![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
NEUROPHARMACOLOGY
Department of Pharmacology and Toxicology, Duquesne University, Pittsburgh, Pennsylvania (O.T.U., C.D.B., S.P., C.K.S.); Medicinal Chemistry Section, Intramural Research Program, National Institute on Drug Abuse, Baltimore, Maryland (A.H.N., S.S.K.); and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois (A.P.K.)
Received March 4, 2005; accepted May 4, 2005.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
). The DAT is therefore a logical target for development of medications for cocaine abuse. Elucidating the relationship between the DAT and its substrates and inhibitors is essential, especially regarding cocaine points of contact with the DAT protein relevant to its inhibition of dopamine uptake.
Because dopamine and all classical DAT inhibitors possess a protonated nitrogen atom, a longstanding model for cocaine inhibition of dopamine uptake at DAT centers on competition between the positive charges of substrate and inhibitor for the negatively charged aspartic acid residue in the first transmembrane domain (TM 1) of the DAT (Carroll et al., 1992; Kitayama et al., 1992). This residue is only one of two negative charges clearly predicted to lie in a transmembrane domain, and the positioning of a fully charged side chain in the hydrophobic lipid bilayer implies its contribution to a hydrophilic enclave found in a ligand or ion binding site. The model is based on the formation of an ion pair between the 
-adrenergic receptor and its agonists (Strader et al., 1988), and although the model seems to generally hold for G protein-coupled receptors that recognize amine-containing agonists and antagonists (Wang et al., 1991; Spalding et al., 1994; Surratt et al., 1994; Li et al., 1999), a parallel among neurotransmitter transporter proteins is unsubstantiated. The premise has been tested at the level of the DAT inhibitor by synthesis of charge-neutral cocaine analogs. Sulfonylation of the tropane nitrogen atom of cocaine to neutralize the positive charge did not compromise the DAT binding affinity or dopamine uptake inhibition potency (DUIP) of the cocaine analog (Kozikowski et al., 1994). Replacement of the same nitrogen atom with an uncharged oxygen atom to yield 8-oxanorcocaine (necessarily removing the N-8 methyl group) resulted in only 4- and 8-fold reductions in DUIP and binding affinity, respectively, relative to norcocaine (Kozikowski et al., 1999). On the other hand, a similar 8-oxa modification of WIN 35,428 sharply reduced binding affinity and DUIP at the DAT. This deficit was fully compensated for by further replacing the p-fluoro substituent of the C-3 phenyl group with a chloride atom (Madras et al., 1996) and further improved with a 3,4-dichloro substitution (Meltzer et al., 1997). Elimination of hydrogen bonding potential at the 8-position of WIN 35,428 by substitution of a carbon atom also substantially compromised affinity at the DAT. This affinity loss was also entirely reversed by additionally replacing the p-fluoro substituent of the C-3 phenyl group with a 3,4-dichloro moiety, or replacing the phenyl group with a 2-naphthyl moiety (Meltzer et al., 2000). The effectiveness of these compounds in dopamine uptake inhibition at DAT was not reported (Meltzer et al., 2000). Similarly modified methylphenidate analogs displayed DAT affinities indistinguishable from methylphenidate itself (Meltzer et al., 2003). In contrast, replacement of the basic tropane nitrogen with a neutral functional group (e.g., N-formyl group or oxygen atom) in the benztropine class of DAT inhibitors substantially reduced DAT binding affinity (Agoston et al., 1997; Meltzer et al., 1997; Simoni et al., 2001).
Alanine, glycine, or glutamate substitution of the TM 1 DAT aspartate, residue D79, was initially reported to substantially decrease DAT affinities for dopamine and WIN 35,428 (Kitayama et al., 1992). In contrast, a recent mutagenesis study by authors of the present work indicated that the glutamate-substituted (D79E) DAT mutation had no effect on dopamine affinity or DUIPs for WIN 35,428 and cocaine, and decreased WIN 35,428 affinity by only 3-fold (Wang et al., 2003). The latter study cast doubt on the necessity of a dopamine-D79 ion pair, but it did not address whether the residue plays a role in recognizing the cocaine pharmacophore. DAT inhibitors containing modifications of three primary components of this pharmacophore—the positively charged tropane nitrogen atom, the seven-carbon tropane ring itself, and the aromatic substituent at the tropane C-3-position—were herein evaluated for binding affinity and dopamine uptake inhibition at the same D79 DAT mutants.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
85 Ci/mmol), [3H]mazindol (24.5 Ci/mmol), and [3H]dopamine (29 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Foster City, CA). Nonradioactive WIN 35,428, cocaine, and methylphenidate were obtained from Research Triangle Institute (Research Triangle Park, NC) via the National Institute on Drug Abuse Division of Basic Research. Nonradioactive benztropine mesylate, dopamine, and ascorbic acid were obtained from Sigma-Aldrich (St. Louis, MO); mazindol and GBR-12,909 were obtained from RBI/Sigma (Natick, MA). All other benztropine analogs and rimcazole were synthesized in the laboratory of Dr. A. H. Newman, and 8-oxa-norcocaine and 4-ARA-127 were synthesized in the laboratory of Dr. A. P. Kozikowski. Scintillation counting materials were from Fisher Scientific Co. (Pittsburgh, PA).
