Oligomerization of Monoamine Transporters.

Transporters of the monoamine transporter (MAT) family regulate the uptake of important neurotransmitters like dopamine, serotonin, and norepinephrine. The MAT family functions using the electrochemical gradient of ions across the membrane and comprises three transporters, dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). MAT transporters have been observed to exist in monomeric states to higher-order oligomeric states. Structural features, allosteric modulation, and lipid environment regulate the oligomerization of MAT transporters. NET and SERT oligomerization are regulated by levels of PIP2 present in the membrane. The kink present in TM12 in the MAT family is crucial for dimer interface formation. Allosteric modulation in the dimer interface hinders dimer formation. Oligomerization also influences the transporters' function, trafficking, and regulation. This chapter will focus on recent studies on monoamine transporters and discuss the factors affecting their oligomerization and its impact on their function.

Dimerization and antidepressant recognition at noradrenaline transporter.

The noradrenaline transporter has a pivotal role in regulating neurotransmitter balance and is crucial for normal physiology and neurobiology1. Dysfunction of noradrenaline transporter has been implicated in numerous neuropsychiatric diseases, including depression and attention deficit hyperactivity disorder2. Here we report cryo-electron microscopy structures of noradrenaline transporter in apo and substrate-bound forms, and as complexes with six antidepressants. The structures reveal a noradrenaline transporter dimer interface that is mediated predominantly by cholesterol and lipid molecules. The substrate noradrenaline binds deep in the central binding pocket, and its amine group interacts with a conserved aspartate residue. Our structures also provide insight into antidepressant recognition and monoamine transporter selectivity. Together, these findings advance our understanding of noradrenaline transporter regulation and inhibition, and provide templates for designing improved antidepressants to treat neuropsychiatric disorders.

Norepinephrine Reuptake Inhibition, an Emergent Treatment for Neurogenic Orthostatic Hypotension.

The NET (norepinephrine transporter) is situated in the prejunctional plasma membrane of noradrenergic neurons. It is responsible for >90% of the norepinephrine uptake that is released in the autonomic neuroeffector junction. Inhibitors of this cell membrane transporter, known as norepinephrine reuptake inhibitors (NRIs), are commercially available for the treatment of depression and attention deficit hyperactivity disorder. These agents increase norepinephrine levels, potentiating its action in preganglionic and postganglionic adrenergic neurons, the latter through activation of α-1 adrenoreceptors. Previous studies found that patients with neurogenic orthostatic hypotension can improve standing blood pressure and reduce symptoms of neurogenic orthostatic hypotension after a single administration of the selective NRI atomoxetine. This effect was primarily observed in patients with impaired central autonomic pathways with otherwise normal postganglionic sympathetic fibers, known as multiple system atrophy. Likewise, patients with normal or high norepinephrine levels may benefit from NRIs. The long-term efficacy of NRIs for the treatment of neurogenic orthostatic hypotension-related symptoms is currently under investigation. In summary, an in-depth understanding of the pathophysiology of neurogenic orthostatic hypotension resulted in the discovery of a new therapeutic pathway targeted by NRI.

Cardiac Uptake of the Adrenergic Imaging Agent meta-Iodobenzylguanidine (mIBG) Is Mediated by Organic Cation Transporter 3 (Oct3).

Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.123I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 +/+ and Oct3 -/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 +/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 +/+ and Oct3 -/- mice. Strikingly, cardiac mIBG was depleted in Oct3 -/- mice, resulting in 83% reduction in overall cardiac exposure (AUC0-24 h: 12.7 vs. 2.1 µg × h/g). mIBG tissue exposure (AUC0-24 h) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 -/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: 123I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.

A Conserved Intramolecular Ion-Pair Plays a Critical but Divergent Role in Regulation of Dimerization and Transport Function among the Monoamine Transporters.

