Interactions of Granisetron with an Agonist-Free 5-HT_3A Receptor Model

Abstract

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.

The binding orientations of structurally-related ligands can differ; A cautionary note

“…For H185 a substitution to cysteine would also conserve its side-chain interactions as both residues are capable of hydrogen bonding. However, it was previously reported that mutations at this location reduce cell-surface expression, and the lower B max value we measured for granisetron (H185C = 123 ± 23 fmol mg −1 ; wild type = 4843 ± 512 fmol mg −1 ; paired samples, n  = 4) is consistent with this (Joshi et al., 2006, Thompson et al., 2008). For tropisetron it is therefore possible that the absence of binding does not reveal interactions but, given the higher levels of non-specific binding, instead reveals expression levels that are below the threshold for detection.…”

Synthesis and Receptor Binding of New Thieno[2,3‐d]‐pyrimidines as Selective Ligands of 5‐HT3 Receptors

“…How can we decide which docking position is relevant in binding? The docking vicinity of the distal piperazinyl tertiary amino group of compounds 2 and 32 to N128 is in agreement with the docking vicinity of the analogous granatane amino group of granisetron, a 5-HT 3 R antagonist (K i = 0.20 nM) [30], to N128 [27]. Overlapping docking positions of active and inactive derivatives were considered artefacts, while the similar dockings of the active ones support similar binding modes associated with high affinity.…”

“…This was based on the building of a homology model of 3A -type 5-HT receptors (5-HT 3A Rs), obtained from the electron microscopic structure of the nACh receptor with agonist-free binding cavities at 4 resolution [27]. This was based on the building of a homology model of 3A -type 5-HT receptors (5-HT 3A Rs), obtained from the electron microscopic structure of the nACh receptor with agonist-free binding cavities at 4 resolution [27].…”

“…The homology model of 5-HT 3A Rs based on the structure of nACh receptors with an agonist-free (empty) binding cavity [27] is an improvement compared to previous models involving an acetylcholine-binding protein [28] where, according to an emerging consensus, bound agonists "pull in" loop C to close the binding cavity. In contrast, the open binding cavity can accommodate 5-HT 3 RAs in more docking positions [27].…”

Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

“…Docking studies using homology models have found multiple energetically favorable poses of the antagonist granisetron within this binding site, and the orientation of the drug and the identities of the interacting residues are not constant across poses. 11–14 For example, Thompson et. al.…”