Faculty Bibliography

Current therapy of depression is less than ideal with remission rates of only 25-35% and response rates of 45-60%. It has been hypothesized that a dysfunctional dopaminergic system in the mesocorticolimbic pathway in depressive disorder may lead to development of anhedonia associated with loss of pleasure and interest along with loss of motivation. The current antidepressants do not address dopamine dysfunction which might explain their low efficacy. In this report, we have described an SAR study on our pyran-based triple reuptake inhibitors (TRIs) which are being investigated as the next-generation antidepressants. In the present work we demonstrate that our lead TRIs can be modified with appropriate aromatic substitutions to display a highly potent SSRI profile for compounds 2a and 4a (Ki (SERT); 0.71 and 2.68nM, respectively) or a potent DNRI profile for compounds 6b and 6h (Ki (DAT/NET); 8.94/4.76 and 13/7.37nM, respectively). Compounds 4g-4i exhibited potencies at all three monoamine transporters. The results provide insights into the structural requirements for developing selective dual- and triple-uptake inhibitors from a unique pyran molecular template for an effective management of depression and related disorders.

Previous studies point to quaternary assembly of dopamine transporters (DATs) in oligomers. However, it is not clear whether the protomers function independently in the oligomer. Is each protomer an entirely separate unit that takes up dopamine and is inhibited by drugs known to block DAT function? In this work, human embryonic kidney 293 cells were co-transfected with DAT constructs possessing differential binding affinities for the phenyltropane cocaine analog, [3 H]WIN35,428. It was assessed whether the binding properties in co-expressing cells capable of forming hetero-oligomers differ from those in preparations obtained from mixed singly transfected cells where such oligomers cannot occur. A method is described that replaces laborious 'mixing' experiments with an in silico method predicting binding parameters from those observed for the singly expressed constructs. Among 5 pairs of constructs tested, statistically significant interactions were found between protomers of wild-type (WT) and D313N, WT and D345N, and WT and D436N. Compared with predicted Kd values of [3 H]WIN35,428 binding to the non-interacting pairs, the observed affinity of the former pair was increased 1.7 fold while the latter two were reduced 2.2 and 4.1 fold, respectively. This is the first report of an influence of protomer composition on the properties of a DAT inhibitor, indicating cooperativity within the oligomer

BACKGROUND: Treatment of stimulant-use disorders remains a formidable challenge, and the dopamine transporter (DAT) remains a potential target for antagonist or agonist-like substitution therapies. METHODS: This review focuses on DAT ligands, such as benztropine, GBR 12909, modafinil, and DAT substrates derived from phenethylamine or cathinone that have atypical DAT-inhibitor effects, either in vitro or in vivo. The compounds are described from a molecular mechanistic, behavioral, and medicinal-chemical perspective. RESULTS: Possible mechanisms for atypicality at the molecular level can be deduced from the conformational cycle for substrate translocation. For each conformation, a crystal structure of a bacterial homolog is available, with a possible role of cholesterol, which is also present in the crystal of Drosophila DAT. Although there is a direct relationship between behavioral potencies of most DAT inhibitors and their DAT affinities, a number of compounds bind to the DAT and inhibit dopamine uptake but do not share cocaine-like effects. Such atypical behavior, depending on the compound, may be related to slow DAT association, combined sigma-receptor actions, or bias for cytosol-facing DAT. Some structures are sterically small enough to serve as DAT substrates but large enough to also inhibit transport. Such compounds may display partial DA releasing effects, and may be combined with release or uptake inhibition at other monoamine transporters. CONCLUSIONS: Mechanisms of atypical DAT inhibitors may serve as targets for the development of treatments for stimulant abuse. These mechanisms are novel and their further exploration may produce compounds with unique therapeutic potential as treatments for stimulant abuse.

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.