The monoamine transporters, including the serotonin transporter (SERT), dopamine transporter (DAT), and norepinephrine transporter (NET), are the therapeutic targets for the treatment of many neuropsychiatric disorders. Despite significant progress in characterizing the structures and transport mechanisms of these transporters, the regulation of their transport functions through dimerization or oligomerization remains to be understood. In the present study, we identified a conserved intramolecular ion-pair at the third extracellular loop (EL3) connecting TM5 and TM6 that plays a critical but divergent role in the modulation of dimerization and transport functions among the monoamine transporters. The disruption of the ion-pair interactions by mutations induced a significant spontaneous cross-linking of a cysteine mutant of SERT and an increase in cell surface expression but with an impaired specific transport activity. On the other hand, similar mutations of the corresponding ion-pair residues in both DAT and NET resulted in an opposite effect on their oxidation-induced dimerization, cell surface expression, and transport function. Reversible biotinylation experiments indicated that the ion-pair mutations slowed down the internalization of SERT but stimulated the internalization of DAT. In addition, cysteine accessibility measurements for monitoring SERT conformational changes indicated that substitution of the ion-pair residues resulted in profound effects on the rate constants for cysteine modification in both the extracellular and cytoplasmatic substrate permeation pathways. Furthermore, molecular dynamics simulations showed that the ion-pair mutations increased the interfacial interactions in a SERT dimer but decreased it in a DAT dimer. Taken together, we propose that the transport function is modulated by the equilibrium between monomers and dimers on the cell surface, which is regulated by a potential compensatory mechanism but with different molecular solutions among the monoamine transporters. The present study provided new insights into the structural elements regulating the transport function of the monoamine transporters through their dimerization.

Perturbations in risk/reward decision making and frontal cortical catecholamine regulation induced by mild traumatic brain injury.

Mild traumatic brain injury (mTBI) disrupts cognitive processes that influence risk taking behavior. Little is known regarding the effects of repetitive mild injury (rmTBI) or whether these outcomes are sex specific. Risk/reward decision making is mediated by the prefrontal cortex (PFC), which is densely innervated by catecholaminergic fibers. Aberrant PFC catecholamine activity has been documented following TBI and may underlie TBI-induced risky behavior. The present study characterized the effects of rmTBI on risk/reward decision making behavior and catecholamine transmitter regulatory proteins within the PFC. Rats were exposed to sham, single (smTBI), or three closed-head controlled cortical impact (CH-CCI) injuries and assessed for injury-induced effects on risk/reward decision making using a probabilistic discounting task (PDT). In the first week post-final surgery, mTBI increased risky choice preference. By the fourth week, males exhibited increased latencies to make risky choices following rmTBI, demonstrating a delayed effect on processing speed. When levels of tyrosine hydroxylase (TH) and the norepinephrine reuptake transporter (NET) were measured within subregions of the PFC, females exhibited dramatic increases of TH levels within the orbitofrontal cortex (OFC) following smTBI. However, both males and females demonstrated reduced levels of OFC NET following rmTBI. These results indicate the OFC is susceptible to catecholamine instability after rmTBI and suggests that not all areas of the PFC contribute equally to TBI-induced imbalances. Overall, the CH-CCI model of rmTBI has revealed time-dependent and sex-specific changes in risk/reward decision making and catecholamine regulation following repetitive mild head injuries.

Feasibility Study of Single-Photon Emission Computed Tomography with Iodine-123 Labeled Metaiodobenzylguanidine for Preclinical Evaluation of Labetalol as a ß-Adrenergic Receptor Blocker.

Among clinically used radiopharmaceuticals, iodine-123 labeled metaiodobenzylguanidine ([123I]mIBG) serves for diagnosing neuroendocrine tumors and obtaining images of myocardial sympathetic innervation. mIBG, a structural analogue of norepinephrine (NE), a neurotransmitter acting in peripheral and central nerves, follows a pathway similar to NE, transmitting signals through the NE transporter (NET) located at synaptic terminals. It moves through the body without decomposing, enabling noninvasive image evaluation. In this study, we aimed to quantify [123I]mIBG uptake in the adrenal glands using small animal single-photon emission computed tomography/computed tomography (SPECT/CT) images post [123I]mIBG administration. We investigated the possibility of assessing the effectiveness of ß-adrenergic receptor blockers by quantifying SPECT/CT images and biodistribution results to determine the degree of [123I]mIBG uptake in the adrenal glands treated with labetalol, a known ß-adrenergic receptor blocker. Upon intravenous administration of [123I]mIBG to mice, SPECT/CT images were acquired over time to confirm the in vivo distribution pattern, revealing a clear uptake in the adrenal glands. Labetalol inhibited the uptake of [123I]mIBG in cell lines expressing NET. A decrease in [123I]mIBG uptake in the adrenal glands was observed in the labetalol-treated group compared with the normal group through SPECT/CT imaging and biodistribution studies. These results demonstrate that SPECT/CT imaging with [123I]mIBG could be applicable for evaluating the preclinical efficacy of new antihypertensive drug candidates such as labetalol, a ß-adrenergic receptor blocker.

