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-HT3 and 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-HT3 versus 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-HT3 receptors by in silico modelling and docking, radioligand binding on cysteine-substituted 5-HT3 receptor 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-HT3 receptor 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.

High-resolution Structures of multiple 5-HT3AR-setron complexes reveal a novel mechanism of competitive inhibition

“…Our MD simulations predict that Arg65 may have a differential effect on the binding of various setrons. In agreement, mutations at the Arg65 position in human 5-HT3AR abolish granisetron binding but tropisetron binding is only reduced 40 . To further assess the role of Arg65 in binding various setrons, we measured the extent of inhibition of serotonin-induced currents.…”

Modeling and Mutational Analysis of the Binding Mode for the Multimodal Antidepressant Drug Vortioxetine to the Human 5-HT3A Receptor

“…In the present study, we have studied the molecular basis of vortioxetine inhibition of human 5-HT 3A (h5-HT 3A ) receptors by constructing a structural model of h5-HT 3A . This model is able to accurately predict the established binding mode of granisetron (Kesters et al, 2013;Ruepp et al, 2017), and was used for docking vortioxetine at the orthosteric binding site. We used pharmacological characterization of mutant receptors to identify and validate a model for vortioxetine in its bioactive conformation bound to the receptor.…”

Mapping the Orthosteric Binding Site of the Human 5-HT3 Receptor Using Photo-cross-linking Antagonists

“…Based upon the experimental findings that 4 and 10 competitively displace [ 3 H]granisetron and crosslink with Met-228 of loop C we sought a computational approach to predict possible binding orientations of the two probes in the orthosteric 5-HT 3 receptor binding site. We used the high-resolution 5HTBP structure (PDB ID: 2YME) as docking template as (i) it has been mutated to mimic the human 5-HT 3 receptor binding site, (ii) the orientation of granisetron in 5HTBP is known to correctly represent the orientation found in the native 5-HT 3 receptor 16 and, (iii) it was co-crystallized with granisetron which is the same recognition moiety of our probes. For the purpose of validation, granisetron was docked (FRED v2.1) into this binding site model and its predicted orientation was found to be similar to the binding pose in the crystal structure ( Figure S6a).…”

“…Ultimately, probes 4 and 10 are photo-crosslinking conjugates of granisetron and caution may be needed when predicting binding orientations for other ligands from the poses that are shown. 16 a b with evidence from mutagenesis and the pharmacophore model for 5-HT 3 receptor ligands. 14,16,47 The -17-peptide chain of the principal face (left) is shown in white and of the complementary face (right) in gray.…”