title: Binding characteristics of GYKI-46 903, a non-competitive ligand at 5-HT3 receptors.
abstract: GYKI-46903 [(+)-(5S,6R)-4-(4-fluorophenyl)-6-propionyloxy-1-aza-bicyclo[3.3.1]-non-3-ene-hydrochloride], a cognition enhancer identified as a non-competitive antagonist of 5-HT3receptors in isolated guinea-pig ileum, was investigated for allosteric action at 5-HT3 receptors in rat cortical membranes by using [3H]granisetron. Equilibrium and kinetic protocols were applied and the competitive antagonist granisetron was included as a negative control. In competition studies, both granisetron and GYKI-46 903 displaced the radioligand with K(i) values of 0.20 +/- 0.02 and 79.84 +/- 0.28 nM, respectively. The inhibition curve for GYKI-46 903 resulted in a Hill slope significantly greater than unity ( 1.37 +/- 0.11), whereas the slope for granisetron was 0.88 +/- 0.08, not different from unity. These results indicate non-competitive and competitive interactions, respectively. Scatchard analysis yielded a linear plot, suggesting a single population of binding sites with a Kd of 0.13 +/- 0.01 nM and a Bmax) of 13.15 +/- 0.34 fmol per mg of protein. Scatchard plots obtained in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-1000 nM) revealed a concentration-dependent increase in Kd values by either of these compounds. Granisetron left the Bmax unchanged, but there was a significant increase in the Bmax by GYKI-46 903, which could point to an atypical allosteric interaction. The Schild plot derived from the Kd shifts induced by granisetron was linear with a slope of 1.02, not different from unity, as expected from a competitive interaction. The Schild regression for GYKI-46 903 was linear with a slope of 1.20, deviating significantly from unity, which may also indicate an allosteric interaction. Both the association and dissociation curves of [3H]granisetron were monoexponential. The dissociation rate constant (K(-1)) and the association rate constant (K(+1)) were 0.32 +/- 0.01 min(-1) and 1.15 min(-1) x nM(-1), respectively. The dissociation driven by an excess concentration of ondansetron ( 1 microM) in the absence and presence of granisetron (0.1-3 nM) or GYKI-46 903 (30-10 000 nM) was not influenced by the compounds under study, as compared with the control, indicating the lack of an allosteric effect on the dissociation. Summing up, the binding profile of GYKI-46 903 may reflect a mixed type of action, including a negative allosteric interaction.
authors.affiliation: Institute for Drug Research Ltd., Budapest, Hungary .
authors.forename: B
authors.initials: B
authors.lastname: Vitális
authors.forename: L
authors.initials: L
authors.lastname: Sebestyén
authors.forename: M
authors.initials: M
authors.lastname: Sike
authors.forename: S
authors.initials: S
authors.lastname: Sólyom
authors.forename: L G
authors.initials: LG
authors.lastname: Hársing
mesh_terms: D000495:Allosteric Site; D000818:Animals; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D017829:Granisetron / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000187:drug effects / Q000378:metabolism*; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists / Q000494:pharmacology*; D013997:Time Factors; D014316:Tritium
chemical_list: D019086:Bridged Bicyclo Compounds, Heterocyclic; D017829:Granisetron; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists; D014316:Tritium; C103284:GYKI 46903