Uncovering Structure-Activity Relationships of Phenethylamines: Paving the Way for Innovative Mental Health Treatments.

The rapidly evolving psychedelic industry has garnered considerable attention due to 3,4-methylenedioxymethamphetamine-assisted psychotherapy's ground-breaking success in treating moderate-to-severe Post-traumatic Stress Disorder in two Phase 3 clinical trials. This has opened Pandora's box for the development of innovative therapeutic modalities. Of particular interest are the phenethylamines and their ability to inhibit monoamine transporters. In this study, we employed the quantitative structure-activity relationship methodology to develop three vigorous models for the reuptake of serotonin, dopamine, and norepinephrine through monoamine transporters. These models were thoroughly validated using various criteria, including fitting (R2DAT = 0.869, R2SERT = 0.828, and R2NET = 0.887), internal (Q2looDAT = 0.795, Q2looSERT = 0.784, and Q2looNET = 0.820), and external (RMSEextDAT = 0.373, R2extDAT = 0.831, RMSEextSERT = 0.200, R2extSERT = 0.955, RMSEextNET = 0.318, and R2extNET = 0.711) criteria.

Relationship between SLC6A2 gene polymorphisms and brain volume in Han Chinese adults who lost their sole child.

BACKGROUND: Norepinephrine transporter (NET) is encoded by the SLC6A2 gene and is a potential target for studying the pathogenesis of PTSD. To the best of our knowledge, no prior investigations have examined SLC6A2 polymorphism-related neuroimaging abnormalities in PTSD patients. METHODS: In 218 Han Chinese adults who had lost their sole child, we investigated the association between the T-182 C SLC6A2 genotype and gray matter volume (GMV). Participants included 57 PTSD sufferers and 161 non-PTSD sufferers, and each group was further separated into three subgroups based on each participant's SLC6A2 genotype (TT, CT, and CC). All participants received magnetic resonance imaging (MRI) and clinical evaluation. To assess the effects of PTSD diagnosis, genotype, and genotype × diagnosis interaction on GMV, 2 × 3 full factorial designs were used. Pearson's correlations were used to examine the association between GMV and CAPS, HAMD, and HAMA. RESULTS: The SLC6A2 genotype showed significant main effects on GMV of the left superior parietal gyrus (SPG) and the bilateral middle cingulate gyrus (MCG). Additionally, impacts of the SLC6A2 genotype-diagnosis interaction were discovered in the left superior frontal gyrus (SFG). The CAPS, HAMA, and HAMD scores, as well as the genotype main effect and diagnostic SLC6A2 interaction, did not significantly correlate with each other. CONCLUSION: These findings indicate a modulatory effect that the SLC6A2 polymorphism exerts on the SPG and MCG, irrespective of PTSD diagnosis. We found evidence to suggest that the SLC6A2 genotype-diagnosis interaction on SFG may potentially contribute to PTSD pathogenesis in adults who lost their sole child.

Resting-state alterations in behavioral variant frontotemporal dementia are related to the distribution of monoamine and GABA neurotransmitter systems.

