Characterizing new fluorescent tools for studying 5-HT3 receptor pharmacology

Jack, Thomas R.; Simonin, Jonathan; Ruepp, Marc‐David; Thompson, Andrew; Gertsch, Jürg; Lochner, Martin

doi:10.1016/j.neuropharm.2014.11.007

Cited by 17 publications

(34 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…2, Fig. 5) and we have previously noted that attachment of bulky fluorophores via long linkers to each position can be accommodated (Jack et al., 2015, Simonin et al., 2012). These results contrast with those for tropisetron, and unlike tropisetron, are consistent with the orientation in the crystal structure 2YME.…”

Section: Discussionmentioning

confidence: 84%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 84%

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

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a second example, the homopentamer of the serotonin type-3 receptor (5HT3A) was prepared in an identical fashion as the P2X1 receptor samples and the unbinding of a fluorescent analog of granisetron (Gr-SiR) (Jack et al, 2015) was followed by single molecule tracking as described above. The homopentamer 5HT 3A has five identical allosteric binding sites and granisetron is a 5HT 3 specific antagonist.…”

Section: Ligand Binding Dynamics and Stochiometrymentioning

confidence: 99%

“…Instead the elementary kinetic rate constants can be determined by following the fluctuation dynamics of individual membrane proteins (Selvin and Ha, 2008). In this special addition of Neuropharmacology,Ruepp et al (2015) present ensemble measurements of binding of a fluorescent analog of ATP (Alexa-647-ATP, or ATP*) at the purinergic receptor P2X1. Single molecule binding studies provide complimentary information for ATP* binding, revealing a strong binding state at short times.…”

mentioning

confidence: 99%

Tracking individual membrane proteins and their biochemistry: The power of direct observation

et al. 2015

Self Cite

View full text Add to dashboard Cite

The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.

show abstract

“…To further test for orthosteric interactions, we measured the binding of a fluorescent granisetron derivative (G-FL) together with the three compounds using flow cytometry (Jack et al, 2015). Both 5-HT and granisetron reduced G-FL binding in a concentration-dependent manner (Fig.…”

Section: Resultsmentioning

confidence: 99%

Noncompetitive Inhibition of 5-HT3 Receptors by Citral, Linalool, and Eucalyptol Revealed by Nonlinear Mixed-Effects Modeling

Jarvis

Barbosa

Thompson

2015

Journal of Pharmacology and Experimental Therapeutics

View full text Add to dashboard Cite

Citral, eucalyptol, and linalool are widely used as flavorings, fragrances, and cosmetics. Here, we examined their effects on electrophysiological and binding properties of human 5-HT3 receptors expressed in Xenopus oocytes and human embryonic kidney 293 cells, respectively. Data were analyzed using nonlinear mixed-effects modeling to account for random variance in the peak current response between oocytes. The oils caused an insurmountable inhibition of 5‐HT–evoked currents (citral IC50 = 120 µM; eucalyptol = 258 µM; linalool = 141 µM) and did not compete with fluorescently labeled granisetron, suggesting a noncompetitive mechanism of action. Inhibition was not use‐dependent but required a 30-second preapplication. Compound washout caused a slow (∼180 seconds) but complete recovery. Coapplication of the oils with bilobalide or diltiazem indicated they did not bind at the same locations as these channel blockers. Homology modeling and ligand docking predicted binding to a transmembrane cavity at the interface of adjacent subunits. Liquid chromatography coupled to mass spectrometry showed that an essential oil extracted from Lippia alba contained 75.9% citral. This inhibited expressed 5‐HT3 receptors (IC50 = 45 µg ml−1) and smooth muscle contractions in rat trachea (IC50 = 200 µg ml−1) and guinea pig ileum (IC50 = 20 µg ml−1), providing a possible mechanistic explanation for why this oil has been used to treat gastrointestinal and respiratory ailments. These results demonstrate that citral, eucalyptol, and linalool inhibit 5-HT3 receptors, and their binding to a conserved cavity suggests a valuable target for novel allosteric modulators.

show abstract

Characterizing new fluorescent tools for studying 5-HT3 receptor pharmacology

Cited by 17 publications

References 49 publications

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

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

Tracking individual membrane proteins and their biochemistry: The power of direct observation

Noncompetitive Inhibition of 5-HT3 Receptors by Citral, Linalool, and Eucalyptol Revealed by Nonlinear Mixed-Effects Modeling

Contact Info

Product

Resources

About