Structural basis of norepinephrine recognition and transport inhibition in neurotransmitter transporters (original) (raw)
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Frontiers in Pharmacology, 2020
The human norepinephrine transporter (hNET) is a member of the neurotransmitter/sodium symporter family, which also includes the neuronal monoamine transporters for serotonin (SERT) and dopamine (DAT). Its involvement in chronic pain and many neurological disorders underlies its pharmaceutical importance. Using the X-ray crystal structures of the human serotonin transporter (hSERT) (PDB 5I6X) and Drosophila melanogaster dopamine transporter (dDAT) (PDB 4M48 and PDB 4XPA) as templates, we developed molecular models for norepinephrine (NE) bound to its high affinity binding site (S1) in the hNET. Our model suggests that the S1 site for NE is deeply buried between transmembrane helices (TMHs) 1, 3, 6, and 8 and overlaps the binding site for leucine in the bacterial leucine transporter (LeuT) and dopamine (DA) in dDAT. Mutational studies identified the functional binding pocket for NE comprised residues A73, A77, N78, V148, N153, I156, G320, F329, N350, S420, G423, and M424, which all influenced NE affinity and/or transport. These effects support a NE-hNET docking model where A73, A77, G320, S420, G423, and M424 form H-bond interactions with NE, V148, I156, and F329 form hydrophobic interactions with NE, whereas N78 affects NE transport and N350 affects NE affinity and transport via an influence on the octahedral coordination of the Na 1 + ion. Consistent with a conserved structure-function amongst sodium-dependent neurotransmitter transporters, S1 residues A73, A77 (G100 in hSERT), N78, V148 (I150 in hSERT), N153, G320, F329 (Y331 in d DAT), N350, and G423 are conserved in DAT and SERT, indicating they likely play conserved functional roles.
A short update on the structure of drug binding sites on neurotransmitter transporters
BMC Research Notes, 2011
Background: The dopamine (DAT), noradrenalin (NET) and serotonin (SERT) transporters are molecular targets for different classes of psychotropic drugs. Cocaine and the SSRI (S)-citalopram block neurotransmitter reuptake competitively, but while cocaine is a non-selective reuptake inhibitor, (S)-citalopram is a selective SERT inhibitor. Findings: Here we present comparisons of the binding sites and the electrostatic potential surfaces (EPS) of DAT, NET and SERT homology models based on two different LeuT Aa templates; with a substrate (leucine) in an occluded conformation (PDB id 2a65), and with an inhibitor (tryptophan) in an open-to-out conformation (PDB id 3f3a). In the occluded homology models, two conserved aromatic amino acids (tyrosine and phenylalanine) formed a gate between the putative binding pockets, and this contact was interrupted in the open to out conformation. The EPS of DAT and NET were generally negative in the vestibular area, whereas the EPS of the vestibular area of SERT was more neutral. Conclusions: The findings presented here contribute as an update on the structure of the binding sites of DAT, NET and SERT. The updated models, which have larger ligand binding site areas than models based on other templates, may serve as improved tools for virtual ligand screening.
ACS Chemical Neuroscience, 2019
16 Octopamine, a trace amine in mammals, is a major neurotransmitter linked to important 17 biological processes in insects. Interestingly, one of the molecular entities responsible for 18 octopamine availability, the octopamine transporter (OAT), has not been identified in 19 certain insect species. For instance, no OAT has been reported in the fly Drosophila 20 melanogaster (Dm), and the molecule involved in octopamine reuptake in Drosophila is not 21 known. Here, we used molecular modeling methodologies to obtain three-dimensional 22 insights for the dopamine transporter (DAT) and OAT in a common agricultural pest insect, 23 Trichoplusia ni (Tni). Our results show several similarities but also significant differences in 24 the general structures of the proteins of Dm and Tni. One important difference is observed 25 in the ligand binding cavity, where a negatively charged amino acid present in both 26 dopamine transporters is replaced by a polar neutral residue in the Trichoplusia OAT. This 27 modification could influence both the binding mode and the driving force involved in the 28 transport mechanism of these amines into neurons of these species. We also obtained data 29 that support the idea that octopamine could bind and possibly be transported by DmDAT. 30 The structural characterization of macromolecules from different insect species is 31 fundamental in the agricultural field to gain insights into the design of new compounds for 32 controlling pests.
PLoS ONE, 2011
Background: The dopamine transporter (DAT), a member of the neurotransmitter:Na + symporter (NSS) family, terminates dopaminergic neurotransmission and is a major molecular target for psychostimulants such as cocaine and amphetamine, and for the treatment of attention deficit disorder and depression. The crystal structures of the prokaryotic NSS homolog of DAT, the leucine transporter LeuT, have provided critical structural insights about the occluded and outward-facing conformations visited during the substrate transport, but only limited clues regarding mechanism. To understand the transport mechanism in DAT we have used a homology model based on the LeuT structure in a computational protocol validated previously for LeuT, in which steered molecular dynamics (SMD) simulations guide the substrate along a pathway leading from the extracellular end to the intracellular (cytoplasmic) end.