Background: Aside to clinical changes, behavioral variant frontotemporal dementia (bvFTD) is characterized by progressive structural and functional alterations in frontal and temporal regions. We examined if there is a selective vulnerability of specific neurotransmitter systems in bvFTD by evaluating the link between disease-related functional alterations and the spatial distribution of specific neurotransmitter systems and their underlying gene expression levels. Methods: Maps of fractional amplitude of low-frequency fluctuations (fALFF) were derived as a measure of local activity from resting-state functional magnetic resonance imaging for 52 bvFTD patients (mean age = 61.5 ± 10.0 years; 14 females) and 22 healthy controls (HC) (mean age = 63.6 ± 11.9 years; 13 females). We tested if alterations of fALFF in patients co-localize with the non-pathological distribution of specific neurotransmitter systems and their coding mRNA gene expression. Furthermore, we evaluated if the strength of co-localization is associated with the observed clinical symptoms. Results: Patients displayed significantly reduced fALFF in frontotemporal and frontoparietal regions. These alterations co-localized with the distribution of serotonin (5-HT1b and 5-HT2a) and γ-aminobutyric acid type A (GABAa) receptors, the norepinephrine transporter (NET), and their encoding mRNA gene expression. The strength of co-localization with NET was associated with cognitive symptoms and disease severity of bvFTD. Conclusions: Local brain functional activity reductions in bvFTD followed the distribution of specific neurotransmitter systems indicating a selective vulnerability. These findings provide novel insight into the disease mechanisms underlying functional alterations. Our data-driven method opens the road to generate new hypotheses for pharmacological interventions in neurodegenerative diseases even beyond bvFTD. Funding: This study has been supported by the German Consortium for Frontotemporal Lobar Degeneration, funded by the German Federal Ministry of Education and Research (BMBF; grant no. FKZ01GI1007A).

Transporters involved in adult rat cortical astrocyte dopamine uptake: Kinetics, expression and pharmacological modulation.

Astrocytes, glial cells in the central nervous system, perform a multitude of homeostatic functions and are in constant bidirectional communication with neuronal cells, a concept named the tripartite synapse; however, their role in the dopamine homeostasis remains unexplored. The aim of this study was to clarify the pharmacological and molecular characteristics of dopamine transport in cultured cortical astrocytes of adult rats. In addition, we were interested in the expression of mRNA of dopamine transporters as well as dopamine receptors D1 and D2 and in the effect of dopaminergic drugs on the expression of these transporters and receptors. We have found that astrocytes possess both Na+-dependent and Na+-independent transporters. Uptake of radiolabelled dopamine was time-, temperature- and concentration-dependent and was inhibited by decynium-22, a plasma membrane monoamine transporter inhibitor, tricyclic antidepressants desipramine and nortriptyline, both inhibitors of the norepinephrine transporter. Results of transporter mRNA expression indicate that the main transporters involved in cortical astrocyte dopamine uptake are the norepinephrine transporter and plasma membrane monoamine transporter. Both dopamine receptor subtypes were identified in cortical astrocyte cultures. Twenty-four-hour treatment of astrocyte cultures with apomorphine, a D1/D2 agonist, induced upregulation of D1 receptor, norepinephrine transporter and plasma membrane monoamine transporter, whereas the latter was downregulated by haloperidol and L-DOPA. Astrocytes take up dopamine by multiple transporters and express dopamine receptors, which are sensitive to dopaminergic drugs. The findings of this study could open a promising area of research for the fine-tuning of existing therapeutic strategies.

Structure-activity relationships for locomotor stimulant effects and monoamine transporter interactions of substituted amphetamines and cathinones.

Substitutions to the phenethylamine structure give rise to numerous amphetamines and cathinones, contributing to an ever-growing number of abused novel psychoactive substances. Understanding how various substitutions affect the pharmacology of phenethylamines may help lawmakers and scientists predict the effects of newly emerging drugs. Here, we established structure-activity relationships for locomotor stimulant and monoamine transporter effects of 12 phenethylamines with combinations of para-chloro, ß-keto, N-methyl, or N-ethyl additions. Automated photobeam analysis was used to evaluate effects of drugs on ambulatory activity in mice, whereas in vitro assays were used to determine activities at transporters for dopamine (DAT), norepinephrine (NET), and 5-HT (SERT) in rat brain synaptosomes. In mouse studies, all compounds stimulated locomotion, except for 4-chloro-N-ethylcathinone. Amphetamines were more potent stimulants than their ß-keto counterparts, while para-chloro amphetamines tended to be more efficacious than unsubstituted amphetamines. Para-chloro compounds also produced lethality at doses on the ascending limbs of their locomotor dose-effect functions. The in vitro assays showed that all compounds inhibited uptake at DAT, NET, and SERT, with most compounds also acting as substrates (i.e., releasers) at these sites. Unsubstituted compounds displayed better potency at DAT and NET relative to SERT. Para-chloro substitution or increased N-alkyl chain length augmented relative potency at SERT, while combined para-chloro and N-ethyl substitutions reduced releasing effects at NET and DAT. These results demonstrate orderly SAR for locomotor stimulant effects, monoamine transporter activities, and lethality induced by phenethylamines. Importantly, 4-chloro compounds produce toxicity in mice that suggests serious risk to humans using these drugs in recreational contexts.

