Structure Modeling of the Norepinephrine Transporter

The norepinephrine transporter (NET) is one of the monoamine transporters. Its X-ray crystal structure has not been obtained yet. Inhibitors of human NET (hNET) play a major role in the treatment of many central and peripheral nervous system diseases. In this study, we focused on the spatial structure of a NET constructed by homology modeling on Drosophila melanogaster dopamine transporter templates. We further examined molecular construction of primary binding pocket (S1) together with secondary binding site (S2) and extracellular loop 4 (EL4). The next stage involved docking of transporter inhibitors: Reboxetine, duloxetine, desipramine, and other commonly used drugs. The procedure revealed the molecular orientation of residues and disclosed ones that are the most important for ligand binding: Phenylalanine F72, aspartic acid D75, tyrosine Y152, and phenylalanine F317. Aspartic acid D75 plays a key role in recognition of the basic amino group present in monoamine transporter inhibitors and substrates. The study also presents a comparison of hNET models with other related proteins, which could provide new insights into their interaction with therapeutics and aid future development of novel bioactive compounds

Structure Based Ligand Design for Monoamine Transporters and Mitogen Activated Kinase 5

Depression is a major psychological disorder that affects a person\u27s mental and physical abilities. The National Institute of Mental Health (NIMH) classified it as a serious medical illness. It causes huge economic, as well as financial impact on the people, and it is also becoming a major public health issue. Antidepressant drugs are prescribed to mitigate the suffering caused by this disorder. Different generations of antidepressants have been developed with dissimilar mechanisms of action. According to the Center for Disease Control, the usage of antidepressants has skyrocketed by 400 percent increase over 2005- 2008 survey period. This dramatic rise in usage indicates that these are the most prescribed drugs in the US. Even with the FDA mandated black box warning of increased suicidal thoughts upon use of selected antidepressants, these drugs are still being used at a higher rate. All classes of antidepressants are plagued by side effects with mainly sexual dysfunction common among them. To avoid the adverse effects, an emphasis is to discover novel structural drug scaffolds that can be further developed as a new generation of antidepressants. The importance of this research is to discover structurally novel antidepressants by performing in silico virtual screening (VS) of chemical databases using the serotonin transporter (SERT). In the absence of a SERT crystal structure, a homology model was developed. The homology model was utilized to develop the first structure-based pharmacophore for the extracellular facing secondary ligand binding pocket. The pharmacophore captured the necessary drug-SERT interaction pattern for SERT inhibitory action. This pharmacophore was employed as one of the filters for VS of candidate ligands. The ten compounds identified were purchased and tested pharmacologically. Out of the ten hits, three structurally novel ligands were identified as lead compounds. Two of these compounds exhibited selectivity towards SERT; the remaining lead compound was selective towards the dopamine transporter and displayed cocaine inhibition. The two SERT selective compounds will provide new opportunities in the development of novel therapeutics to treat depression. For dopamine transporter (DAT), the study was based on recently developed structurally diverse photo probes. In an effort to better understand the binding profile similarities among these different scaffolds, the photo probes were docked into DAT. The finger print analysis of the interaction pattern of docked poses was performed to identify the inhibitor-binding sites. For mitogen activated protein kinase 5 (MEK5), given the lack of structural information, a homology model of MEK5 was developed to guide the rational design of inhibitors. Docking of known MEK5 inhibitors into the homology model was performed to understand the inhibitory interaction profile. Several series of analogues were designed utilizing the generated interaction profile

Understanding The Structure-Function Relationships Between Monoamine Neurotransmitter Transporters And Their Cognate Ions And Ligands

