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) 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 benztropine 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 benztropine 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 rats, 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. As such, we have described this class of compounds as atypical dopamine uptake inhibitors. By varying the structures of the parent compounds and thereby modifying their physical properties, pharmacokinetics (PK) as well as pharmacodynamics (PD) are 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 benztropine analogues has demonstrated that these compounds readily penetrate the blood brain barrier, but compared to cocaine, they typically have a slower onset and longer 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 PD of this class of compounds and developing these agents as potential cocaine-abuse therapeutics is ongoing. In this regard, we have extended the studies on our previously characterized cocaine antagonist, JHW 007 (N-n-butyl-4, 4-diF-benztropine), and discovered that JHW 007 and structurally related benztropine analogues were not self administered, in rats and indeed could selectively attenuate self administration of cocaine while having no effect on food administration. These studies further demonstrate the lack of abuse liability for these agents and strengthen their potential for development as therapeutics. Further evaluation of these and other N-substituted benztropines 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. Further investigation has shown that several additional analogues of JHW 007 show similar profiles in vivo and have been identified as lead candidates for development as medications to treat cocaine abuse, as well as attention deficit hyperactivity disorder (ADHD). Recent studies using site-directed mutagenesis have revealed differences in binding domains between the benztropines, cocaine and other structurally diverse dopamine uptake inhibitors. Interestingly, experimental evidence using the DAT inhibitors cocaine, WIN 35,428, and several benztropine analogues and comparing them to the substrates dopamine and MDMA has provided evidence, at the molecular level, of binding interaction differences that correlate with their distinctive behavioral profiles. Of note, cocaine binds to an outward facing conformation of the DAT, whereas the benztropines as well as the substrates, prefer an inward facing closed conformation. These conformational studies provide evidence at the molecular level that the atypical dopamine uptake inhibitors are indeed functioning differently than cocaine at the DAT and this is correlated with their distinct behavioral profiles. Our newest synthetic studies are focused on novel 2-substituted benztropine and benztropinamine analogues that incorporate oxazole functionality into the parent structure. Several of these compounds demonstrate high affinity and selectivity for the DAT and are currently being tested in the Y156F and Y335A mutants to determine preference for the outward or inward facing conformations of the DAT. Behavioral evaluation of selected analogues will follow. In addition, modafinil binds to the DAT and is currently used clinically for the treatment of sleep disorders. Recently, modafinil has been evaluated as a potential medication to treat methamphetamine and cocaine abuse and is also being used off-label for the treatment of ADHD. As modafinil has structural and pharmacological features that resemble the benztropines, we have embarked on a SAR study to further characterize these compounds at DAT, NET and SERT and to explore novel compounds with improved water solubility. The first series of compounds demonstrated a unique SAR profile and efforts to separate enantiomers of selected analogues are underway. In addition, computational studies support empirical studies in the mutant DATs that suggest modafinil prefers an inward facing conformation of the DAT. However, it is binding in a way that is unique to both cocaine and the benztropines. Further characterization of modafinil and its novel analogues will help elucidate the mechanisms underlying modafinils therapeutic action and thus far support the further development of the R-enantiomer as a psychostimulant medication strategy. In addition to developing agents for in vivo studies, we have also synthesized a number of important molecular tools in the form of fluorescent-derivatives of our tropane based DAT inhibitors. One such compound, JHC 1-064, has been used to characterize the trafficking and cellular distribution of DAT in living neuronal cells. Indeed, recent experiments in living neurons with JHC 1-064 have provided data that challenge mechanistic dogma for transporter translocation, as determined in DAT transfected heterologous cells, which is one area of ongoing research with these agents. Further, JHC 1-064 binds with high affinity to the serotonin transporter (SERT), as well, and we are conducting analogous studies, first in cell lines to study trafficking and cellular distribution of SERT. This project has led to a new project in which we have designed and synthesized novel analogues of the SERT inhibitor and antidepressant, citalopram. We have recently prepared a fluorescent citalopram analogue that is currently being used to visualize SERT in living cells. Citalopram is a racemic mixture of (+)- and (-) enantiomers, of which the latter (S-(+)-citalopram) is the more active antidepressant and currently marketed as Escitalopram. Previously obtained data suggest that the S-(+)-citalopram also binds to an allosteric site (S2) that can affect binding at the primary S1 site. To further explore this S2 site, we have designed and synthesized several series of citalopram analogues and separated several sets of enantiomers to further characterize and compare binding profiles at both the SERT and the other monoamine transporters, with the parent ligand. Our goal is to ultimately use these citalopram analogues to characterize the role of this putative allosteric S2 site. Collectively, these novel SERT compounds are being used to further elucidate structure and function of the SERT by characterizing primary and secondary binding domains that are related in an as of yet undetermined way to behavior.
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