Mechanism for Cocaine Blocking the Transport of Dopamine: Insights from Molecular Modeling and Dynamics Simulations (original) (raw)
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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.
The binding sites for cocaine and dopamine in the dopamine transporter overlap
Nature Neuroscience, 2008
Cocaine is a widely abused substance with psychostimulant effects that are attributed to inhibition of the dopamine transporter (DAT). We present molecular models for DAT binding of cocaine and cocaine analogs constructed from the high-resolution structure of the bacterial transporter homolog LeuT. Our models suggest that the binding site for cocaine and cocaine analogs is deeply buried between transmembrane segments 1, 3, 6 and 8, and overlaps with the binding sites for the substrates dopamine and amphetamine, as well as for benztropine-like DAT inhibitors. We validated our models by detailed mutagenesis and by trapping the radiolabeled cocaine analog [ 3 H]CFT in the transporter, either by cross-linking engineered cysteines or with an engineered Zn 21 -binding site that was situated extracellularly to the predicted common binding pocket. Our data demonstrate the molecular basis for the competitive inhibition of dopamine transport by cocaine.
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.
Journal of Biological Chemistry, 2003
Recently we showed evidence that mutation of Tyr-335 to Ala (Y335A) in the human dopamine transporter (hDAT) alters the conformational equilibrium of the transport cycle. Here, by substituting, one at a time, 16 different bulky or charged intracellular residues, we identify three residues, Lys-264, Asp-345, and Asp-436, the mutation of which to alanine produces a phenotype similar to that of Y335A. Like Y335A, the mutants (K264A, D345A, and D436A) were characterized by low uptake capacity that was potentiated by Zn 2؉. Moreover, the mutants displayed lower affinity for cocaine and other inhibitors, suggesting a role for these residues in maintaining the structural integrity of the inhibitor binding crevice. The conformational state of K264A, Y335A, and D345A was investigated by assessing the accessibility to MTSET ([2-(trimethylammonium)ethyl]methanethiosulfonate) of a cysteine engineered into position 159 (I159C) in transmembrane segment 3 of the MTSET-insensitive "E2C" background (C90A/C306A). Unlike its effect at the corresponding position in the homologous norepinephrine transporter (NET I155C), MTSET did not inhibit uptake mediated by E2C I159C. Furthermore, no inhibition was observed upon treatment with MTSET in the presence of dopamine, cocaine, or Zn 2؉. Without Zn 2؉ , E2C I159C/K264A, E2C I159C/ Y335A, and E2C I159C/D345A were also not inactivated by MTSET. In the presence of Zn 2؉ (10 M), however, MTSET (0.5 mM) caused up to ϳ60% inactivation. As in NET I155C, this inactivation was protected by dopamine and enhanced by cocaine. These data are consistent with a Zn 2؉-dependent partial reversal of a constitutively altered conformational equilibrium in the mutant transporters. They also suggest that the conformational equilibrium produced by the mutations resembles that of the NET more than that of the DAT. Moreover, the data provide evidence that the cocaine-bound state of both DAT mutants and of the NET is structurally distinct from the cocaine-bound state of the DAT.
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.
ACS chemical neuroscience, 2017
Cocaine, a widely abused psychostimulant, inhibits the dopamine transporter (DAT) by trapping the protein in an outward-open conformation, whereas atypical DAT inhibitors such as benztropine have low abuse liability and prefer less outward-open conformations. Here, we use a spectrum of computational modeling and simulation approaches to obtain the underlying molecular mechanism in atomistic detail. Interestingly, our quantum mechanical calculations and molecular dynamics (MD) simulations suggest that a benztropine derivative JHW007 prefers a different stereoisomeric conformation of tropane in binding to DAT compared to that of a cocaine derivative, CFT. To further investigate the different inhibition mechanisms of DAT, we carried out MD simulations in combination with Markov state modeling analysis of wild-type and Y156F DAT in the absence of any ligand or the presence of CFT or JHW007. Our results indicate that the Y156F mutation and CFT shift the conformational equilibrium toward ...
Biochemical pharmacology, 2017
Dopamine transporter (DAT) blockers like cocaine and many other abused and therapeutic drugs bind and stabilize an inactive form of the transporter inhibiting reuptake of extracellular dopamine (DA). The resulting increases in DA lead to the ability of these drugs to induce psychomotor alterations and addiction, but paradoxical findings in animal models indicate that not all DAT antagonists induce cocaine-like behavioral outcomes. How this occurs is not known, but one possibility is that uptake inhibitors may bind at multiple locations or in different poses to stabilize distinct conformational transporter states associated with differential neurochemical endpoints. Understanding the molecular mechanisms governing the pharmacological inhibition of DAT is therefore key for understanding the requisite interactions for behavioral modulation and addiction. Previously, we leveraged complementary computational docking, mutagenesis, peptide mapping, and substituted cysteine accessibility st...
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.