Faculty Bibliography

In this study we have generated a pharmacophore model of triple uptake inhibitor compounds based on novel asymmetric pyran derivatives and the newly developed asymmetric furan derivatives. The model revealed features important for inhibitors to exhibit a balanced activity against dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). In particular, a 'folded' conformation was found common to the active pyran compounds in the training set and was crucial to triple uptake inhibitory activity. Furthermore, the distances between the benzhydryl moiety and the N-benzyl group as well as the orientation of the secondary nitrogen were also important for TUI activity. We have validated our findings by synthesizing and testing novel asymmetric pyran analogs. The present work has also resulted in the discovery of a new series of asymmetric tetrahydrofuran derivatives as novel TUIs. Lead compounds 41 and 42 exhibited moderate TUI activity. Interestingly, the highest TUI activity by lead tetrahydrofuran compounds for example, 41 and 42, was exhibited in a stereochemical preference similar to pyran TUI for example, D-161.

Major depressive disorder (MDD) is a debilitating disease affecting a wide cross section of people around the world. The current therapy for depression is less than adequate and there is a considerable unmet need for more efficacious treatment. Dopamine has been shown to play a significant role in depression including production of anhedonia which has been one of the untreated symptoms in MDD. It has been hypothesized that drugs acting at all three monoamine transporters including dopamine transporter should provide more efficacious antidepressants activity. This has led to the development of triple reuptake inhibitor D-473 which is a novel pyran based molecule and interacts with all three monoamine transporters. The monoamine uptake inhibition activity in the cloned human transporters expressed in HEK-293 cells (70.4, 9.18 and 39.7 for DAT, SERT and NET, respectively) indicates a serotonin preferring triple reuptake inhibition profile for this drug. The drug D-473 exhibited good brain penetration and produced efficacious activity in rat forced swim test under oral administration. The optimal efficacy dose did not produce any locomotor activation. Microdialysis experiment demonstrated that systemic administration of D-473 elevated extracellular level of the three monoamines DA, 5-HT, and NE efficaciously in the dorsal lateral striatum (DLS) and the medial prefrontal cortex (mPFC) area, indicating in vivo blockade of all three monoamine transporters by D-473. Thus, the current biological data from D-473 indicate potent antidepressant activity of the molecule.

The dopamine transporter (DAT) is a sodium-coupled symporter protein responsible for modulating the concentration of extraneuronal dopamine in the brain. The DAT is a principle target of various psychostimulant, nootropic, and antidepressant drugs, as well as certain drugs used recreationally, including the notoriously addictive stimulant cocaine. DAT ligands have traditionally been divided into two categories: cocaine-like inhibitors and amphetamine-like substrates. Whereas inhibitors block monoamine uptake by the DAT but are not translocated across the membrane, substrates are actively translocated and trigger DAT-mediated release of dopamine by reversal of the translocation cycle. Because both inhibitors and substrates increase extraneuronal dopamine levels, it is often assumed that all DAT ligands possess an addictive liability equivalent to that of cocaine. However, certain recently developed ligands, such as atypical benztropine-like DAT inhibitors with reduced or even a complete lack of cocaine-like rewarding effects, suggest that addictiveness is not a constant property of DAT-affecting compounds. These atypical ligands do not conform to the classic preconception that all DAT inhibitors (or substrates) are functionally and mechanistically alike. Instead, they suggest the possibility that the DAT exhibits some of the ligand-specific pleiotropic functional qualities inherent to G-protein-coupled receptors. That is, ligands with different chemical structures induce specific conformational changes in the transporter protein that can be differentially transduced by the cell, ultimately eliciting unique behavioral and psychological effects. The present overview discusses compounds with conformation-specific activity, useful not only as tools for studying the mechanics of dopamine transport, but also as leads for medication development in addictive disorders.

The study reported by Wenge and Bonisch in this issue provides critical structural information regarding extracellular loop 2 (EL2) of the human norepinephrine transporter (NET). A systematic search among all 10 cysteine and 13 histidine residues in NET led to His222 in EL2 as the target for N-ethylmaleimide: its alkylation interferes with [(3)H]nisoxetine binding, indicating the part of EL2 containing His 222 reaches back into the protein interior where it prevents access by nisoxetine to its binding site. Thus, EL2 in human NET does much more than conformationally assisting substrate translocation. The present study underscores the importance of site-directed mutagenesis approaches to elucidate structural features that cannot be deduced from crystals of homolog proteins. In the case of NET, the closest crystal structure is that of the homolog LeuT, but EL2 is difficult to align with 22 less loop residues in LeuT than in NET. The present results could only be achieved by the systematic mutagenesis study of all cysteines and all histidines in NET.

The goal of the present study was to explore, in our previously developed hybrid template, the effect of introduction of additional heterocyclic rings (mimicking catechol hydroxyl groups as bioisosteric replacement) on selectivity and affinity for the D3 versus D2 receptor. In addition, we wanted to explore the effect of derivatization of functional groups of the agonist binding moiety in compounds developed by us earlier from the hybrid template. Binding affinity (Ki) of the new compounds was measured with tritiated spiperone as the radioligand and HEK-293 cells expressing either D2 or D3 receptors. Functional activity of selected compounds was assessed in the GTPgammaS binding assay. In the imidazole series, compound 10a exhibited the highest D3 affinity whereas the indole derivative 13 exhibited similar high D3 affinity. Functionalization of the amino group in agonist (+)-9d with different sulfonamides derivatives improved the D3 affinity significantly with (+)-14f exhibiting the highest affinity. However, functionalization of the hydroxyl and amino groups of 15 and (+)-9d, known agonist and partial agonist, to sulfonate ester and amide in general modulated the affinity. In both cases loss of agonist potency resulted from such derivatization.