Antagonist and partial agonist modulators of the dopamine D3 receptor (D3R) have emerged as promising therapeutics for the treatment of substance abuse and neuropsychiatric disorders. However, development of druglike lead compounds with selectivity for the D3 receptor has been challenging because of the high sequence homology between the D3R and the dopamine D2 receptor (D2R). In this effort, we synthesized a series of acylaminobutylpiperazines incorporating aza-aromatic units and evaluated their binding and functional activities at the D3 and D2 receptors. Docking studies and results from evaluations against a set of chimeric and mutant receptors suggest that interactions at the extracellular end of TM7 contribute to the D3R versus D2R selectivity of these ligands. Molecular insights from this study could potentially enable rational design of potent and selective D3R ligands.

We have developed a series of dihydroxy compounds and related analogues based on our hybrid D2/D3 agonist molecular template to develop multifunctional drugs for symptomatic and neuroprotective treatment for Parkinson's disease (PD). The lead compound (-)-24b (D-520) exhibited high agonist potency at D2/D3 receptors and produced efficacious activity in the animal models for PD. The data from thioflavin T (ThT) assay and from transmission electron microscopy (TEM) analysis demonstrate that D-520 is able to modulate aggregation of alpha-synuclein (alphaSN). Additionally, coincubation of D-520 with alphaSN is able to reduce toxicity of preformed aggregates of alphaSN compared to control alphaSN alone. Finally, in a neuroprotection study with dopaminergic MN9D cells, D-520 clearly demonstrated the effect of neuroprotection from toxicity of 6-hydroxydopamine. Thus, compound D-520 possesses properties characteristic of multifunctionality conducive to symptomatic and neuroprotective treatment of PD.

Dopamine transporter deficiency syndrome due to SLC6A3 mutations is the first inherited dopamine 'transportopathy' to be described, with a classical presentation of early infantile-onset progressive parkinsonism dystonia. In this study we have identified a new cohort of patients with dopamine transporter deficiency syndrome, including, most significantly, atypical presentation later in childhood with a milder disease course. We report the detailed clinical features, molecular genetic findings and in vitro functional investigations undertaken for adult and paediatric cases. Patients presenting with parkinsonism dystonia or a neurotransmitter profile characteristic of dopamine transporter deficiency syndrome were recruited for study. SLC6A3 mutational analysis was undertaken in all patients. The functional consequences of missense variants on the dopamine transporter were evaluated by determining the effect of mutant dopamine transporter on dopamine uptake, protein expression and amphetamine-mediated dopamine efflux using an in vitro cellular heterologous expression system. We identified eight new patients from five unrelated families with dopamine transporter deficiency syndrome. The median age at diagnosis was 13 years (range 1.5-34 years). Most significantly, the case series included three adolescent males with atypical dopamine transporter deficiency syndrome of juvenile onset (outside infancy) and progressive parkinsonism dystonia. The other five patients in the cohort presented with classical infantile-onset parkinsonism dystonia, with one surviving into adulthood (currently aged 34 years) and labelled as having 'juvenile parkinsonism'. All eight patients harboured homozygous or compound heterozygous mutations in SLC6A3, of which the majority are previously unreported variants. In vitro studies of mutant dopamine transporter demonstrated multifaceted loss of dopamine transporter function. Impaired dopamine uptake was universally present, and more severely impacted in dopamine transporter mutants causing infantile-onset rather than juvenile-onset disease. Dopamine transporter mutants also showed diminished dopamine binding affinity, reduced cell surface transporter, loss of post-translational dopamine transporter glycosylation and failure of amphetamine-mediated dopamine efflux. Our data series expands the clinical phenotypic continuum of dopamine transporter deficiency syndrome and indicates that there is a phenotypic spectrum from infancy (early onset, rapidly progressive disease) to childhood/adolescence and adulthood (later onset, slower disease progression). Genotype-phenotype analysis in this cohort suggests that higher residual dopamine transporter activity is likely to contribute to postponing disease presentation in these later-onset adult cases. Dopamine transporter deficiency syndrome remains under-recognized and our data highlights that dopamine transporter deficiency syndrome should be considered as a differential diagnosis for both infantile- and juvenile-onset movement disorders, including cerebral palsy and juvenile parkinsonism.