at C3, at least some portions of the tropane ring, and the presence of the C2 methyl ester group in the ~ conformation. A variety of psychoactive drugs are known to inhibit the active neurotransmitter uptake process and as well as llgand binding to transporter sites. Cocaine, in particular, has been shown to inhibit the transport of dop~nine, norepinephrine and serotonin (i, 2, 3, 4, 5) and to interact with some receptors (6, 7, 8). Recently, Ritz et al. (9) presented evidence that the cocaine inhibition of 3H-mazindol binding to the dopamine transporter may be related to the reinforcing properties of cocaine and related drugs.
Journal of Neurochemistry, 2008
The widely abused psychostimulant cocaine is thought to elicit its reinforcing effects primarily via inhibition of the neuronal dopamine transporter (DAT). However, not all DAT inhibitors share cocaine's behavioral profile, despite similar or greater affinity for the DAT. This may be due to differential molecular interactions with the DAT. Our previous work using transporter mutants with altered conformational equilibrium (W84L and D313N) indicated that benztropine and GBR12909 interact with the DAT in a different manner than cocaine. Here, we expand upon these previous findings, studying a number of structurally different DAT inhibitors for their ability to inhibit [ 3 H]CFT binding to wildtype, W84L and D313N transporters. We systematically tested structural intermediates between cocaine and benztropine, structural hybrids of benztropine and GBR12909 and a number of other structurally heterologous inhibitors. Derivatives of the stimulant desoxypipradrol (2-benzhydrylpiperidine) exhibited a cocaine-like binding profile with respect to mutation, whereas compounds possessing the diphenylmethoxy moiety of benztropine and GBR12909 were dissimilar to cocaine-like compounds. In tests with specific isomers of cocaine and tropane analogues, compounds with 3α stereochemistry tended to exhibit benztropine-like binding, whereas those with 3β stereochemistry were more cocaine-like. Our results point to the importance of specific molecular features-most notably the presence of a diphenylmethoxy moiety-in determining a compound's binding profile. This study furthers the concept of using DAT mutants to differentiate cocaine-like inhibitors from atypical inhibitors in vitro. Further studies of the molecular features that define inhibitor-transporter interaction could lead to the development of DAT inhibitors with differential clinical utility.
Probes for the cocaine receptor. Potentially irreversible ligands for the dopamine transporter
Journal of Medicinal Chemistry, 1992
Several potentially irreversible ligands (Le., wash-resistant binding inhibitors) for the cocaine receptor site on the dopamine transporter, derived from (-)-cocaine or 3B-phenyltropan-28-carboxylic acid methyl ester (WIN 35,065-2), were prepared and shown to produce wash-resistant inhibition of [3H]-3&(p-fluorophenyl)tropan-2~-carboxylic acid methyl ester ([3H]WIN 35,428) binding. All the compounds prepared had the game absolute configuration as cocaine; they include analogues possessing chemically reactive groups such as the isothiocyanato and bromoacetamido as well as photoactive azido groups. The potentially irreversible ligands, as well as all the intermediates prepared in this study, were evaluated for their ability to inhibit the binding of SH]WIN 35,428 in coincubation experiments. Of the potentially irreversible ligands, 3 & ( p c h l o r o p h e n y l ) t r~~~~l i c acid 2-[p(bromoacetamido)phenyl]ethyl ester (6c) had the higheat apparent potency. The potentially irreversible ligands were also preincubated, and inhibition of [3H]WIN 35,428 binding was determined both before and after washing the ligand-exposed tissues. The most effective ligands in this regard were 3~-(3-iodo-4-azidophenyl)tropan-2/3-carboxylic acid methyl ester (5) and 38-@-chlorophenyl)hpan-ropan-2gcarboxylic acid 2-(3-iodo4azidophenyl)ethyl ester (Sa). The structureadivity relationships of these data are discussed. Ritz, M. C.; Lamb, R. J.; Goldberg, S. R.; Kuhar, M. J. Cocaine Receptors on Dopamine Transporters Are Related to Self-administration of Cocaine. Science 1987,237, 1219-1223. Bergman, J.; Madras, B. K.; Johnson, S. E.; Spealman, R. D. Effecta of Cocaine and Related Drug in Nonhuman Primates. 111. Self-administration by Squirrel Monkeys. J. Pharrnacol. Exp. Ther. 1989,251, 150-155. Carroll, F. I.; Lewin, A. H.; Boja, J. W.; Kuhar, M. J. Cocaine Receptor: Biochemical Characterization and Structure-Activity Relationships for the Dopamine Transporter. J. Med. Chern., in press. Boja, J. W.; Rahman, M. R.; Philip, A.; Lewin, A. H.; Carroll, F. I.; Kuhar, M. J. Isothiocyanate Derivatives of Cocaine: Irreversible Inhibition of Ligand Binding at the Dopamine Transporter. Mol. Phormacol. 1991,39,339-345. Lew, R.; Boja, J. W.; Simantov, R.; Carroll, F. I.; Lewin, A.; Kuhar, M. J. New Photoaffinity Probes for the Cocaine Receptor. SOC. Neurosci. Abstr. 1990, 16, 746. Lewin, A. H.; Gao, Y.; Abraham, P.; Boja, J. W.; Kuhar, M. J.; Carroll, F. I. P&Substituted Analogs of Cocaine. Syntheais and Inhibition of Binding to the Cocaine Receptor.
Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3b-aryltropanes with 2b-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2a-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2b-Ph 2 COCH 2-3b-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2b compounds increased locomotion, only the 2b-(4-ClPh)PhCOCH 2-3b-4-Cl-Ph analog had cocaine-like efficacy. None of the 2a-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)-compared with inward-facing confor-mation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2b-and 2a-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.
Advances in Pharmacology, 2009
Stimulant drugs acting at the dopamine transporter (DAT), like cocaine, are widely abused, yet effective medical treatments for this abuse have not been found. Analogs of benztropine that, like cocaine, act at the DAT have effects that differ from cocaine, and in some situations block the behavioral, neurochemical, and reinforcing actions of cocaine. Neurochemical studies of dopamine levels in brain and behavioral studies have demonstrated that benztropine analogs have a relatively slow onset and reduced maximal effects compared to cocaine. Pharmacokinetic studies, however, indicated that the benztropine analogs access the brain at concentrations above their in vitro binding affinities while binding in vivo demonstrates apparent association rates for benztropine analogs lower than that for cocaine. Additionally, the off-target effects of these compounds do not fully explain their differences from cocaine. Initial structure-activity studies indicated that benztropine analogs bind to DAT differently from cocaine and these differences have been supported by site-directed mutagenesis studies of the DAT. In addition, benztropine analog-mediated inhibition of uptake was more resistant to mutations producing inward conformational DAT changes than cocaine analogs. The benztropine analogs have provided new insights into the relation between the molecular and behavioral actions of cocaine, and the diversity of effects produced by dopamine transport inhibitors. Novel interactions of benztropine analogs with the DAT suggest that these drugs may have a pharmacology that would be useful in their development as treatments for cocaine abuse.
Identification of a Dopamine Transporter Ligand That Blocks the Stimulant Effects of Cocaine
Journal of Neuroscience, 2005
There is a large unmet medical need for cocaine addiction treatments. Studies have indicated that the dopamine transporter (DAT) is the primary biological target of cocaine, and most drugs that have DAT affinity have behavioral effects like those of cocaine. However, analogs of benztropine have high DAT affinity and behavioral effects that show varying degrees of similarity to cocaine. We now report the discovery that a benztropine analog, JHW007, with high affinity for the DAT does not have cocaine-like behavioral effects and antagonizes the effects of cocaine. JHW007 occupied the DAT in vivo more slowly than did cocaine and had not reached an apparent plateau up to 270 min after injection. The in vivo binding of cocaine to the DAT suggested rate of DAT occupancy as an important contributor to its behavioral effects, and the slow association with the DAT may provide an explanation for JHW007 being relatively devoid of cocaine-like behavioral effects. The antagonism of cocaine suggests that DAT ligands with reduced cocaine-like activity can function as cocaine antagonists and suggests JHW007 as a lead for discovery of cocaine-abuse pharmacotherapeutics.
