title: The binding orientations of structurally-related ligands can differ; A cautionary note.
abstract: Crystal structures can identify ligand-receptor interactions and assist the development of novel therapeutics, but experimental challenges sometimes necessitate the use of homologous proteins. Tropisetron is an orthosteric ligand at both 5-HT3and α7 nACh receptors and its binding orientation has been determined in the structural homologue AChBP (pdbid: 2WNC). Co-crystallisation with a structurally-related ligand, granisetron, reveals an almost identical orientation (pdbid; 2YME). However, there is a >1000-fold difference in the affinity of tropisetron at 5-HT3versus α7 nACh receptors, and α7 nACh receptors do not bind granisetron. These striking pharmacological differences prompt questions about which receptor the crystal structures most closely represent and whether the ligand orientations are correct. Here we probe the binding orientation of tropisetron and granisetron at 5-HT3receptors by in silico modelling and docking, radioligand binding on cysteine-substituted 5-HT3receptor mutants transiently expressed in HEK 293 cells, and synthetic modification of the ligands. For 15 of the 23 cysteine substitutions, the effects on tropisetron and granisetron were different. Structure-activity relationships on synthesised derivatives of both ligands were also consistent with different orientations, revealing that contrary to the crystallographic evidence from AChBP, the two ligands adopt different orientations in the 5-HT3receptor binding site. Our results show that even quite structurally similar molecules can adopt different orientations in the same binding site, and that caution may be needed when using homologous proteins to predict ligand binding.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Marc-David
authors.initials: MD
authors.lastname: Ruepp
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK.
authors.forename: Hao
authors.initials: H
authors.lastname: Wei
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland.
authors.forename: Michele
authors.initials: M
authors.lastname: Leuenberger
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland; Institute of Biochemistry and Molecular Medicine, University of Bern, Bern, Switzerland. Electronic address: martin.lochner@ibmm.unibe.ch.
authors.forename: Martin
authors.initials: M
authors.lastname: Lochner
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, UK. Electronic address: ajt44@cam.ac.uk.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
mesh_terms: D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects* / Q000235:genetics; D018360:Crystallography, X-Ray; D017829:Granisetron / Q000737:chemistry / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000737:chemistry / Q000494:pharmacology; D008024:Ligands; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics*; D012702:Serotonin Antagonists / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014316:Tritium / Q000494:pharmacology; D000077526:Tropisetron
chemical_list: D017829:Granisetron; D007211:Indoles; D008024:Ligands; D044406:Receptors, Serotonin, 5-HT3; D012702:Serotonin Antagonists; D014316:Tritium; D000077526:Tropisetron
keywords: 5-HT(3); Agonist; Antagonist; Binding; Crystal; Cys-loop; Granisetron; Ion channel; Ligand; Radioligand; Receptor; Structure; Synthesis; Tropisetron