Novel Probes For The Dopamine Transporter

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The inhibition of dopamine reuptake via the dopamine transporter (DAT) has been characterized as the primary mechanism by which cocaine produces its psychomotor stimulant and reinforcing actions. In order to understand further the molecular mechanisms underlying cocaine abuse, structure-function studies have been directed toward characterizing the DAT protein at a molecular level. The design, synthesis and evaluation of 3-alpha-(diphenylmethoxy)tropane (benztropine, BZT) analogs have provided potent and selective probes for the DAT. Structure-activity relationships (SAR) have been developed that contrast with those described for cocaine, despite significant structural similarity. Furthermore, behavioral evaluation of many of the BZT analogs, in animal models of cocaine abuse, has suggested that these two classes of tropane-based dopamine uptake inhibitors have distinct pharmacological profiles. In general, our previous studies have shown that the BZT analogs, do not demonstrate efficacious locomotor stimulation in mice, do not fully substitute for a cocaine discriminative stimulus and are not appreciably self-administered in rhesus or squirrel monkeys. These compounds are generally more potent than cocaine as dopamine uptake inhibitors, in vitro, although their actions in vivo are not consistent with this action. By varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) is directly affected. Evaluating these compounds in both in vitro and in vivo models to obtain PK and PD profiles on these agents, in comparison to cocaine, with a series of N-substituted BZT analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they have a slower onset and duration of action, which is a suitable profile for development as pharmacotherapeutics and may be directly related to their lack of cocaine-like behavioral profiles. Further investigation into correlating structure, pharmacological action and pharmacodynamics of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In this regard, we have further characterized one of our novel DAT ligands, JHW 007 (N-n-butyl-4?4?-diF-BZT), and discovered that this compound has a unique pharmacological profile in that it slowly associates with the dopamine transporter and not only demonstrates no cocaine-like actions in mice, rats or monkeys, but fully blocks cocaine-induced locomotor activity and the cocaine discriminative stimulus in mice. These studies are the first to show that a dopamine uptake inhibitor can prevent cocaine from producing its psychostimulant actions and further support the hypothesis that the rate of in vivo occupancy of the DAT can dictate the behavioral actions of these compounds. Further evaluation of this and other N-substituted BZTs using microdialysis has allowed us to relate the rate and levels of increasing extracellular dopamine, in vivo, with binding of these compounds to the DAT. Recent studies using site-directed mutagenesis has revealed differences in binding domains between the BZTs, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, the BZTs showed a unique binding profile in the WT and D79E DAT mutants as compared to other cocaine-like DAT inhibitors providing evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of radioactive and/or irreversible ligands. Radioiodinated analogues of both azido (photoactivated) and isothiocyanato-derivatives of our tropane based DAT inhibitors have been synthesized and are currently being utilized in combination with proteolysis and MALDI MS to elucidate transmembrane domains at which these compounds bind covalently to both DAT and SERT. We have also synthesized a high affinity fluorescent DAT ligand that is currently being used to characterize the DAT in living neuronal cells and as a fluoroprobe for visualization of dopamine neurons in stem cells.