Mutagenesis and Cell Transfections. All site-directed mutagenesis was conducted either as described previously (Surratt et al., 1994) or via the QuikChange (Stratagene, La Jolla, CA) method, following the kit protocol. Complementary DNA fragments containing the desired mutation were subcloned into a pIRESneo plasmid containing the WT DAT open reading frame minus the subcloned fragment, and the presence of an intact coding region lacking extraneous mutations was confirmed by DNA sequencing (University of Pittsburgh core facility) of the shuttled region and its splice junctions. When the wild-type DAT cDNA was originally subcloned into a plasmid, an HA epitope tag sequence was included immediately after the N-terminal methionine residue. Antibodies were not directed against the HA tag in the present study. This construct was pharmacologically indistinguishable from wild-type DAT (data not shown) and is referred to as "wild-type (WT) DAT" hereafter. A WT or mutant DAT plasmid was used to transfect COS-7 or CHO-K1 cells. COS-7 cells were cultured in "DMEM" (DMEM containing 20 mM L-glutamine plus 10% fetal bovine serum, 100 units/ml penicillin, and 100 mg/ml streptomycin) at 37°C in 5% CO2 in 750-cm2 flasks. For CHO-K1 cells, DMEM was replaced with Ham's/F-12 medium. Stably transfected WT and D79E DAT CHO cell lines were prepared and maintained as described previously (Wang et al., 2003).
Transient transfection of COS cells either used a modification of the calcium phosphate method or was achieved with the Polyfect (QIAGEN, Valencia, CA) system. In the former, a 1.4-ml solution of 1 mM Tris-HCl buffer, pH 8.0, containing 1 mM EDTA, 250 mM CaCl2, and 20 µg of plasmid DNA was added dropwise to a vortexed 1.4-ml solution of 50 mM HEPES buffer, pH 7.12, containing 280 mM NaCl and 1.5 mM Na2HPO4. After incubating at room temperature for 1 min without vortexing, the Ca3(PO4)2-DNA fine precipitant was suspended by rapid pipetting, and 200 ml of the suspension was quickly added to each 35-mm well of a six-well polylysine-coated culture plate. Cell monolayers in each well were 30 to 40% confluent and were supplied with fresh DMEM 4 h before transfection; the DMEM was again replaced 12 to 18 h after transfection to remove Ca2+ and unincorporated DNA. Transfected cell monolayers were used in pharmacological studies 72 h after initiation of transfection.
Immunocytochemistry and Confocal Microscopy. COS-7 cells were seeded on coverslips placed in six-well plates and grown to 40 to 60% confluence. Cells were transiently transfected on the following day with WT or D79 mutant DAT plasmids, or the vector control plasmid, using PolyFect reagent (QIAGEN). After 48 h, cells were fixed in 4% paraformaldehyde solution in PBS at room temperature for 15 min, rinsed once with PBS, and incubated with blocking-permeabilizing solution (5% goat serum, 1% bovine serum albumin, and 0.1% Triton X-100 in PBS buffer solution) for 45 min at room temperature. Cells were next incubated with rat monoclonal anti-DAT antibody (MAB369; Chemicon International, Temecula, CA) at 1:1000 dilution for 1 h. The anti-DAT antibody solution was aspirated and cells were washed five times with PBS containing 0.1% Triton X-100 and incubated with a mixture of secondary antibody (goat anti-rat Alexa Fluor 488; Molecular Probes, Eugene, OR) at 1:500 dilution and rhodamine phalloidin (Molecular Probes) at 1:250 dilution for 1 h. After three washes in PBS containing 0.1% Triton X-100 followed by two washes in PBS, coverslips were mounted on slides using GVA mounting solution (Zymed Laboratories, South San Francisco, CA) and left to dry overnight in the dark at 4°C. DAT protein was visualized using the Leica TCS-SP2 confocal laser microscope with a glycerin immersion 63x objective. Alexa 488 was excited at 488 nm with an argon/krypton laser, and emission photons from 500 to 600 nm were accumulated by the photomultiplier tube. Rhodamine phalloidin was excited at 543 nm with a helium/neon laser, and emission photons from 550 to 650 nm were accumulated. The fluorophores were detected separately and overlay images were generated automatically by the imaging software.
