Equilibrium Assays Are Required to Accurately Characterize the Activity Profiles of Drugs Modulating Gq-Protein-Coupled Receptors

Abstract

This paper discusses the process of determining the activity of candidate molecules targeting Gq-protein activation through G-protein-coupled receptors for possible therapeutic application with two functional assays; calcium release and inositol phosphate metabolism [inositol monophosphate (IP1)]. While both are suitable for detecting ligand activity (screening), differences are seen when these assays are used to quantitatively measure ligand parameters for therapeutic activity. Specifically, responses for Gq-related pathways present different and dissimulating patterns depending on the functional assay used to assess them. To investigate the impact of functional assays on the accuracy of compound pharmacological profiles, five exemplar molecules [partial agonist, antagonist, inverse agonist, positive allosteric modulator (PAM) agonist, and positive -PAM] targeting either muscarinic M1 or ghrelin receptors were tested using two functional assays (calcium release and IP1) and the results were compared with theoretical pharmacological models. The IP1 assay is an equilibrium assay that is able to determine the correct (i.e., internally consistent) pharmacological profiles of all tested compounds. In contrast, the nonequilibrium nature of calcium assays yields misleading classification of most of the tested compounds. Our study suggests that the use of an equilibrium assay, such as IP1, is mandatory for the optimal use of pharmacological models that can both identify mechanisms of action and also convert descriptive-to-predictive data for therapeutic systems. Such assays allow the identification of consistent and simple scales of activity that can guide medicinal chemistry in lead optimization of candidate molecules for therapeutic use.

Identification of higenamine as a novel α1‐adrenergic receptor antagonist

“…IP3 is very transient and difficult to quantitate (Trinquet, Bouhelal, & Dietz, 2011). But IP1 can be used as a surrogate for IP3 for detecting the intracellular accumulation of a metabolite of IP3 and discovering or characterizing new compounds targeting GPCRs (Bdioui et al, 2018;Trinquet et al, 2011). Therefore, HG was assessed by its effect on IP1 production in HEK293 cells transfected with α 1A -, α 1B -, and α 1D -AR, respectively.…”

A kinetic operational model of agonism incorporating receptor desensitization for G-protein-coupled receptors

“…While likely not an issue for low potency ligands (affinity in the µM range), lack of equilibration is likely to affect the time course of response for high affinity ligands, leading to erroneous estimates of ligand affinity. This problem is exacerbated for rapid responses, such as calcium mobilization (Bdioui et al, 2018;Charlton and Vauquelin, 2010). In principal, the problem can be avoided by incorporating receptor binding kinetics of the agonist into the model equations.…”

“…In classical pharmacological models, it is implicitly assumed that agonist-receptor binding is at equilibrium at the time point at which the response is measured (Bdioui et al, 2018;Black and Leff, 1983;Colquhoun, 1998;Rang, 2006;Slack and Hall, 2012). In a kinetic model, it cannot be generally assumed that agonist is at equilibrium with the receptor at all time points of response measurement (unless a maximally-effective concentration of agonist is employed -see below).…”

“…The equilibrium assumption is probably reasonable for low potency ligands when the response progresses over timescales of at least several minutes, for example second messenger generation and gene expression (Bdioui et al, 2018). Such ligands (EC50 in the micromolar range) equilibrate rapidly even at the low concentrations required to span the concentration-response curve 11 (equilibration time < 1 min) .…”

“…However, high potency agonists (EC50 in the low nM range) equilibrate more slowly, especially at the low concentrations required to span the concentration-response curve (at least several minutes). In addition, lack of equilibration is likely to be an issue for responses that are extremely rapid (Bdioui et al, 2018;Charlton and Vauquelin, 2010). For example, the rise of intracellular Ca 2+ occurs within a few seconds of agonist application.…”