The SLC6 family of secondary active transporters is made up of integral membrane solute carrier proteins characterized by the Na+-dependent translocation of small amino acid or amino acid-like substrates. SLC6 transporters, particularly the monoamine transporters (MATs) of serotonin, dopamine and norepinephrine, are some of the most heavily studied proteins today due to their association with a number of human diseases and disorders, making MATs a critical target for therapeutic development. In addition, MATs are directly involved in the action of drugs of abuse such as cocaine, amphetamines, and ecstasy. Following the first cloning of a MAT gene in the early 1990s, much has been uncovered about the structure and function of these proteins. Early studies developed an understanding of the kinetic parameters by which MATs operate and also yielded enough information to model the basic structural characteristics of MATs. This was greatly improved upon within the last decade, as crystallographic and computational advances have provided structural insights that have vastly accelerated our ability to study these proteins and their involvement in complex biological processes. However, despite a wealth of knowledge concerning the structural and kinetic characteristics of MATs, little is understood as to how these features are interrelated and much is still unclear as to the how regulation (and maybe more importantly, dysregulation) of MATs alters the functionality of these proteins at the molecular and synaptic levels. The overall goal of this dissertation was to comprehensively examine the relationship between MAT structure and the ions and ligands that bind to MATs to promote/prevent transporter function. This was done using a comprehensive approach that included biological, electrophysiological and computational techniques to target and elucidate the roles of specific amino acid residues in ion/ligand binding and/or mediation of the substrate translocation process. In successfully examining a number of specific MAT residues, this work has lead to the deduction of basic roles for each of the ion binding sites in the translocation mechanism (chapters II and III), as well as detailed the importance of specific structural components of MATs that are vital for functionality (chapters IV and V). Furthermore, this dissertation includes work highlighting the development of several photo-labeled, radio-iodinated antagonist analogues that will be used to further improve the understanding of how inhibitors bind to and block MAT function at the molecular level (chapter VI). In total, the work outlined in this dissertation provides a clearer understanding as to the molecular interactions that are necessary for MAT function and contributes an improved appreciation for the underlying mechanisms of substrate translocation and pharmacological intervention

In vivo analysis of a salt bridge at the external gate of the Drosophila melanogaster serotonin transporter in response to amphetamines

Monoamine neurotransmitter transporters are membrane proteins responsible for the clearing of biogenic amines from a synapse. These transporters are targets for many important pharmaceuticals including antidepressants, as well as psychostimulant drugs such as cocaine and amphetamines. Amphetamines are believed to elicit their psychostimulant activity primarily by inducing a reversal of the transport cycle and increasing neurotransmitter release into the synapse, though the mechanism of this activity is incompletely understood. Previous in vitro research has suggested functional significance of a conserved salt bridge in the serotonin transporter (SERT) in amphetamine-induced 5-HT efflux. This salt bridge is disrupted in the Drosophila melanogaster SERT. Here, a mutant line of D. melanogaster expressing a SERT with a restored salt bridge (dSERT N484D) was studied. Changes in neurochemistry induced by methamphetamine (METH) or 3, 4-methylenedioxymethamphetamine (MDMA) were examined in vivo. HPLC/Mass spectrometry was used to quantify brain concentrations of neurotransmitters in fly tissue after drug treatment. N484D flies were found to have significantly depleted 5-HT in response to 0.05% MDMA relative to wild-type. This depletion of 5-HT was not observed after treatment with 0.6% MDMA or 0.6% METH. No significant drug-induced changes were observed in concentrations of other neurotransmitters examined. The results show that the presence of the salt bridge at the external gate of SERT may be important for amphetamine-induced 5-HT efflux, and helps explain pharmacological differences observed between hSERT and dSERT

Synthesis and Biological Evaluation of Novel GBR 12909 Tropane and Azetidine Hybrid Analogues

The high affinity, selective dopamine transporter ligand GBR 12909 has served as a template for the design of two novel classes of dopamine transporter ligands. A series of 3-[2- (diarylmethoxyethyidenyl)]-N-substituted tropane derivatives were synthesized and the binding affinities of these compounds were determined at the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in rat brain tissue preparations. The tropane derivatives were found to exhibit more potent affinity and selectivity for DAT than GBR 12909. From the SAR of the tropane analogues and GBR 12909, a novel series of 3-[2-(diarylmethoxyethylidenyl)]-Nsubstituted azetidine derivatives has been developed

Synthesis and Biological Evaluation of Novel GBR 12909 Tropane and Azetidine Hybrid Analogues

The high affinity, selective dopamine transporter ligand GBR 12909 has served as a template for the design of two novel classes of dopamine transporter ligands. A series of 3-[2- (diarylmethoxyethyidenyl)]-N-substituted tropane derivatives were synthesized and the binding affinities of these compounds were determined at the dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in rat brain tissue preparations. The tropane derivatives were found to exhibit more potent affinity and selectivity for DAT than GBR 12909. From the SAR of the tropane analogues and GBR 12909, a novel series of 3-[2-(diarylmethoxyethylidenyl)]-Nsubstituted azetidine derivatives has been developed

Exploring The Ligand Binding Site On The Dopamine Transporter By Photoaffinity Labeling And Site-Directed Mutagenesis