[3H]Dopamine Uptake. Assays were conducted with cell monolayers in six-well plates. The monolayer was washed 2 x 2 ml with KRH buffer (25 mM HEPES, pH 7.3, 125 mM NaCl, 4.8 mM KCl, 1.3 mM CaCl2, 1.2 mM Mg2SO4, 1.2 mM KH2PO4, and 5.6 mM glucose), and uptake was initiated by addition of 1 ml of 10 nM [3H]dopamine and 50 mM ascorbic acid in KRH to duplicate or triplicate cell monolayers. A time course was conducted for each DAT construct to determine when uptake velocity was in the linear phase, typically occurring at 5 min at 22°C. Uptake was quenched by washing the monolayer with 2 x 2 ml of KRH + ascorbic acid. Cell monolayers were solubilized in 0.5 ml of 1% SDS and transferred to scintillation vials for determination of incorporated tritium. Nonspecific uptake was assessed by inclusion of 30 µM cocaine or 10 µM mazindol, as appropriate. Uptake inhibition experiments included nonradioactive DAT uptake blockers at the following concentration ranges: cocaine, 10 nM to 100 µM; WIN 35,428, 3 nM to 10 µM; 8-oxa-norcocaine, 10 nM to 1 mM; 4-ARA-127, 10 nM to 100 µM; mazindol, 3 nM to 10 µM; methylphenidate, 3 nM to 10 µM; benztropine, 0.1 nM to 30 µM; 3-4'-chlorobenztropine, 1 nM to 30 µM; 4'-chlorobenztropine, 0.1 nM to 30 µM; 4',4''-difluorobenztropine, 0.1 nM to 10 µM; GBR-12,909, 0.1 nM to 10 µM; rimcazole, 1 nM to 30 µM; N-formyl-4,4'-difluorobenztropine, 10 nM to 1 mM; and 4-trifluoromethylbenztropine, 1 nM to 30 µM. All inhibitors were preincubated 10 min with the cell monolayer before adding [3H]dopamine. Ki values for uptake inhibition were determined with GraphPad Prism (GraphPad Software Inc., San Diego, CA) 3.0 software.
Ligand Binding Assays. [3H]WIN 35,428 was the radioligand used for all experiments. Binding assays were conducted exactly as described above for the dopamine uptake assay except that [3H]dopamine was replaced with 1 nM [3H]WIN 35,428, and radioligand and nonradioactive competitor were incubated with cells for 15 min (the same incubation period allowed for an uptake blocker in the uptake assay). Nonradioactive competitor concentrations were as indicated above for uptake inhibition. Nonspecific binding was assessed by addition of 10 µM mazindol except when mazindol was the drug tested, in which case, 30 µM cocaine was substituted. For all binding assays, data were analyzed with GraphPad Prism 3.0 software to obtain Kd, Ki, and Bmax values.
Molecular Modeling. Molecular modeling studies used SYBYL 7.0 (Tripos, St. Louis, MO) package software using a Silicon Graphics workstation. Standard Tripos forcefield and Gasteiger Marsili charges were used for all energy calculations. The X-ray crystal structure of 4,4'-difluorobenztropine was used to generate scenarios for all other compounds by superimposition of the nonhydrogen atoms of the tropane ring (Kulkarni et al., 2004). The electrostatic potentials were mapped for Connolly surfaces using the MOLCAD module of the SYBYL software, and the MVOLUME routine was used to compute essential regions of the "receptor".
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). The present study adds a DAT mutant with a leucine substitution at this position (D79L), directly addresses binding of a radiolabeled inhibitor at WT and D79 DAT mutants, and pharmacologically characterizes functionally active mutants with a broader spectrum of DAT blockers. Relative to WT DAT, only the D79E DAT mutant displayed detectable specific binding of [3H]WIN 35,428 (Fig. 1). All DAT protein constructs were observed to colocalize with a marker at the cell plasma membrane (Fig. 2). Previous biotinylation/Western blotting experiments confirmed that WT and D79 mutant DAT proteins were expressed and glycosylated to a similar extent and that only D79N DAT sustained a notable decrease in cell surface expression (Wang et al., 2003). With the possible exception of D79N DAT, the radioligand binding deficits for the DAT mutants were not due to cell surface targeting defects. The D79A, D79L, and D79N DAT mutants could not be further characterized due to the inability to detect either radioligand specific binding (Fig. 1) or [3H]dopamine uptake (data not shown; Wang et al., 2003). The D79L mutation provides a leucine side chain substitution that approximates the length and steric bulk of aspartate but removes hydrogen bonding potential and the negative charge. The D79N mutation additionally provides hydrogen bonding potential similar to aspartate. Thus, it seems that a negative charge at position 79 of DAT is necessary for recognition of the cocaine analog [3H]WIN 35,428.
|
|
). Additional structurally diverse DAT inhibitors (Fig. 3) were similarly tested at WT and D79E DAT to better assess key functional groups for DAT recognition. Ki values for binding affinity and DUIP were derived from displacement of [3H]WIN 35,428 binding and inhibition of cellular incorporation of [3H]dopamine, respectively; both assays were conducted under identical conditions.