Norepinephrine transporter and vesicular monoamine transporter 2 tumor expression as a predictor of response to 131 I-MIBG in patients with relapsed/refractory neuroblastoma.

BACKGROUND: Prior studies suggest that norepinephrine transporter (NET) and vesicular monoamine transporter 2 (VMAT2) mediate meta-iodobenzylguanidine (MIBG) uptake and retention in neuroblastoma tumors. We evaluated the relationship between NET and VMAT2 tumor expression and clinical response to 131 I-MIBG therapy in patients with neuroblastoma. METHODS: Immunohistochemistry (IHC) was used to evaluate NET and VMAT2 protein expression levels on archival tumor samples (obtained at diagnosis or relapse) from patients with relapsed or refractory neuroblastoma treated with 131 I-MIBG. A composite protein expression H-score was determined by multiplying a semi-quantitative intensity value (0-3+) by the percentage of tumor cells expressing the protein. RESULTS: Tumor samples and clinical data were available for 106 patients, of whom 28.3% had partial response (PR) or higher. NET H-score was not significantly associated with response (≥PR), though the percentage of tumor cells expressing NET was lower among responders (median 80% for ≥PR vs. 90% for

Chirality Matters: Fine-Tuning of Novel Monoamine Reuptake Inhibitors Selectivity through Manipulation of Stereochemistry.

The high structural similarity, especially in transmembrane regions, of dopamine, norepinephrine, and serotonin transporters, as well as the lack of all crystal structures of human isoforms, make the specific targeting of individual transporters rather challenging. Ligand design itself is also rather limited, as many chemists, fully aware of the synthetic and analytical challenges, tend to modify lead compounds in a way that reduces the number of chiral centers and hence limits the potential chemical space of synthetic ligands. We have previously shown that increasing molecular complexity by introducing additional chiral centers ultimately leads to more selective and potent dopamine reuptake inhibitors. Herein, we significantly extend our structure-activity relationship of dopamine transporter-selective ligands and further demonstrate how stereoisomers of defined absolute configuration may fine-tune and direct the activity towards distinct targets. From the pool of active compounds, using the examples of stereoisomers 7h and 8h, we further showcase how in vitro activity significantly differs in in vivo drug efficacy experiments, calling for proper validation of individual stereoisomers in animal studies. Furthermore, by generating a large library of compounds with defined absolute configurations, we lay the groundwork for computational chemists to further optimize and rationally design specific monoamine transporter reuptake inhibitors.

Preliminary Evaluation of 18F-Labeled Benzylguanidine Analogs as NET Tracers for Myocardial Infarction Diagnosis.

PURPOSE: Heart failure (HF) remains a major cause of late morbidity and mortality after myocardial infarction (MI). To date, no clinically established 18F-labeled sympathetic nerve PET tracers for monitoring myocardial infarction are available. Therefore, in this study, we synthesized a series of 18F-labeled benzyl guanidine analogs and evaluated their efficacy as cardiac neuronal norepinephrine transporter (NET) tracers for myocardial imaging. We also investigated the preliminary diagnostic capabilities of these tracers in myocardial infarction animal models, as well as the structure-activity relationship of these tracers. PROCEDURES: Three benzyl guanidine-NET tracers, including [18F]1, [18F]2, and [18F]3, were synthesized and evaluated in vivo as PET tracers in a myocardial infarction mouse model. [18F]LMI1195 was used as a positive control for the tracers. H&E staining of the isolated myocardial infarction heart tissue sections was performed to verify the efficacy of the selected PET tracer. RESULTS: Our data show that [18F]3 had a moderate decay corrected labeling yield (~10%) and high radiochemical purity (>95%) compared to other tracers. The uptake of [18F]3 in normal mouse hearts was 1.7±0.1%ID/cc at 1 h post-injection (p. i.), while it was 2.4±0.1, 2.6±0.9, and 2.1±0.4%ID/cc in the MI mouse hearts at 1, 2, and 3 days after surgery, respectively. Compared with [18F]LMI1195, [18F]3 had a better myocardial imaging effect in terms of the contrast between normal and MI hearts. The area of myocardial infarction shown by PET imaging corresponded well with the infarcted tissue demonstrated by H&E staining. CONCLUSIONS: With an obvious cardiac uptake contrast between normal mice and the myocardial infarction mouse model, [18F]3 appears to be a potential tool in the diagnosis of myocardial infarction. Therefore, it is necessary to conduct further structural modification studies on the chemical structure of [18F]3 to improve its in vivo stability and diagnostic detection ability to achieve reliable and practical imaging effects.

