title: Functional human 5-HT6 receptor assay for high throughput screening of chemical ligands and binding proteins.
abstract: Continuous identification and validation of novel drug targets require the development of rapid, reliable, and sensitive cell-based high-throughput screening (HTS) methods for proposed targets. Recently, the 5-HT(6) receptor (5-HT(6)R), a member of the class of recently discovered 5-HT receptors, has received considerable attention for its possible implications in depression, cognition, and anxiety. However, the cellular signaling mechanisms of 5-HT(6)R are poorly understood due to the lack of selective 5-HT(6)R ligands. In the present study, we examined functional coupling of the human 5-HT(6)R, 5-HT(7A)R, or 5-HT(7B)R with various Galpha-proteins (Galpha(15), Galpha(qs5), or Galpha(qG66Ds5)) to develop a reliable cell-based HTS method for 5-HT receptors. Among variable couplings between 5-HT receptors and G-proteins, we found that functional coupling of human 5-HT(6)R with Galpha(qG66Ds5) produced the highest levels of Ca(2+) signaling in HEK293 cells as measured by the fluorescence-based HTS plate reader, FDSS6000. After validation of this new 5-HT(6)R HTS system (Z'-factor = 0.56) in 96-well plates and characterization of the pharmacological profile of the 5-HT(6)R, we screened approximately 500 synthetic chemical compounds including butanamide and benzenesulfonamide derivatives. Based on this preliminary screening, we found that the butanamide derivative LSG11104 produced an IC(50) value of 6.3 microM. This compound will serve as a lead structure for further chemical modification to develop novel 5-HT(6)R ligands. Furthermore, we demonstrated that this HTS method can be utilized to identify proteins that modulate 5-HT(6)R function and present Fyn tyrosine kinase as an example, which is already known as a 5-HT(6)R interacting protein. Taken together, these results suggest that the 5-HT(6)R/Galpha(qG66Ds5) FDSS6000 system can be utilized to screen for selective 5-HT(6)R ligands and to examine any functional relationships between 5-HT(6)R and its binding proteins.
authors.affiliation: Life Sciences Division, Korea Institute of Science and Technology (KIST), Seoul 136-791, Korea.
authors.forename: Hyun-Ji
authors.initials: HJ
authors.lastname: Kim
authors.forename: Hyung-Mun
authors.initials: HM
authors.lastname: Yun
authors.forename: Taehyun
authors.initials: T
authors.lastname: Kim
authors.forename: Ghilsoo
authors.initials: G
authors.lastname: Nam
authors.forename: Eun Joo
authors.initials: EJ
authors.lastname: Roh
authors.forename: Evi
authors.initials: E
authors.lastname: Kostenis
authors.forename: Hea-Young Park
authors.initials: HY
authors.lastname: Choo
authors.forename: Ae Nim
authors.initials: AN
authors.lastname: Pae
authors.forename: Hyewhon
authors.initials: H
authors.lastname: Rhim
mesh_terms: D008735:5-Methoxytryptamine / Q000494:pharmacology; D000814:Aniline Compounds / Q000737:chemistry; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D002460:Cell Line; D003024:Clozapine / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D044385:GTP-Binding Protein alpha Subunits / Q000235:genetics / Q000378:metabolism; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D010879:Piperazines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism; D011985:Receptors, Serotonin / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D012701:Serotonin / Q000031:analogs & derivatives / Q000494:pharmacology; D012702:Serotonin Antagonists / Q000737:chemistry / Q000494:pharmacology*; D017366:Serotonin Receptor Agonists / Q000737:chemistry / Q000494:pharmacology*; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013449:Sulfonamides / Q000494:pharmacology; D014162:Transfection; D014363:Tryptamines / Q000494:pharmacology; D014966:Xanthenes / Q000737:chemistry
chemical_list: D000814:Aniline Compounds; C409648:Fluo 4; D044385:GTP-Binding Protein alpha Subunits; D010879:Piperazines; D011985:Receptors, Serotonin; C418570:SB 258585; D012702:Serotonin Antagonists; D017366:Serotonin Receptor Agonists; D054852:Small Molecule Libraries; D013449:Sulfonamides; D014363:Tryptamines; D014966:Xanthenes; C097216:serotonin 6 receptor; C104425:serotonin 7 receptor; D012701:Serotonin; D008735:5-Methoxytryptamine; C030820:tryptamine; C047087:5-carboxamidotryptamine; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; C112631:G protein alpha 16; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D003024:Clozapine; D002118:Calcium