|
The DUIPs of additional DAT inhibitors including 8-oxanorcocaine, the piperidine-based WIN 35,428 analog 4-ARA-127, rimcazole, and GBR-12909 were unaffected by the D79E DAT mutation (Fig. 4; Table 1). The result mirrored those for the previously studied inhibitors WIN 35,428, mazindol, and methylphenidate (cocaine sustained only a 2-fold potency decrease) (Wang et al., 2003). The exception to this trend was benztropine, the DUIP of which increased 8-fold with the D79E substitution (Table 1). The pattern of binding affinities 3- to 4-fold higher than the DUIPs at WT DAT displayed by cocaine, WIN 35,428, mazindol, and methylphenidate also held for 8-oxa-norcocaine and rimcazole. Benztropine, in contrast, displayed general agreement between the WT DAT binding and uptake Ki values, and binding affinity was 2-fold lower than DUIP for 4-ARA-127 and GBR-12,909. Binding affinities and DUIPs matched at D79E DAT for cocaine, WIN 35,428, mazindol, methylphenidate, and 4-ARA-127. Binding affinity was 2- to 4-fold higher than DUIP for 8-oxa-norcocaine and rimcazole at D79E DAT, and 3-fold lower than DUIP for GBR-12,909 and benztropine at this mutant transporter.
|
|
Because only the DUIP of benztropine was affected by the D79E DAT mutation, the pharmacology of structural analogs of this drug was further investigated at WT and D79E DAT. Interestingly, widely divergent DUIPs ranging from 15 to 964 nM at the wild-type transporter were unified to approximately 20 nM by the conservative D79E substitution (Fig. 5; Table 2). Within this series, only 4',4''-difluoroBZT and GBR-12,909 were unaffected by the mutation with respect to DUIP or binding affinity. These compounds share the 4',4''-difluorophenylmethoxy moiety previously demonstrated to provide optimal DAT binding affinity (van der Zee et al., 1980; Newman et al., 1995). Replacement of the N-methyl group of 4',4''-difluoroBZT with the charge-neutral N-formyl group, however, dramatically reduced DUIPs at both WT and D79E DAT, and this reduction was 13-fold more severe at the WT DAT. The benztropine analog 4'-trifluoromethylBZT, possessing a larger and electronically rich aryl substituent, also diverged from the "20 nM DUIP" D79E DAT pattern. The 3-4-chlorobenztropine compound, unique among the benztropines tested for its opposite stereochemistry at the 3-position of the tropane ring of the diphenylmethoxy moiety, yielded a DUIP of approximately 20 nM at D79E DAT, which required a 37-fold DUIP increase over that of the WT DAT.
|
|
In contrast to the DUIP values, apparent binding affinities of the benztropine analogs were typically not dramatically altered by the D79E mutation. Only 3-4-chloroBZT and N-formyl-difluoroBZT registered 3- and 5-fold increases, respectively (Fig. 5; Table 2). Rimcazole, a distant benztropine relative, exhibited a unique pharmacological profile in that the D79E DAT mutation impacted neither DUIP nor binding affinity, yet DUIP was 3- to 4-fold lower than binding affinity at each construct (Table 1). As evidenced by benztropine and its analogs, a correlation was therefore detected between modifications of the tropane ring C-3-position of cocaine and modifications of the DAT D79 side chain with respect to the potency of DAT blocking drugs in eliminating DAT-mediated translocation of [3H]dopamine into the cell.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Alanine, leucine, asparagine, or glutamate substitutions at this position were generated to assess the importance of side chain length, hydrogen bonding potential, and negative charge for recognition of DAT inhibitors. Unfortunately, only the D79E DAT mutant displayed detectable specific binding of [3H]WIN 35,428 (Fig. 1), and at a level such that creation of a stably transfected D79E DAT CHO cell line was achieved only by assaying the clonal cells for [3H]dopamine uptake (Wang et al., 2003). This stably transfected D79E DAT cell line was used with a range of DAT blockers either structurally dissimilar to cocaine or containing variations of the three primary components of the cocaine pharmacophore: the positively charged tropane nitrogen atom, the tropane ring, and the C-3 aromatic substituent.
To eliminate the positive charge of cocaine, N-8 of the tropane ring was replaced with an oxygen atom, a modification previously shown to spare binding and DUIP at WT DAT (Kozikowski et al., 1999). The DUIP of 8-oxa-norcocaine was unaffected by the D79E mutation, and binding affinity decreased by less than 4-fold (Table 1). By extending the side chain by one carbon, some steric hindrance should be introduced to the ligand binding cavity if indeed this residue is so situated. That the DUIP of this charge-neutral cocaine analog is not altered by an allegedly more crowded binding pocket is inconsistent with (but not exclusive of) the postulate that a salt bridge between the D79 residue and the N-8 atom is a critical DAT-cocaine interaction. The 8-oxa-norcocaine compound mirrored cocaine in that Ki values suggest its affinity to be severalfold greater than its DUIP at WT DAT and in that its affinity and DUIP values were less than 2-fold apart at D79E DAT.
To address the criticality of the cocaine tropane ring with respect to the D79 side chain, a piperidine ring was substituted. Kozikowski and colleagues have demonstrated that piperidine-based cocaine analogs are effective in blocking binding of DAT ligands or dopamine uptake at the DAT (Kozikowski et al., 1998), indicating that the tropane ring of cocaine is not absolutely required for cocaine action at the transporter. The compound used in the present work, 4-ARA-127, is a WIN 35,428 analog that lacks the 6,7-bridgehead of the tropane ring and contains a p-chlorophenyl instead of a p-fluorophenyl substituent on what would be C-3 of the tropane ring (Fig. 3). The D79E mutation had little or no effect on the pharmacology of this piperidine-based cocaine analog (Table 1). GBR-12,909 and rimcazole also contain a structurally more flexible piperazine ring in place of the tropane ring, and again, the mutation did not affect the DAT pharmacology of these drugs (Table 1). These findings suggest that the role of the D79 residue may not include recognition of the tropane ring (or piperidine/piperazine ring replacement) of these DAT inhibitors.