Proceedings of the National Academy of Sciences, 2011
The norepinephrine transporter (NET) transports norepinephrine from the synapse into presynaptic neurons, where norepinephrine regulates signaling pathways associated with cardiovascular effects and behavioral traits via binding to various receptors (e.g., β2-adrenergic receptor). NET is a known target for a variety of prescription drugs, including antidepressants and psychostimulants, and may mediate off-target effects of other prescription drugs. Here, we identify prescription drugs that bind NET, using virtual ligand screening followed by experimental validation of predicted ligands. We began by constructing a comparative structural model of NET based on its alignment to the atomic structure of a prokaryotic NET homolog, the leucine transporter LeuT. The modeled binding site was validated by confirming that known NET ligands can be docked favorably compared to nonbinding molecules. We then computationally screened 6,436 drugs from the Kyoto Encyclopedia of Genes and Genomes (KEGG DRUG) against the NET model. Ten of the 18 high-scoring drugs tested experimentally were found to be NET inhibitors; five of these were chemically novel ligands of NET. These results may rationalize the efficacy of several sympathetic (tuaminoheptane) and antidepressant (tranylcypromine) drugs, as well as side effects of diabetes (phenformin) and Alzheimer's (talsaclidine) drugs. The observations highlight the utility of virtual screening against a comparative model, even when the target shares less than 30% sequence identity with its template structure and no known ligands in the primary binding site.
Journal of Biological Chemistry, 2016
Folding-defective mutants of the human dopamine transporter (DAT) cause a syndrome of infantile dystonia/parkinsonism. Here, we provide a proof-of-principle that the folding deficit is amenable to correction in vivo by two means, the cognate DAT ligand noribogaine and the HSP70 inhibitor, pifithrin-. We examined the Drosophila melanogaster (d) mutant dDAT-G108Q, which leads to a sleepless phenotype in flies harboring this mutation. Molecular dynamics simulations suggested an unstable structure of dDAT-G108Q consistent with a folding defect. This conjecture was verified; heterologously expressed dDAT-G108Q and the human (h) equivalent hDAT-G140Q were retained in the endoplasmic reticulum in a complex with endogenous folding sensors (calnexin and HSP70-1A). Incubation of the cells with noribogaine (a DAT ligand selective for the inward-facing state) and/or pifithrin-(an HSP70 inhibitor) restored folding of, and hence dopamine transport by, dDAT-G108Q and hDAT-G140Q. The mutated versions of DAT were confined to the cell bodies of the dopaminergic neurons in the fly brain and failed to reach the axonal compartments. Axonal delivery was restored, and sleep time was increased to normal length (from 300 to 1000 min/day) if the dDAT-G108Q-expressing flies were treated with noribogaine and/or pifithrin-. Rescuing misfolded versions of DAT by pharmacochaperoning is of therapeutic interest; it may provide opportunities to remedy disorders arising from folding-defective mutants of human DAT and of other related SLC6 transporters.
Drug and alcohol dependence, 2015
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. 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. 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...
Structure–activity relationships for substrate recognition by the human dopamine transporter
Biochemical Pharmacology, 2004
Information is available on the structure-activity relationships for dopamine as a substrate for uptake by the dopamine transporter. However, dopamine transport is a complex process involving substrate binding, translocation, release as well as transporter reorientation. The present study examines only the substrate recognition step by assessment of the potency of various dopamine-related compounds in inhibiting the binding of the cocaine analog [ 3 H]2b-carbomethoxy-3b-(4-fluorophenyl)tropane ([ 3 H]WIN 35,428) to human dopamine transporters expressed in HEK-293 cells. a-Methylation of the side chain, the presence of the amine, and the 2-carbon-length of the side chain were found to be important for binding affinity, whereas b-hydroxylation of the side chain and methoxylation at the phenyl ring generated weaker compounds. In addition, the presence of both m-and p-OH at the phenyl ring bestowed an increase in potency but the presence of p-OH alone a decrease. N-alkylation (propylation or methylation) had little or an even slightly beneficial effect on affinity, whereas a-carbonylation and a-methanoylation reduced affinity. Amino naphthalene compounds with a fused benzenoid ring system retained some potency consonant with the extended (i.e. b-rotameric) trans (¼anti) form of the side chain in dopamine when interacting with the transporter. In a second series of experiments, the interaction between dopamine and structural variants was assessed by monitoring the capability of a compound to shift the dopamine inhibition curve to the right as expected for a competitive inhibitor acting at the same site. Appreciable deviation from competitive interaction was observed by removal of the amine from the side chain, by acarbonylation, and by a-methanoylation. Two blocker-type compounds, semi-rigid variants of cocaine, also displayed significant deviation. A substrate-based compound, inhibiting cocaine analog binding without interfering with dopamine recognition, could be a cocaine antagonist allowing conformational changes to occur during dopamine uptake. #