Relationships of in vivo brain norepinephrine transporter and age, BMI, and gender.

Previous research reported an age-related decline in brain norepinephrine transporter (NET) using (S, S)-[11C]O-methylreboxetine ([11C]MRB) as a radiotracer. Studies with the same tracer have been mixed in regard to differences related to body mass index (BMI). Here, we investigated potential age-, BMI-, and gender-related differences in brain NET availability using [11C]MRB, the most selective available radiotracer. Forty-three healthy participants (20 females, 23 males; age range 18-49 years), including 12 individuals with normal/lean weight, 15 with overweight, and 16 with obesity were scanned with [11C]MRB using a positron emission tomography (PET) high-resolution research tomograph (HRRT). We evaluated binding potential (BPND ) in brain regions with high NET availability using multilinear reference tissue model 2 (MRTM2) with the occipital cortex as a reference region. Brain regions were delineated with a defined anatomic template applied to subjects' structural MR scans. We found a negative association between age and NET availability in the locus coeruleus, raphe nucleus, and hypothalamus, with a 17%, 19%, and 14% decrease per decade, respectively, in each region. No gender or BMI relationships with NET availability were observed. Our findings suggest an age-related decline, but no BMI- or gender-related differences, in NET availability in healthy adults.

Synthesis and preliminary evaluation of benzylaminoimidazoline derivatives as novel norepinephrine transporter ligands.

A series of benzylaminoimidazoline derivatives was synthesized and evaluated for norepinephrine transporter (NET) targeting. Among them, N-(3-iodobenzyl)-4,5-dihydro-1H-imidazol-2-amine (Compound 9) displayed the highest affinity for NET (IC50 = 5.65 ± 0.97 µM). The corresponding radiotracer [125 I]9 was further prepared by copper-mediated radioiodination and evaluated both in vitro and in vivo. The cellular uptake results suggested that [125 I]9 was specifically taken up by the NET-expressing SK-N-SH cell line. Biodistribution studies showed that [125 I]9 accumulated in the heart (5.54 ± 1.24 %ID/g at 5 min p.i. and 0.79 ± 0.08 %ID/g at 2 h p.i.) and adrenal gland (14.83 ± 3.47 %ID/g at 5 min p.i. and 3.87 ± 0.24 %ID/g at 2 h p.i.). The uptake in the heart and adrenal gland could be significantly inhibited by preinjection of desipramine (DMI). These results indicated that the benzylaminoimidazoline derivatives retained affinity for NET, which could provide structure-activity relationship data for further studies.

An exploratory analysis of the performance of methylphenidate regimens based on a PKPD model of dopamine and norepinephrine transporter occupancy.

Methylphenidate (MPH) is a psychostimulant which inhibits the uptake of dopamine and norepinephrine transporters, DAT and NET, and is mostly used to treat Attention Deficit/Hyperactivity Disorder. The current dose optimization is done through titration, a cumbersome approach for patients. To assess the therapeutic performance of MPH regimens, we introduce an in silico framework composed of (i) a population pharmacokinetic model of MPH, (ii) a pharmacodynamic (PD) model of DAT and NET occupancy, (iii) a therapeutic box delimited by time and DAT occupancy, and (iv) a performance score computation. DAT occupancy data was digitized (n = 152) and described with Emax models. NET occupancy was described with a KPD model. We used this integrative framework to simulate the performance of extended-release (18-99 mg) and tid MPH regimens (25-40 mg). Early blood samples of MPH seem to lead to higher DAT occupancy, consistent with an acute tolerance observed in clinical rating scales. An Emax model with a time-dependent tolerance was fitted to available data to assess the observed clockwise hysteresis. Peak performance is observed at 63 mg. While our analysis does not deny the existence of an acute tolerance, data precision in terms of formulation and sampling times does not allow a definite confirmation of this phenomenon. This work justifies the need for a more systematic collection of DAT and NET occupancy data to further investigate the presence of acute tolerance and assess the impact of low MPH doses on its efficacy.