The aromatic group at the tropane C-3 position was anticipated to be most sensitive to alterations of the DAT D79 side chain. Recent work from this laboratory (Wang et al., 2003) indicates that the D79 residue may be more likely to contribute to binding of the aromatic moiety of dopamine, contrary to the postulate that the aspartate side chain found at this position in the monoamine transporters forms an ionic bond with the protonated amine of the substrate (Kitayama et al., 1992; Barker et al., 1999). In this scenario, ionic/hydrogen bonds formed between D79 and one or more TM domain residues or pore loops extending into the lipid bilayer would serve as "struts" supporting an aromatic binding site. An "aromatic pocket" role for the DAT D79 residue is consistent with the fact that the aspartate is conserved among the Na+/Cl–-dependent neurotransmitter transporters of substrates bearing an aromatic moiety. A glycine residue replaces the aspartate for members of the same transporter family that recognize nonaromatic substrates that nevertheless retain the protonated amino group (Wang et al., 2003). For an inhibitor that directly blocks dopamine binding at a DAT site in which the D79 residue is crucial, an aromatic ring would be the more logical primary pharmacophore for this role. An aromatic moiety is necessary at the tropane C-3 position of cocaine for DAT inhibition, and even for inhibitors lacking the tropane structure an aromatic ring is required (Carroll et al., 1992; Newman and Kulkarni, 2002). It was therefore of interest to explore in depth the contribution of the tropane C-3 substituent to DAT inhibition by testing benztropine, possessing one of the more profound structural variations of this portion of the pharmacophore, with the D79E mutant. Indeed, of nine structurally diverse DAT blockers initially studied, only the DUIP of benztropine was notably altered by the D79E mutation (Table 1).
That benztropine and several of its analogs yielded the same DUIP at D79E DAT despite often very different DUIPs at WT DAT (Table 2) may suggest that the mutation influences recognition of the common diphenylmethoxy pharmacophore. Unlike WT DAT, the D79E DAT protein was generally tolerant of diphenylmethoxy ring substituents, especially surprising considering the marked reorientation of the diphenylmethoxy moiety in the 3-4'-chlorobenztropine derivative. Via one of several possible mechanisms (discussed below), the D79E substitution seems to have enhanced DAT interaction with the diphenylmethoxy moiety and at the same time provided a "roomier" binding pocket for this functional group that accommodates different halogen substituents and ring orientations. Both of the phenyl rings of benztropine may simultaneously contribute to inhibition of D79E DAT, but likely not when in the same plane. The structurally constrained 3-benzsuberone analog of benztropine displayed substantially lower affinity than the parent compound (Kline et al., 1997). Similarly, the phenyl rings of rimcazole are fused and thus constrained in space by a bond that creates an intervening pyrrole ring. Comparing results (Table 1) for rimcazole to those for GBR-12,909, its closest structural analog in this study, suggests that the phenyl rings must be able to rotate freely for potent dopamine uptake inhibition at the DAT. Comparative molecular field analysis modeling of benztropines also argues that the relative orientation of the phenyl rings is important for DAT affinity (Kline et al., 1997; Kulkarni et al., 2004).
The pharmacological distinctions observed here between the benztropine analogs and other DAT inhibitors are consistent with reports that benztropine uses a DAT binding site distinguishable from the other blockers. Newman and colleagues have identified dissimilarities between benztropine and cocaine actions at the DAT via distinctive structure-activity relationship profiles (Newman and Kulkarni, 2002). In addition, benztropine altered the accessibility of alkylation agents to wild-type DAT cysteine residues in a pattern distinct from the pattern generated by cocaine, WIN 35,428, and mazindol (Reith et al., 2001). The latter study demonstrated a primary alkylation pattern difference at C90, a DAT extracellular loop cysteine residue that immediately follows TM 1. Moreover, benztropine and GBR-12,909 affinities, but not those of cocaine or WIN 35,428, were Na+-dependent at W84L DAT, a mutation of a TM 1 residue only five positions away from D79 (Chen et al., 2004). Finally, subsequent proteolysis of wild-type DAT protein crosslinked to photoaffinity-radiolabeled analogs of benztropine and a GBR series compound yielded a radioactive polypeptide fragment containing TM domains 1 and 2. In contrast, a photoaffinity-radiolabeled WIN series compound lacking the diphenylmethoxy moiety yielded a DAT proteolysis fragment that included TM domains 4 to 7 (Vaughan et al., 1999).
