Computational and biochemical docking of the irreversible cocaine analog RTI 82 directly demonstrates ligand positioning in the dopamine transporter central substrate-binding site (original) (raw)
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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...
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.
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 Journal of Physical Chemistry B, 2009
Molecular modeling and dynamics simulations have been performed to study how cocaine inhibits dopamine transporter (DAT) for the transport of dopamine. The computationally determined DATligand binding mode is totally different from previously proposed overlap binding mode in which cocaine-and dopamine-binding sites are the same (Beuming, T. et al. Nature Neurosci. 2008, 11, 780-789). The new cocaine-binding site does not overlap with, but close to, the dopamine-binding site. Analysis of all results reveals that when cocaine binds to DAT, the initial binding site is likely the one modeled in this study, as this binding site can naturally accommodate cocaine. Then, cocaine may move to the dopamine-binding site after DAT makes some necessary conformational change and expands the binding site cavity. It has been demonstrated that cocaine may inhibit the transport of dopamine through both blocking the initial DAT-dopamine binding and reducing the kinetic turnover of the transporter following the DAT-dopamine binding. The relative contributions to the phenomenological inhibition of the transport of dopamine from blocking the initial binding and reducing the kinetic turnover can be different in different types of assays. The obtained general structural and mechanistic insights are consistent with available experimental data and could be valuable for guiding future studies towards understanding cocaine inhibiting other transporters.
Differential binding of tropane-based photoaffinity ligands on the dopamine transporter
The Journal of …, 1999
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, [ 125 I]N-[n-butyl-4-(4ٞ-azido-3ٞ-iodophenyl)]-4Ј,4Љ-difluoro-3␣-(diphenylmethoxy)tropane ([ 125 I]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, [ 125 I]1-[2-(diphenylmethoxy)ethyl]-4-[2-(4-azido-3-iodophenyl)ethyl]piperazine ([ 125 I]DEEP), and a cocaine analog, [ 125 I]3-( p-chlorophenyl)tropane-2-carboxylic acid, 4Јazido-3Ј-iodophenylethyl ester ([ 125 I]RTI 82), which have been shown previously to interact with different regions of the primary sequence of the protein. [ 125 I]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 [ 125 I]DEEP, whereas the binding site for [ 125 I]RTI 82 occurs closer to the C terminal in a domain containing transmembrane helices 4-7.
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.
Neuropharmacology, 2011
Cocaine binds and inhibits dopamine transporter (DAT), norepinephrine transporter (NET) and serotonin transporter. The residues forming cocaine binding sites are unknown. RTI-113, a cocaine analog, is 100x more potent at inhibiting DAT than inhibiting NET. Here we show that removing the hydroxyl group from residue Tyr151 in NET by replacing it with Phe, the corresponding residue in DAT, increased the sensitivity of NET to RTI-113, while the reverse mutation in DAT decreased the sensitivity of DAT to RTI-113. In contrast, RTI-31, another cocaine analog having the same structure as RTI-113 but with the phenyl group at the 2β position replaced by a methyl group, inhibits the transporter mutants equally well whether a hydroxyl group is present at the residue or not. The data suggest that this residue contributes to cocaine binding site and is close to the 2β position of cocaine analogs. These results are consistent with our previously proposed cocaine-DAT binding model where cocaine initially binds to a site that does not overlap with, but is close to, the dopamine-binding site. Computational modeling and molecular docking yielded a binding model that explains the observed changes in RTI-113 inhibition potencies.
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.