Proteochemometric Modeling Identifies Chemically Diverse Norepinephrine Transporter Inhibitors.

Solute carriers (SLCs) are relatively underexplored compared to other prominent protein families such as kinases and G protein-coupled receptors. However, proteins from the SLC family play an essential role in various diseases. One such SLC is the high-affinity norepinephrine transporter (NET/SLC6A2). In contrast to most other SLCs, the NET has been relatively well studied. However, the chemical space of known ligands has a low chemical diversity, making it challenging to identify chemically novel ligands. Here, a computational screening pipeline was developed to find new NET inhibitors. The approach increases the chemical space to model for NETs using the chemical space of related proteins that were selected utilizing similarity networks. Prior proteochemometric models added data from related proteins, but here we use a data-driven approach to select the optimal proteins to add to the modeled data set. After optimizing the data set, the proteochemometric model was optimized using stepwise feature selection. The final model was created using a two-step approach combining several proteochemometric machine learning models through stacking. This model was applied to the extensive virtual compound database of Enamine, from which the top predicted 22,000 of the 600 million virtual compounds were clustered to end up with 46 chemically diverse candidates. A subselection of 32 candidates was synthesized and subsequently tested using an impedance-based assay. There were five hit compounds identified (hit rate 16%) with sub-micromolar inhibitory potencies toward NET, which are promising for follow-up experimental research. This study demonstrates a data-driven approach to diversify known chemical space to identify novel ligands and is to our knowledge the first to select this set based on the sequence similarity of related targets.

Levodopa responsive freezing of gait is associated with reduced norepinephrine transporter binding in Parkinson's disease.

BACKGROUND: Freezing of gait (FOG) is a major cause of falling in Parkinson's disease (PD) and can be responsive or unresponsive to levodopa. Pathophysiology is poorly understood. OBJECTIVE: To examine the link between noradrenergic systems, the development of FOG in PD and its responsiveness to levodopa. METHODS: We examined norepinephrine transporter (NET) binding via brain positron emission tomography (PET) to evaluate changes in NET density associated with FOG using the high affinity selective NET antagonist radioligand [11C]MeNER (2S,3S)(2-[α-(2-methoxyphenoxy)benzyl]morpholine) in 52 parkinsonian patients. We used a rigorous levodopa challenge paradigm to characterize PD patients as non-freezing (NO-FOG, N = 16), levodopa responsive freezing (OFF-FOG, N = 10), and levodopa-unresponsive freezing (ONOFF-FOG, N = 21), and also included a non-PD FOG group, primary progressive freezing of gait (PP-FOG, N = 5). RESULTS: Linear mixed models identified significant reductions in whole brain NET binding in the OFF-FOG group compared to the NO-FOG group (-16.8%, P = 0.021) and regionally in the frontal lobe, left and right thalamus, temporal lobe, and locus coeruleus, with the strongest effect in right thalamus (P = 0.038). Additional regions examined in a post hoc secondary analysis including the left and right amygdalae confirmed the contrast between OFF-FOG and NO-FOG (P = 0.003). A linear regression analysis identified an association between reduced NET binding in the right thalamus and more severe New FOG Questionnaire (N-FOG-Q) score only in the OFF-FOG group (P = 0.022). CONCLUSION: This is the first study to examine brain noradrenergic innervation using NET-PET in PD patients with and without FOG. Based on the normal regional distribution of noradrenergic innervation and pathological studies in the thalamus of PD patients, the implications of our findings suggest that noradrenergic limbic pathways may play a key role in OFF-FOG in PD. This finding could have implications for clinical subtyping of FOG as well as development of therapies.