From these structure-activity studies and the data herein, the influence of particular functional groups within the C-3 position of the pharmacophore seems to be weighted toward the aromatic ring system. In the benztropine class, the 4',4''-difluorophenyl ether provides optimal binding affinity at WT DAT. Molecular modeling of benztropine and its most divergent analogs with respect to WT versus D79E DAT pharmacology suggests that C-3 aromatic substituents that reduce DUIP at DAT fall outside of the optimal binding pocket. The same is true for the neutral tropane N-formyl group of the N-formyl-difluoroBZT compound (Fig. 6). Considering the significant-to-profound DUIP increases for these compounds with the D79E mutation, it may be that the benztropine binding pocket is modified by the mutation to increase tolerance for these C-3 or tropane nitrogen modifications. Regarding the latter, it is possible that the cocaine and benztropine recognition sites of DAT differ in that D79 contributes to recognition of the C-3 aryl ring of cocaine, but the tropane nitrogen of benztropine.
|
). The 8-oxanorcocaine results are also in agreement with this hypothesis (Table 1). For benztropine and structurally analogous DAT inhibitors, however, binding affinities and DUIPs at WT DAT were lower than, or at best, equal to, those at D79E DAT (Table 2). Moreover, binding affinities and DUIPs for these compounds at D79E DAT were nonidentical. For these drugs, the DAT site/conformation/population responsible for higher affinity inhibitor binding is likely to be different from that for cocaine and may be affected differently by the D79E substitution. Conceivably, benztropine and its analogs displace [3H]WIN 35,428 at a DAT population unrelated to that at which they primarily inhibit uptake of [3H]dopamine, explaining why the binding affinity and DUIP values are not identical within the same DAT construct.
The idea of separate DAT populations, as opposed to simply multiple binding sites or conformations, is consistent with the observations that the DAT and other monoamine transporter proteins form functional homo-oligomeric complexes (Kilic and Rudnick, 2000; Hastrup et al., 2003) as well as complexes with membrane and cytoskeletal proteins (Torres et al., 2001; Lee et al., 2004). At least one of these proteins, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor SNARE protein syntaxin 1A, has been shown to inhibit substrate uptake via direct interaction with GABA, glycine, serotonin, and norepinephrine transporters, all members of the Na+/Cl–-dependent transporter family that includes the DAT (Geerlings et al., 2000; Sung et al., 2003; Hansra et al., 2004). It is possible that the D79E mutation yields a DAT conformation that disallows or promotes complexes with endogenous factors (Ramsey and DeFelice, 2002). Such TM 1 mutations have been observed to alter Na+ or Cl– binding, in turn altering inhibitor affinities (Mager et al., 1996; Barker et al., 1999; Chen et al., 2004); thus, the D79E mutation could manipulate availability of DAT sites, conformations, or populations in this way. The "DAT populations" explanation for the present findings is also preferable to one merely involving multiple ligand binding sites or conformations of the same DAT molecule because the DUIP for cocaine at CHO cells stably transfected with WT DAT fluctuated with cell state, yet cocaine binding affinity was static (O. T. Ukairo., C. D. Bondi, and C. K. Surratt, manuscript in preparation).
Clinically useful therapeutics that would combat the actions of cocaine, amphetamines, and other psychostimulants at the level of the dopamine transporter are currently unavailable and desperately needed. The development of drugs, including cocaine and benztropine analogs, that inhibit the DAT and yet do not display behavioral profiles similar to cocaine in animal models (Newman et al., 1994; Newman and Kulkarni, 2002; Woolverton et al., 2002; Kozikowski et al., 2003; Desai et al., 2005) suggest that such a medication is attainable. By combining medicinal chemistry efforts with DAT structure-function molecular pharmacology, computer-aided rational design of agents that counter the actions of abused psychostimulants should be feasible.
|
Acknowledgements |
|---|
|
Footnotes |
|---|
ABBREVIATIONS: DAT, dopamine transporter; TM, transmembrane domain; DUIP, dopamine uptake inhibition potency; WIN 35,428, (–)-3-(4-fluorophenyl)tropan-2-carboxylic acid methyl ester tartrate; GBR 12,909, 1-{2-[di(4-fluorophenyl)-methoxy]-ethyl}-4-(3-phenylpropyl)piperazine; 4-ARA-127, methyl-4-(4'-chlorophenyl)-1-methylpiperidine-3
-carboxylic acid; WT, wild type, CHO, Chinese hamster ovary; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; KRH, Krebs-Ringer-HEPES.
Address correspondence to: Dr. Christopher K. Surratt, Division of Pharmaceutical Sciences, Duquesne University, Mellon Hall, Room 453, 600 Forbes Ave., Pittsburgh, PA 15282. E-mail: surratt{at}duq.edu
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Agoston GE, Wu JH, Izenwasser S, George C, Katz J, Kline RH, and Newman AH (1997) Novel N-substituted 3 alpha-[bis(4'-fluorophenyl)methoxy]tropane analogues: selective ligands for the dopamine transporter. J Med Chem 40: 4329–4339.[CrossRef][Medline]
Barker EL, Moore KR, Rakhshan F, and Blakely RD (1999) Transmembrane domain I contributes to the permeation pathway for serotonin and ions in the serotonin transporter. J Neurosci 19: 4705–4717.
