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J. Biol. Chem., Vol. 280, Issue 21, 20476-20482, May 27, 2005

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Locating an Antagonist in the 5-HT₃ Receptor Binding Site Using Modeling and Radioligand Binding^*

Andrew J. Thompson, Kerry L. Price, David C. Reeves, S. Ling Chan¶, P.-L. Chau||**, and Sarah C. R. Lummis

From the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom, the Albert Einstein College of Medicine, New York, New York 10461, ^¶Chemical Computing Group, Inc., Montreal H3A 2R7, Canada, the ^||Bioinformatique Structurale, Institut Pasteur, Paris, France, and the Neurobiology Division, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom

We have used a homology model of the extracellular domain of the 5-HT₃ receptor to dock granisetron, a 5-HT₃ receptor antagonist, into the binding site using AUTODOCK. This yielded 13 alternative energetically favorable models. The models fell into 3 groups. In model type A the aromatic rings of granisetron were between Trp-90 and Phe-226 and its azabicyclic ring was between Trp-183 and Tyr-234, in model type B this orientation was reversed, and in model type C the aromatic rings were between Asp-229 and Ser-200 and the azabicyclic ring was between Phe-226 and Asn-128. Residues located no more than 5 Å from the docked granisetron were identified for each model; of 26 residues identified, 8 were found to be common to all models, with 18 others being represented in only a subset of the models. To identify which of the docking models best represents the ligand-receptor complex, we substituted each of these 26 residues with alanine and a residue with similar chemical properties. The mutant receptors were expressed in human embryonic kidney (HEK)293 cells and the affinity of granisetron determined using radioligand binding. Mutation of 2 residues (Trp-183 and Glu-129) ablated binding, whereas mutation of 14 other residues caused changes in the [³H]granisetron binding affinity in one or both mutant receptors. The data showed that residues both in and close to the binding pocket can affect antagonist binding and overall were found to best support model B.

Received for publication, December 3, 2004 , and in revised form, March 3, 2005.

^* This work was supported by the Wellcome Trust (to S. C. R. L. and D. C. R.), the BBSRC (to P.-L. C.), the MRC (to K. L. P.), the Royal Society (to S. C. R. L. and P.-L. C.), and New Hall, Cambridge (to P.-L. C.) for funding. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** Held a David Phillips Research Fellowship and Senior Research Fellowship at New Hall, Cambridge.

A Wellcome Trust Senior Research Fellow in Basic Biomedical Studies. To whom correspondence should be addressed: Dept. of Biochemistry, Tennis Court Rd., Cambridge CB2 1QW, United Kingdom. Tel.: 44-1223-765950; Fax: 44-1223-333345; E-mail: sl120{at}cam.ac.uk.

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