Differential Binding of Tropane-Based Photoaffinity Ligands on the Dopamine Transporter
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Benztropine and its analogs are tropane ring-containing dopamine uptake inhibitors that produce behavioral effects markedly different from cocaine and other dopamine transporter blockers. We investigated the benztropine binding site on dopamine transporters by covalently attaching a benztropine-based photoaffinity ligand, [125I]N-[n-butyl-4-(4"'-azido-3"'-iodophenyl)]-4', 4"-difluoro-3alpha-(diphenylmethoxy)tropane ([125I]GA II 34), to the protein, followed by proteolytic and immunological peptide mapping. The maps were compared with those obtained for dopamine transporters photoaffinity labeled with a GBR 12935 analog, [125I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethy l]p iperazine ([125I]DEEP), and a cocaine analog, [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), which have been shown previously to interact with different regions of the primary sequence of the protein. [125I]GA II 34 became incorporated in a membrane-bound, 14 kDa fragment predicted to contain transmembrane domains 1 and 2. This is the same region of the protein that binds [125I]DEEP, whereas the binding site for [125I]RTI 82 occurs closer to the C terminal in a domain containing transmembrane helices 4-7. Thus, although benztropine and cocaine both contain tropane rings, their binding sites are distinct, suggesting that dopamine transport inhibition may occur by different mechanisms. These results support previously derived structure-activity relationships suggesting that benztropine and cocaine analogs bind to different domains on the dopamine transporter. These differing molecular interactions may lead to the distinctive behavioral profiles of these compounds in animal models of drug abuse and indicate promise for the development of benztropine-based molecules for cocaine substitution therapies.Keywords:
Tropane
Photoaffinity labeling
We recently reported a new class of tropanes, based on benztropine, that bind uniquely, in the S-configuration, to the dopamine transporter. We have now extended this series to evaluate the effects of substituents on the nitrogen and the diarylmethoxy group. Herein we have described the synthesis and biological evaluation of a series of 2-carbomethoxy-3-(diarylmethoxy)-1αH,5αH-tropane (2-carbomethoxybenztropine) analogs. Examination of the binding data obtained for these compounds shows that while the 4,4'-difluoro compound is potent and selective for the dopamine transporter, introduction of larger groups such as 4,4'-dichloro, 4,4'-dibromo, 4,4'-diiodo, or 4,4'-dimethyl on the 3-diphenylmethoxy moiety reduces this potency. However, although introduction of only one group (e.g., 4-chloro, 4-bromo, 4-iodo, or 4-methyl) leads to a similar reduction of binding affinity, these monosubstituted 2-carbomethoxybenztropines are significantly more potent than the related disubstituted compounds. Finally, from the data for the N-substituted 2-carbomethoxybenztropine analogs, it is evident that steric bulk can be tolerated at the nitrogen site. A comparison of structure−activity relationship data for the tropanes, GBR analogs, and these benztropines indicates that the 2-carbomethoxybenztropine analogs may be more like the GBR analogs in their mode of binding to the dopamine transporter than like the tropanes. This conclusion supports the notion that the binding site for (−)-cocaine [and the (1R)-tropanes] may differ from that of the 2-carbomethoxybenztropine analogs.
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Benztropine
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A novel photoaffinity label for the dopamine transporter (DAT) based on N-substituted 3α-[bis(4′-fluorophenyl)methoxy]tropane has been synthesized in five steps and has been characterized. Preliminary binding studies indicated this ligand bound irreversibly to the dopamine transporter. Preparation of the 125I analog and its photoactivation in the presence of membrane bound DAT demonstrated it covalently binds to the DAT.
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Dopamine Uptake Inhibitors
Photoaffinity labeling
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The design, synthesis and pharmacological evaluation of novel dopamine transporter ligands, based on Benztropine [3a-(diphenylmethoxy) tropane], has been a focus of our research efforts toward the development of novel cocaine-abuse pharmacotherapeutics. Structure-activity relationships at the dopamine transporter, for this series of compounds, have been derived and compared to those of cocaine and GBR 12909. These studies suggest that structurally diverse dopamine uptake inhibitors may access different binding domains on the dopamine transporter. The distinctive behavioral profile displayed in this series of compounds, as compared to cocaine and other dopamine uptake inhibitors, is of particular interest and is proposed to be relevant to the pharmacodynamic and pharmacokinetic properties of this class of tropane-based molecules.