OBJECTIVE: The iboga alkaloids are a class of small molecules defined structurally on the basis of a common ibogamine skeleton, some of which modify opioid withdrawal and drug self-administration in humans and preclinical models. These compounds may represent an innovative approach to neurobiological investigation and development of addiction pharmacotherapy. In particular, the use of the prototypic iboga alkaloid ibogaine for opioid detoxification in humans raises the question of whether its effect is mediated by an opioid agonist action, or if it represents alternative and possibly novel mechanism of action. The aim of this study was to independently replicate and extend evidence regarding the activation of mu-opioid receptor (MOR)-related G proteins by iboga alkaloids. METHODS: Ibogaine, its major metabolite noribogaine, and 18-methoxycoronaridine (18-MC), a synthetic congener, were evaluated by agonist-stimulated guanosine-5 -O-(gamma-thio)-triphosphate ([(35)S]GTPgammaS) binding in cells overexpressing the recombinant MOR, in rat thalamic membranes, and autoradiography in rat brain slices. RESULTS AND SIGNIFICANCE: In rat thalamic membranes ibogaine, noribogaine and 18-MC were MOR antagonists with functional Ke values ranging from 3 uM (ibogaine) to 13 uM (noribogaine and 18MC). Noribogaine and 18-MC did not stimulate [(35)S]GTPgammaS binding in Chinese hamster ovary cells expressing human or rat MORs, and had only limited partial agonist effects in human embryonic kidney cells expressing mouse MORs. Ibogaine did not did not stimulate [(35)S]GTPgammaS binding in any MOR expressing cells. Noribogaine did not stimulate [(35)S]GTPgammaS binding in brain slices using autoradiography. An MOR agonist action does not appear to account for the effect of these iboga alkaloids on opioid withdrawal. Taken together with existing evidence that their mechanism of action also differs from that of other non-opioids with clinical effects on opioid tolerance and withdrawal, these findings suggest a novel mechanism of action, and further justify the search for alternative targets of iboga alkaloids.

The conformation and function of the dopamine transporter (DAT) can be affected by manipulating membrane cholesterol, yet there is no agreement as to the impact of cholesterol on the activity of lipid-raft localized DATs compared with non-raft DATs. Given the paucity of information regarding the impact of cholesterol on substrate efflux by the DAT, this study explores its influence on the kinetics of DAT-mediated DA efflux induced by dextroamphetamine, as measured by rotating disk electrode voltammetry (RDEV). Treatment with methyl-beta-cyclodextrin (mbetaCD), which effectively depletes total membrane cholesterol--uniformly affecting cholesterol-DAT interactions in both raft and non-raft membrane domains--reduced both DA uptake and efflux rate. In contrast, disruption of raft-localized DAT by cholesterol chelation with nystatin had no effect, arguing against a vital role for raft-localized DAT in substrate uptake or efflux. Supranormal repletion of cholesterol-depleted cells with the analog desmosterol, a non-raft promoting sterol, was as effective as cholesterol itself in restoring transport rates. Further studies with Zn(2+) and the conformationally biased W84L DAT mutant supported the idea that cholesterol is important for maintaining the outward-facing DAT with normal rates of conformational interconversions. Collectively, these results point to a role for direct cholesterol-DAT interactions in regulating DAT function.

To further explore the basic structural motifs (3S,6S)-6-benzhydryl-N-benzyltetrahydro-2H-pyran-3-amine and (2S,4R,5R)-2-benzhydryl-5-(benzylamino)tetrahydro-2H-pyran-4-ol, developed by our research group, for monoamine transport inhibition, we designed and synthesized various structurally altered analogues. The new compounds were tested for their affinities for the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET) in rat brain by measuring their capacity to inhibit the uptake of [(3) H]DA, [(3) H]5-HT, and [(3) H]NE, respectively. Our results point to novel compounds with a TUI, DNRI, SNRI, or SSRI profile. Among the TUIs, compound 2 g exhibited a balanced potency for all three monoamine transporters (K(i) : 60, 79, and 70.3 nM for DAT, SERT, and NET, respectively). In the rat forced swim test, compound 2 g produced a significant decrease in immobility in drug-treated rats relative to vehicle, indicating a potential antidepressant property.

Despite a wealth of information on cocaine-like compounds, there is no information on cocaine analogs with substitutions at C-1. Here, we report on (R)-(-)-cocaine analogs with various C-1 substituents: methyl (2), ethyl (3), n-propyl (4), n-pentyl (5), and phenyl (6). Analog 2 was equipotent to cocaine as an inhibitor of the dopamine transporter (DAT), whereas 3 and 6 were 3- and 10-fold more potent, respectively. None of the analogs, however, stimulated mouse locomotor activity, in contrast to cocaine. Pharmacokinetic assays showed compound 2 occupied mouse brain rapidly, as cocaine itself; moreover, 2 and 6 were behaviorally active in mice in the forced-swim test model of depression and the conditioned place preference test. Analog 2 was a weaker inhibitor of voltage-dependent Na(+) channels than cocaine, although 6 was more potent than cocaine, highlighting the need to assay future C-1 analogs for this activity. Receptorome screening indicated few significant binding targets other than the monoamine transporters. Benztropine-like "atypical" DAT inhibitors are known to display reduced cocaine-like locomotor stimulation, presumably by their propensity to interact with an inward-facing transporter conformation. However, 2 and 6, like cocaine, but unlike benztropine, exhibited preferential interaction with an outward-facing conformation upon docking in our DAT homology model. In summary, C-1 cocaine analogs are not cocaine-like in that they are not stimulatory in vivo. However, they are not benztropine-like in binding mechanism and seem to interact with the DAT similarly to cocaine. The present data warrant further consideration of these novel cocaine analogs for antidepressant or cocaine substitution potential.