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UNLABELLED - Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development.
IMPORTANCE - Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Quantum dot conjugates of compounds capable of inhibiting the serotonin transporter (SERT) could form the basis of fluorescent probes for live cell imaging of membrane bound SERT. Additionally, quantum dot-SERT antagonist conjugates may be amenable to fluorescence-based, high-throughput assays for this transporter. This Letter describes the synthesis of SERT-selective ligands amenable to conjugation to quantum dots via a biotin-streptavidin binding interaction. SERT selectivity and affinity were incorporated into the ligand via a tetrahydropyridine or cyclohexylamine derivative and the affinity of these compounds for SERT was measured by their ability to produce SERT-dependent currents in Xenopus laveis oocytes.
Copyright © 2011 Elsevier Ltd. All rights reserved.
To identify potential determinants of substrate selectivity in serotonin (5-HT) transporters (SERT), models of human and Drosophila serotonin transporters (hSERT, dSERT) were built based on the leucine transporter (LeuT(Aa)) structure reported by Yamashita et al. (Nature 2005;437:215-223), PBDID 2A65. Although the overall amino acid identity between SERTs and the LeuT(Aa) is only 17%, it increases to above 50% in the first shell of the putative 5-HT binding site, allowing de novo computational docking of tryptamine derivatives in atomic detail. Comparison of hSERT and dSERT complexed with substrates pinpoints likely structural determinants for substrate binding. Forgoing the use of experimental transport and binding data of tryptamine derivatives for construction of these models enables us to critically assess and validate their predictive power: A single 5-HT binding mode was identified that retains the amine placement observed in the LeuT(Aa) structure, matches site-directed mutagenesis and substituted cysteine accessibility method (SCAM) data, complies with support vector machine derived relations activity relations, and predicts computational binding energies for 5-HT analogs with a significant correlation coefficient (R = 0.72). This binding mode places 5-HT deep in the binding pocket of the SERT with the 5-position near residue hSERT A169/dSERT D164 in transmembrane helix 3, the indole nitrogen next to residue Y176/Y171, and the ethylamine tail under residues F335/F327 and S336/S328 within 4 A of residue D98. Our studies identify a number of potential contacts whose contribution to substrate binding and transport was previously unsuspected.
(c) 2008 Wiley-Liss, Inc.
The serotonin (5-hydroxytryptamine, 5-HT) transporter (SERT) is responsible for the inactivation of synaptic 5-HT and is also a target for multiple psychostimulants. Despite the critical role of SERT in 5-HT inactivation and psychostimulant response, many aspects of the transporter's recognition of ligands are poorly defined. We took advantage of sequence divergence of SERT species variants to identify structural determinants of substrate recognition. Tryptamine derivatives with substitutions at the 4 and 7 positions on the phenyl ring, the indole nitrogen, and the beta position show up to 40-fold potency differences for inhibiting [(3)H]5-HT transport in cells transfected with either human or Drosophila melanogaster SERT cDNAs. Species selectivities of these derivatives were largely recapitulated in antagonist binding. Human/D. melanogaster SERT chimera studies implicated the first two SERT transmembrane domains (TMDs) in the potency of the indole nitrogen-substituted compounds N-isopropyltryptamine (NIT), 5-methoxy-N-isopropyltryptamine (5-MNIT), and the 7-substituted compound 7-benzyloxytryptamine (7BT). Potency differences of analogs with substitutions at the 4 and beta positions are influenced by sequences distal to this region. Within TMD I-II, species-scanning mutagenesis implicated a single residue (Y95 in human SERT, F90 in D. melanogaster SERT) in the recognition of NIT, 5-MNIT, and 7BT. Remarkably, this is the same site we established previously in species-specific recognition of the antagonists citalopram and mazindol. These findings support a critical role for TMD I residues in defining shared aspects of SERT substrate and antagonist recognition.