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Benztropine
Dopamine Uptake Inhibitors
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Extensive structure−activity relationships at the dopamine transporter (DAT) have been developed around two classes of tropane-based ligands. Opposing stereoselectivity and divergent structural requirements for optimal DAT binding suggest that these tropane-based DAT inhibitors may not access identical binding domains. To further investigate this hypothesis, a series of (S)-2β-carboalkoxy-3α-(bis[4-fluorophenyl]methoxy)tropanes (11a−f, 13−16) and their identically (R)-2β-substituted 3β-(3,4-dichlorophenyl)tropanes (3, 5a−d) were prepared and evaluated for binding at the DAT and for inhibition of [3H]dopamine uptake in rat brain. These studies showed that most of the identically 2-carboalkoxy-substituted analogues, within the two classes of compounds, bind with high affinity to DAT (Ki = 5.5−100 nM), albeit with opposite stereochemistry. However, the larger azido- (15) and isothiocyanato- (16) (S)-2β-carbophenylethoxy-3α-(bis[4-fluorophenyl]methoxy)tropanes demonstrated a significant decrease in DAT binding potency (IC50 = 210 and 537 nM, respectively), suggesting that the DAT does not tolerate 2-position steric bulk in the benztropine class, as it does with the 2-substituted 3-aryltropanes. In addition, binding affinities at the serotonin transporter, norepinephrine transporter, and muscarinic receptors were evaluated and compared for compounds 2, 3, 11a−e, and 13. Together, the binding profiles across these systems demonstrated significant differences between these two classes of tropane-based ligands, which may be exploited toward the discovery of a cocaine-abuse pharmacotherapeutic.
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Benztropine
Norepinephrine transporter
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In a continuing effort to further characterize the role of the dopamine transporter in the pharmacological effects of cocaine, a series of chiral and achiral N-substituted analogues of 3α-[bis(4'-fluorophenyl)methoxy]tropane (5) has been prepared as potential selective dopamine transporter ligands. These novel compounds displaced [3H]WIN 35,428 binding from the dopamine transporter in rat caudate putamen with Ki values ranging from 13.9 to 477 nM. Previously, it was reported that 5 demonstrated a significantly higher affinity for the dopamine transporter than the parent drug, 3α-(diphenylmethoxy)tropane (3; benztropine). However, 5 remained nonselective over muscarinic m1 receptors (dopamine transporter, Ki = 11.8 nM; m1, Ki = 11.6 nM) which could potentially confound the interpretation of behavioral data, for this compound and other members of this series. Thus, significant effort has been directed toward developing analogues that retain high affinity at the dopamine transporter but have decreased affinity at muscarinic sites. Recently, it was discovered that by replacing the N-methyl group of 5 with the phenyl-n-butyl substituent (6) retention of high binding affinity at the dopamine transporter (Ki = 8.51 nM) while decreasing affinity at muscarinic receptors (Ki = 576 nM) was achieved, resulting in 68-fold selectivity. In the present series, a further improvement in the selectivity for the dopamine transporter was accomplished, with the chiral analogue (S)-N-(2-amino-3-methyl-n-butyl)-3α-[bis(4'-fluorophenyl)methoxy]tropane (10b) showing a 136-fold selectivity for the dopamine transporter versus muscarinic m1 receptors (Ki = 29.5 nM versus Ki = 4020 nM, respectively). In addition, a comparative molecular field analysis (CoMFA) model was derived to correlate the binding affinities of all the N-substituted 3α-[bis(4'-fluorophenyl)methoxy]tropane analogues that we have prepared with their 3D-structural features. The best model (q2 = 0.746) was used to accurately predict binding affinities of compounds in the training set and in a test set. The CoMFA coefficient contour plot for this model, which provides a visual representation of the chemical environment of the binding domain of the dopamine transporter, can now be used to design and/or predict the binding affinities of novel drugs within this class of dopamine uptake inhibitors.
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Dopamine Uptake Inhibitors
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The development of novel Tc-99m-labeled tropane derivatives as dopamine transporter imaging agents is reported. A series of neutral and lipophilic conjugated complexes, containing N-(alkylthiolato)tropane, aminobis(ethylthiolato), and a [99mTc]TcO3+ center core, were prepared and evaluated as central nervous system (CNS) dopamine transporter imaging agents in rats. One of the compounds, [99mTc]technetium, [methyl 3-(4-chlorophenyl)-8-(2-mercaptoethyl)-8-azabicyclo[3.2.1]octane-2-carboxylato-S][[2,2'-(methylimino)bis[ethanethiolato]](2-)-N,S,S']oxo (25), displayed low initial uptake in rat brain (0.1% at 2 min post iv injection); the striatal/cerebellar (ST/CB) ratio reached 3.50 at 60 min after an iv injection. The specific uptake can be blocked by pretreating rats with a competing dopamine transporter binding agent, β-CIT (RTI-55, N-methyl-2β-carbomethoxy-3β-(4-iodophenyl)tropane; iv, 1 mg/kg), which reduced the regional brain uptake ratio (ST/CB) to 1.0. In contrast, the specific uptake in striatum was not affected by pretreating rats with a noncompeting ligand, haldol (iv, 1 mg/kg). In vitro autoradiography of rat brain sections exhibited elevated labeling in striatum, major islands of Calleja, and olfactory tubercle regions, where dopamine neurons are known to be concentrated. This series of compounds is the first example of technetium-99m labeled CNS receptor-specific imaging agents and may provide a convenient source of short-lived imaging agents for routine diagnosis of CNS abnormality in conjunction with single photon emission computed tomography.
