The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors

Abstract

VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [3H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [3H]VUF10166 displayed saturable binding with a Kd of 0.18 nM. Kinetic measurements gave monophasic association (6.25 × 107 M−1 min−1) and dissociation (0.01 min−1) rates that yielded a similar Kd value (0.16 nM). At 5-HT3AB receptors two association (6.15 × 10−7, 7.23 M−1 min−1) and dissociation (0.024, 0.162 min−1) rates were seen, yielding Kd values (0.38 nM and 22 nM) that were consistent with values obtained in saturation (Kd = 0.74 nM) and competition (Ki = 37 nM) binding experiments respectively. At both receptor types, specific binding was inhibited by classical 5-HT3 receptor-selective orthosteric ligands (5-HT, allosetron, d-tubocurarine, granisetron, mCPBG, MDL72222, quipazine), but not by non-competitive antagonists (bilobalide, ginkgolide B, picrotoxin) or competitive ligands of other Cys-loop receptors (ACh, bicuculline, glycine, gabazine). To explore VUF10166 ligand–receptor interactions we used in silico modelling and docking, and tested the predictions using site directed mutagenesis. The data suggest that VUF10166 adopts a similar orientation to 5-HT3 receptor agonists bound in AChBP (varenicline) and 5HTBP (5-HT) crystal structures.

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

“…As a similar side chain orientation is seen in 4PIR, ligand interactions with this residue are also likely in the 5-HT 3 receptor binding site and are supported by the large effects we see for both ligands (Kesters et al., 2013). Substitution of another aromatic residue, W90 in loop D, also abolished the binding of both tropisetron and granisetron, similar to reports for other 5-HT 3 receptor ligands (Thompson et al., 2014, Venkataraman et al., 2002, Yan et al., 1999). Again, the aromatic character of W90 is important as removal of the aromatic ring (W90A, W90S) eliminates granisetron binding, but conservative substitutions (W90Y) have reduced effects (Price and Lummis, 2004, Spier and Lummis, 2000, Thompson et al., 2005, Yan and White, 2005).…”

“…From the crystal structure 4PIR it is clear that a hydrogen bond between E129 and T179 is structural (Hassaine et al., 2014). Such an interaction is supported by our finding that E129C abolishes binding of both ligands and by other reports that describe similar effects on granisetron, GR65630 and VUF10166 (Boess et al., 1997, Price et al., 2008, Thompson et al., 2005, Thompson et al., 2014). For T179C the effects on the two ligands were not as pronounced, but as both threonine and cysteine can act as hydrogen bond donors this property may be retained following substitution; consistent with this, substitution to serine also preserves granisetron binding (Thompson et al., 2005).…”

“…In previous work, a similar absence of effects on granisetron has also been reported for range of other substitutions at this location (Price et al., 2008, Sullivan et al., 2006). However, the effects of N128C we measured on the affinity of tropisetron (7-fold) and previous reports of changes to the EC 50 of 5-HT, m CPBG and the binding affinity of VUF10166 suggests that effects may depend upon the ligand studied (Price et al., 2008, Sullivan et al., 2006, Thompson et al., 2014). In P4IR, N128 lies close to L126 on loop A, and E236 in loop C, none of which caused significant effects on granisetron binding.…”

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

“…The role of these residues is extensively studied in the 5-HT 3 receptor (Beene et al, 2002a(Beene et al, ,b, 2004Price et al, 2008), and multiple models exist for their role in binding of setrons and agonists. Early models, and more recently also X-ray structures of 5-HTBP in complex with setrons, collectively show three main factors necessary for setron binding and effect: 1) hydrogen bonds and cation/p interactions with Trp178 in the aromatic box (Kesters et al, 2013;Lochner and Thompson, 2016;Price et al, 2016); 2) cation/p and/or p/p interactions with Tyr136, Tyr138, Tyr148, and Arg87 (Beene et al, 2004;Thompson et al, 2005Thompson et al, , 2014Yan and White, 2005;Joshi et al, 2006); and 3) setron occupation of a subsite beyond Arg87 near loops D and F (Fig. 8) (Kesters et al, 2013;Lochner and Thompson, 2016;Price et al, 2016).…”

The binding orientation of epibatidine at α7 nACh receptors

“…High-affinity epibatidine binding has also been reported at 5-HT 3 receptors, similar to the binding of ACh, nicotine, tropisetron and d -tubocurarine at the two receptor types (ChavezNoriega et al., 1997, Drisdel et al., 2008, Thompson et al., 2014). In light of this we also measured binding at the 5-HT 3 receptor to establish whether epibatidine could have utility for studying this other member of the Cys-loop receptor family.…”