Psychopharmacology, 2001
Rationale: Previous SAR studies demonstrated that small halogen substitutions on the diphenylether system of benztropine (BZT), such as a para-Cl group, retained high affinity at the cocaine binding site on the dopamine transporter. Despite this high affinity, the compounds generally had behavioral effects different from those of cocaine. However, compounds with meta-Cl substitutions had effects more similar to those of cocaine. Objectives: A series of phenyl-ring analogs of benztropine (BZT) substituted with 3′-, 4′-, 3′,4′′- and 4′,4′′-position Cl-groups were synthesized and their pharmacology was evaluated in order to assess more fully the contributions to pharmacological activity of substituents in these positions. Methods: Compounds were synthesized and their pharmacological activity was assessed by examining radioligand binding and behavioral techniques. Results: All of the compounds displaced [3H]WIN 35,428 binding with affinities ranging from 20 to 32.5 nM. Affinities at norepinephrine ([3H]nisoxetine) and serotonin ([3H]citalopram) transporters, respectively, ranged from 259 to 5120 and 451 to 2980 nM. Each of the compounds also inhibited [3H]pirenzepine binding to muscarinic M1 receptors, with affinities ranging from 0.98 to 47.9 nM. Cocaine and the BZT analogs produced dose-related increases in locomotor activity in mice. However, maximal effects of the BZT analogs were uniformly less than those produced by cocaine, and were obtained 2–3 h after injection compared to the relatively rapid onset (within 30 min) of cocaine effects. In rats trained to discriminate IP saline from 29 µmol/kg cocaine (10 mg/kg), cocaine produced a dose-related increase in responding on the cocaine lever, reaching 100% at the training dose; however, none of the BZT analogs fully substituted for cocaine, with maximum cocaine responding from 20 to 69%. Despite their reduced efficacy compared to cocaine in cocaine discrimination, none of the analogs antagonized the effects of cocaine. As has been reported previously for 4′-Cl-BZT, the cocaine discriminative-stimulus effects were shifted leftward by co-administration of the present BZT analogs. Conclusions: The present results indicate that although the BZT analogs bind with relatively high affinity and selectivity at the dopamine transporter, their behavioral profile is distinct from that of cocaine. The present results suggest that analogs of BZT may be useful as treatments for cocaine abuse in situations in which an agonist treatment is indicated. These compounds possess features such as reduced efficacy compared to cocaine and a long duration of action that may render them particularly useful leads for the development of therapeutics for cocaine abusers.
Molecular Pharmacology, 2007
Cocaine exerts its stimulatory effect by inhibiting the dopamine transporter (DAT). However, novel benztropine-and rimcazolebased inhibitors show reduced stimulant effects compared with cocaine, despite higher affinity and selectivity for DAT. To investigate possible mechanisms, we compared the subjective effects of different inhibitors with their molecular mode of interaction at the DAT. We determined how different inhibitors affected accessibility of the sulfhydryl-reactive reagent [2-(trimethylammonium)ethyl]-methanethiosulfonate to an inserted cysteine (I159C), which is accessible when the extracellular transporter gate is open but inaccessible when it is closed. The data indicated that cocaine analogs bind an open conforma-
Neurochemistry International, 2014
The dopamine transporter (DAT), a member of the neurotransmitter:sodium symporter family, mediates the reuptake of dopamine at the synaptic cleft. DAT is the primary target for psychostimulants such as cocaine and amphetamine. We previously demonstrated that cocaine binding and dopamine transport alter the accessibility of Cys342 in the third intracellular loop (IL3). To study the conformational changes associated with the functional mechanism of the transporter, we made cysteine substitution mutants, one at a time, from Phe332 to Ser351 in IL3 of the background DAT construct, X7C, in which 7 endogenous cysteines were mutated. The accessibility of the 20 engineered cysteines to polar charged sulfhydryl reagents was studied in the absence and presence of cocaine or dopamine. Of the 11 positions that reacted with methanethiosulfonate ethyl ammonium, as evidenced by inhibition of ligand binding, 5 were protected against this inhibition by cocaine and dopamine (S333C, S334C, N336C, M342C and T349C), indicating that reagent accessibility is affected by conformational changes associated with inhibitor and substrate binding. In some of the cysteine mutants, transport activity is disrupted, but can be rescued by the presence of zinc, most likely because the distribution between inward-and outward-facing conformations is restored by zinc binding. The experimental data were interpreted in the context of molecular models of DAT in both the inward-and outward-facing conformations. Differences in the solvent accessible surface area for individual IL3 residues calculated for these states correlate well with the experimental accessibility data, and suggest that protection by ligand binding results from the stabilization of the outward-facing configuration. Changes in the residue interaction networks observed from the molecular dynamics simulations also revealed the critical roles of several positions during the conformational transitions. We conclude that the IL3 region of DAT undergoes significant conformational changes in transitions necessary for both cocaine binding and substrate transport.