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Human and bovine dopamine transporters (DAT) demonstrate discrete functional differences in dopamine (DA), 1-methyl-4-phenylpyridium (MPP(+)) transport, and cocaine analog binding. In a previous study, the functional analyses on the chimeras of human and bovine DAT have revealed that the region from residues 133 through 186 (encompassing the third transmembrane domain) is responsible for the substrate transport and cocaine analog binding. The present study has been carried out to determine the specific amino acid(s) conferring DAT functions by interchanging the amino acid residues in the corresponding region between human and bovine DAT. As described previously, the DA, MPP(+) transport, and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) binding almost disappeared in chimera hb3 in which the region from residues 133 through 186 of bovine DAT was substituted into human DAT. Replacement of isoleucine, residue 152 of chimera hb3 (bovine DAT sequence), with valine, the human DAT residue at the identical position, remarkably restored the substrate transport and CFT binding to 76% to 98% of the human DAT values. Similarly, substitution of isoleucine for valine at position 152 in the human DAT reduced the substrate transport and CFT binding by 57% to 97%. Among other amino acids tested at position 152 of the chimera hb3, only alanine resulted in small but significant increases in the DAT functions ranging from 16 to 34%. Thus, valine at position 152 plays a crucial role for molecular mechanisms underlying the interactions of DA, MPP(+), and CFT with human DAT.
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Isoleucine
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The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a marker for Parkinson's disease (Pd) and attention deficit hyperactivity disorder (ADHD). In ADHD, DAT density levels are elevated, while in Pd these levels are depleted. The depletion of DAT levels also corresponds with the loss of dopamine. We now describe the design, synthesis, biology, and SPECT imaging in nonhuman primates of second-generation 99mtechnetium-based tropane ligands that bind potently and selectively to the DAT. We demonstrate that improved selectivity and biological stability allows sufficient agent to enter the brain and label the DAT in vivo to provide a quantitative measure of DAT density in nonhuman primates. We introduce FLUORATEC (N-[(2-((3'-N'-propyl-(1' 'R)-3' 'α-(4-fluorophenyl)tropane-2' 'β-1-propanoyl)(2-mercaptoethyl)amino)acetyl)-2-aminoethanethiolato]technetium(V) oxide), a DAT imaging agent that has emerged from these studies and is now in phase 1 clinical trials in the U.S.
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Emission computed tomography
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Benztropine and its analogs are tropane ring-containing dopamine uptake inhibitors that produce behavioral effects markedly different from cocaine and other dopamine transporter blockers. We investigated the benztropine binding site on dopamine transporters by covalently attaching a benztropine-based photoaffinity ligand, [125I]N-[n-butyl-4-(4"'-azido-3"'-iodophenyl)]-4', 4"-difluoro-3alpha-(diphenylmethoxy)tropane ([125I]GA II 34), to the protein, followed by proteolytic and immunological peptide mapping. The maps were compared with those obtained for dopamine transporters photoaffinity labeled with a GBR 12935 analog, [125I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethy l]p iperazine ([125I]DEEP), and a cocaine analog, [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), which have been shown previously to interact with different regions of the primary sequence of the protein. [125I]GA II 34 became incorporated in a membrane-bound, 14 kDa fragment predicted to contain transmembrane domains 1 and 2. This is the same region of the protein that binds [125I]DEEP, whereas the binding site for [125I]RTI 82 occurs closer to the C terminal in a domain containing transmembrane helices 4-7. Thus, although benztropine and cocaine both contain tropane rings, their binding sites are distinct, suggesting that dopamine transport inhibition may occur by different mechanisms. These results support previously derived structure-activity relationships suggesting that benztropine and cocaine analogs bind to different domains on the dopamine transporter. These differing molecular interactions may lead to the distinctive behavioral profiles of these compounds in animal models of drug abuse and indicate promise for the development of benztropine-based molecules for cocaine substitution therapies.
Tropane
Photoaffinity labeling
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Citations (74)