Carroll FI, Lewin AH, Boja JW, and Kuhar MJ (1992) Cocaine receptor: biochemical characterization and structure-activity relationships of cocaine analogues at the dopamine transporter. J Med Chem 35: 969–981.[CrossRef][Medline]
Chen N, Zhen J, and Reith ME (2004) Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine. J Neurochem 89: 853–864.[CrossRef][Medline]
Desai RI, Kopajtic TA, Koffarnus M, Newman AH, and Katz JL (2005) Identification of a dopamine transporter ligand that blocks the stimulant effects of cocaine. J Neurosci 25: 1889–1893.
Geerlings A, Lopez-Corcuera B, and Aragon C (2000) Characterization of the interactions between the glycine transporters GLYT1 and GLYT2 and the SNARE protein syntaxin 1A. FEBS Lett 470: 51–54.[CrossRef][Medline]
Hansra N, Arya S, and Quick MW (2004) Intracellular domains of a rat brain GABA transporter that govern transport. J Neurosci 24: 4082–4087.
Hastrup H, Sen N, and Javitch JA (2003) The human dopamine transporter forms a tetramer in the plasma membrane: cross-linking of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs. J Biol Chem 278: 45045–45048.
Kilic F and Rudnick G (2000) Oligomerization of serotonin transporter and its functional consequences. Proc Natl Acad Sci USA 97: 3106–3111.
Kitayama S, Shimada S, Xu H, Markham L, Donovan DM, and Uhl GR (1992) Dopamine transporter site-directed mutations differentially alter substrate transport and cocaine binding. Proc Natl Acad Sci USA 89: 7782–7785.
Kline RH, Izenwasser S, Katz JL, Joseph DB, Bowen WD, and Newman AH (1997) 3'-Chloro-3 alpha-(diphenylmethoxy)tropane but not 4'-chloro-3 alpha-(diphenylmethoxy)tropane produces a cocaine-like behavioral profile. J Med Chem 40: 851–857.[CrossRef][Medline]
Kozikowski AP, Araldi GL, Boja J, Meil WM, Johnson KM, Flippen-Anderson JL, George C, and Saiah E (1998) Chemistry and pharmacology of the piperidine-based analogues of cocaine. Identification of potent DAT inhibitors lacking the tropane skeleton. J Med Chem 41: 1962–1969.[CrossRef][Medline]
Kozikowski AP, Eddine-Saiah MK, Bergmann JS, and Johnson KM (1994) Structure-activity relationship studies of N-sulfonyl analogs of cocaine: role of ionic interactions in cocaine binding. J Med Chem 37: 3440–3442.[CrossRef][Medline]
Kozikowski AP, Johnson KM, Deschaux O, Bandyopadhyay BC, Araldi GL, Carmona G, Munzar P, Smith MP, Balster RL, Beardsley PM, et al. (2003) Mixed cocaine agonist/antagonist properties of (+)-methyl 4-(4-chlorophenyl)-1-methylpiperidine-3-carboxylate, a piperidine-based analog of cocaine. J Pharmacol Exp Ther 305: 143–150.
Kozikowski AP, Simoni D, Roberti M, Rondanin R, Wang S, Du P, and Johnson KM (1999) Synthesis of 8-oxa analogues of norcocaine endowed with interesting cocaine-like activity. Bioorg Med Chem Lett 9: 1831–1836.[CrossRef][Medline]
Kulkarni SS, Grundt P, Kopajtic T, Katz JL, and Newman AH (2004) Structure-activity relationships at monoamine transporters for a series of N-substituted 3alpha-(bis[4-fluorophenyl]methoxy)tropanes: comparative molecular field analysis, synthesis and pharmacological evaluation. J Med Chem 47: 3388–3398.[CrossRef][Medline]
Lee KH, Kim MY, Kim DH, and Lee YS (2004) Syntaxin 1A and receptor for activated C kinase interact with the N-terminal region of human dopamine transporter. Neurochem Res 29: 1405–1409.[CrossRef][Medline]
Li JG, Chen C, Yin J, Rice K, Zhang Y, Matecka D, de Riel JK, DesJarlais RL, and Liu-Chen L-Y (1999) Asp147 in the third transmembrane helix of the rat mu opioid receptor forms ion-pairing with morphine and naltrexone. Life Sci 65: 175–185.[CrossRef][Medline]
Madras BK, Pristupa ZB, Niznik HB, Liang AY, Blundell P, Gonzalez MD, and Meltzer PC (1996) Nitrogen-based drugs are not essential for blockade of monoamine transporters. Synapse 24: 340–348.[CrossRef][Medline]
Mager S, Kleinberger-Doron N, Keshet GI, Davidson N, Kanner BI, and Lester HA (1996) Ion binding and permeation at the GABA transporter GAT1. J Neurosci 16: 5405–5414.