The dopamine transporter (DAT) is a neuronal presynaptic transmembrane protein that clears released dopamine (DA) from the synaptic space, regulating the neurotransmitter concentration and availability, DAT and the related serotonin (SERT) and norepinephrine (NET) transporters belong to the SLC6 family of Na+ and CI- dependent symporters, and are major targets for the action of several drugs, including the psychostimulant cocaine. DAT is predicted to possess 12 transmembrane spanning domains (TMs), with both N- and C-termini located intracellularly. Extensive research efforts to gain insight on the molecular aspects of DAT have been attempted, but the three-dimensional arrangement of the protein as well as the molecular mechanisms involved in substrate translocation remain undiscovered. Cocaine and other structurally diverse compounds bind to DAT with high affinity and inhibit its transport activity, but their binding site within the protein and the mechanism by which they block DA transport remain unclear. Two distinct ligand interaction sites at transmembrane domains (TMs) 1-2 and 4-6 have been identified using irreversible uptake-blockers. The current studies explore the incorporation site of the novel cocaine analogue [125I]MFZ-2-24, which is structurally similar to the previously characterized [125 I]RTI 82, but the reactive azido (N3) group is differentially positioned within the cocaine pharmacophore. Trypsin and cyanogen bromide (CNBr) proteolytic maps of [125I]MFZ-2-24 labeled rat and human DATs, followed by epitope-specific immunoprecipitation were used to localize the incorporation site of the ligand to a 13 amino acid stretch in TM1, between residues I67 and L80. This highly conserved region harbors the functionally essential D79 and residues involved in substrate and inhibitor binding. In marked contrast, incorporation of [125I]RTI 82 occurs in TM6, demonstrating that differential positioning of the N 3 group on the cocaine pharmacophore leads to distinct incorporation patterns. This further indicates that TMs 1 and 6 are in dose proximity three dimensionally and participate in the reversible binding of cocaine to DAT

Understanding Addiction, Depression, And Autism Spectrum Disorder Through Structure-Function Analyses Of The Dopamine And Serotonin Transporters

The dopamine (DAT) and serotonin (SERT) transporters are monoamine neurotransmitter transporters (MATs) responsible for the reuptake of dopamine (DA) or serotonin (5-HT) from the synapse following vesicular release, effectively regulating synaptic neurotransmission. Blockade of these transporters by antagonists such as psychostimulant drugs or transporter mutations that affect function can compromise DA or 5-HT homeostasis and impact fundamental brain processes including movement, emotion, behavior, motivation, and memory. To address these issues, my dissertation research focused on: (1) identifying and characterizing the cocaine binding site in DAT, (2) determining the influence of membrane depolarization on DAT trafficking, (3) investigating antidepressant metabolite action in an antidepressant-insensitive mouse model, and (4) understanding the link between the structural and functional changes induced by mutations in SERT associated with autism spectrum disorder. Cocaine binding site in DAT – Through the development and utilization of several high affinity, photoactivatable cocaine ligands we identified a cocaine binding site in the core of DAT, a site that overlaps with the putative DA binding site, supporting a competitive mechanism for cocaine inhibition of DA uptake. (Chapters II-IV) Influence of membrane depolarization on DAT trafficking – Trafficking of mature DAT to and from the cell membrane is a highly regulated process. Within this process, we demonstrated that membrane depolarization alone could induce a CaMKIIα- and dynamin-dependent rapid reversible reduction in membrane DAT. (Chapter V) Characterization of antidepressant metabolites in the blockade of 5-HT reuptake – As studies of the antidepressant selective serotonin reuptake inhibitors (SSRIs) have revealed discrepancies between acute and chronic dosing treatments, we evaluated the sensitivity of SSRI metabolites in a mouse model of depression and identified a role for metabolites in antidepressant administration that may confound study conclusions. (Chapter VI) Autism-associated SERT mutations – Previously, several rare SERT coding variants were identified in humans with autism spectrum disorder that augment 5-HT transport function. We studied the structure of these variants and discovered alterations in SERT tertiary structure, which likely impact the catalytic activity or surface expression of SERT. (Chapter VII

Computational techniques to illuminate secrets of the monoamine transporters

The solute carrier family regulates the flow of various substances such as drugs, amino acids, sugars and inorganic ions across the cell membrane. In particular, the monoamine transporters (MATs) are responsible for the regulation of synaptic neurotransmitter levels. Their structures and conformational rearrangements associated with transport remain unsolved. We employed novel computational techniques to identify the binding pocket of cocaine in the dopamine transporter (DAT) and two intracellular pathways for substrate egress in LeuT and DAT. We review possible coarse grained molecular dynamics techniques to extend the temporal scale accessible in simulations of the MATs. Finally, we present the first computational study of DAT in the presence of an explicit electrochemical gradient. In these simulations, we identify a water wire in DAT that may suggest that DAT does not follow an alternating access mechanism