Meltzer PC, Blundell P, Yong YF, Chen Z, George C, Gonzalez MD, and Madras BK (2000) 2-Carbomethoxy-3-aryl-8-bicyclo[3.2.1]octanes: potent non-nitrogen inhibitors of monoamine transporters. J Med Chem 43: 2982–2991.[CrossRef][Medline]
Meltzer PC, Liang AY, Blundell P, Gonzalez MD, Chen Z, George C, and Madras BK (1997) 2-Carbomethoxy-3-aryl-8-oxabicyclo[3.2.1]octanes: potent non-nitrogen inhibitors of monoamine transporters. J Med Chem 40: 2661–2673.[CrossRef][Medline]
Meltzer PC, Wang P, Blundell P, and Madras BK (2003) Synthesis and evaluation of dopamine and serotonin transporter inhibition by oxacyclic and carbacyclic analogues of methylphenidate. J Med Chem 46: 1538–1545.[CrossRef][Medline]
Newman AH, Allen AC, Izenwasser S, and Katz JL (1994) Novel 3-(diphenylmethoxy)tropane analogs: potent dopamine uptake inhibitors without cocaine-like behavioral profiles. J Med Chem 37: 2258–2261.[CrossRef][Medline]
Newman AH, Kline RH, Allen AC, Izenwasser S, George C, and Katz JL (1995) Novel 4'-substituted and 4',4''-disubstituted 3 alpha-(diphenylmethoxy)tropane analogs as potent and selective dopamine uptake inhibitors. J Med Chem 38: 3933–3940.[CrossRef][Medline]
Newman AH and Kulkarni S (2002) Probes for the dopamine transporter: new leads toward a cocaine-abuse therapeutic–A focus on analogues of benztropine and rimcazole. Med Res Rev 22: 429–464.[CrossRef][Medline]
Ramsey IS and DeFelice LJ (2002) Serotonin transporter function and pharmacology are sensitive to expression level: evidence for an endogenous regulatory factor. J Biol Chem 277: 14475–14482.
Reith ME, Berfield JL, Wang LC, Ferrer JV, and Javitch JA (2001) The uptake inhibitors cocaine and benztropine differentially alter the conformation of the human dopamine transporter. J Biol Chem 276: 29012–29018.
Ritz MC, Lamb RJ, Goldberg SR, and MJK (1987) Cocaine receptors on dopamine transporters are related to self-administration of cocaine. Science (Wash DC) 237: 1219–1223.
Simoni D, Roberti M, Rondanin R, Baruchello R, Rossi M, Invidiata FP, Merighi S, Varani K, Gessi S, Borea PA, et al. (2001) Effects of two-carbon bridge region methoxylation of benztropine: discovery of novel chiral ligands for the dopamine transporter. Bioorg Med Chem Lett 11: 823–827.[CrossRef][Medline]
Spalding TA, Birdsall NJ, Curtis CA, and Hulme EC (1994) Acetylcholine mustard labels the binding site aspartate in muscarinic acetylcholine receptors. J Biol Chem 269: 4092–4097.
Strader CD, Sigal IS, Candelore MR, Rands E, Hill WS, and Dixon RAF (1988) Conserved aspartic acid residues 79 and 113 of the -adrenergic receptor have different roles in receptor function. J Biol Chem 263: 10267–10271.
Sung U, Apparsundaram S, Galli A, Kahlig KM, Savchenko V, Schroeter S, Quick MW, and Blakely RD (2003) A regulated interaction of syntaxin 1A with the antidepressant-sensitive norepinephrine transporter establishes catecholamine clearance capacity. J Neurosci 23: 1697–1709.
Surratt CK, Johnson PS, Moriwaki A, Seidleck BK, Blaschak CJ, Wang JB, and Uhl GR (1994) µ opiate receptor: charged transmembrane domain amino acids are critical for agonist recognition and intrinsic activity. J Biol Chem 269: 20548–20553.
Torres GE, Yao WD, Mohn AR, Quan H, Kim KM, Levey AI, Staudinger J, and Caron MG (2001) Functional interaction between monoamine plasma membrane transporters and the synaptic PDZ domain-containing protein PICK1. Neuron 30: 121–134.[CrossRef][Medline]
van der Zee P, Koger HS, Gootjes J, and Hespe W (1980) Aryl 1,4-dialk(en)ylpiperazines as selective and very potent inhibitors of dopamine uptake. Eur J Med Chem 15: 363–370.
Vaughan RA, Agoston GE, Lever JR, and Newman AH (1999) Differential binding of tropane-based photoaffinity ligands on the dopamine transporter. J Neurosci 19: 630–636.
Wang CD, Buck MA, and Fraser CM (1991) Site-directed mutagenesis of 2A-adrenergic receptors: identification of amino acids involved in ligand binding and receptor activation by agonists. Mol Pharmacol 40: 168–179.[Abstract]
Wang W, Sonders MS, Ukairo OT, Scott H, Kloetzel MK, and Surratt CK (2003) Dissociation of high-affinity cocaine analog binding and dopamine uptake inhibition at the dopamine transporter. Mol Pharmacol 64: 430–439.
Woolverton WL, Ranaldi R, Wang Z, Ordway GA, Paul IA, Petukhov P, and Kozikowski A (2002) Reinforcing strength of a novel dopamine transporter ligand: pharmacodynamic and pharmacokinetic mechanisms. J Pharmacol Exp Ther 303: 211–217.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
