Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2018/06/28/mol.118.112573.DC1 1521-0111/94/3/992–1006$35.00 https://doi.org/10.1124/mol.118.112573 MOLECULAR PHARMACOLOGY Mol Pharmacol 94:992–1006, September 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Equilibrium Assays Are Required to Accurately Characterize the Activity Profiles of Drugs Modulating Gq-Protein-Coupled Receptors s Sara Bdioui, Julien Verdi, Nicolas Pierre, Eric Trinquet, Thomas Roux, and Terry Kenakin Cisbio Bioassays, Codolet, France (S.B., J.V., N.P., E.T., T.R.); and Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, North Carolina (T.K.) Received March 25, 2018; accepted June 11, 2018 Downloaded from ABSTRACT This paper discusses the process of determining the activity of or ghrelin receptors were tested using two functional assays candidate molecules targeting Gq-protein activation through (calcium release and IP1) and the results were compared with G-protein-coupled receptors for possible therapeutic applica- theoretical pharmacological models. The IP1 assay is an tion with two functional assays; calcium release and inositol equilibrium assay that is able to determine the correct (i.e., molpharm.aspetjournals.org phosphate metabolism [inositol monophosphate (IP1)]. While internally consistent) pharmacological profiles of all tested both are suitable for detecting ligand activity (screening), compounds. In contrast, the nonequilibrium nature of calcium differences are seen when these assays are used to quantita- assays yields misleading classification of most of the tested tively measure ligand parameters for therapeutic activity. compounds. Our study suggests that the use of an equilibrium Specifically, responses for Gq-related pathways present dif- assay, such as IP1, is mandatory for the optimal use of ferent and dissimulating patterns depending on the functional pharmacological models that can both identify mechanisms assay used to assess them. To investigate the impact of of action and also convert descriptive-to-predictive data for functional assays on the accuracy of compound pharmacolog- therapeutic systems. Such assays allow the identification of ical profiles, five exemplar molecules [partial agonist, antago- consistent and simple scales of activity that can guide nist, inverse agonist, positive allosteric modulator (PAM) medicinal chemistry in lead optimization of candidate mole- at ASPET Journals on September 27, 2021 agonist, and positive b-PAM] targeting either muscarinic M1 cules for therapeutic use. Introduction in all functional systems. A corollary to this approach is that these same models often provide internal checks, whereby The process of drug lead optimization involves the character- certain predictions must coincide with the observations to ization of the molecular profile of a drug candidate with system- correctly ascribe drug mode of action. For this procedure to be independent scales of drug activity, i.e., the profile ideally should successful, it is imperative that the assays used to determine not be linked only to the experimental system where it is drug function accurately reflect the molecular activity of the observed. This is important since drugs are almost always molecule at the receptor. studied and developed in test experimental systems and not There are two fundamentally different types of assay used the end therapeutic tissue. Therefore, a scale of activity must in drug discovery: detection of drug activity (screening) and bridge the gap between these two systems. Pharmacologically characterization of activity (lead optimization). Detection this is done by comparing experimental data to mathematical assays should be robust and sensitive; however, they only models of drug-receptor interaction and deriving universal need to reveal ligand-receptor interaction and not necessarily parameters such as equilibrium dissociation constants [equilib- quantitatively correct drug parameters. In contrast, lead rium dissociation constant of the agonist-receptor complex (K ) A optimization assays must provide predictive and accurate and equilibrium dissociation constant of antagonist (or allosteric measures of drug activity that can be used to characterize modulator)-receptor complex (KB)], measures of efficacy (t), or activity in therapeutic systems. The activation of Gq-protein- allosteric parameters describing effects on affinity (a)and coupled receptors results in increases in inositol triphosphate efficacy (b), which then can be used to predict agonist activity metabolism and calcium release in cells (Berridge et al., 2003). There are two main and well-described fluorescent-based https://doi.org/10.1124/mol.118.112573. s This article has supplemental material available at molpharm. assays that can be used to monitor the activation of the Gq aspetjournals.org. pathway. On the one hand, calcium assays measure the release ABBREVIATIONS: ACh, acetylcholine; BQCA, benzyl quinolone carboxylic acid [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid]; CHO, Chinese hamster ovary; DR, dose ratio; GHSR1a, growth hormone secretagogue receptor type 1a; HEK, human embryonic kidney; HR, heart rate; IP1, inositol monophosphate; KA, equilibrium dissociation constant of the agonist-receptor complex; KB, equilibrium dissociation constant of antagonist (or allosteric modulator)-receptor complex; NECA, 59-N-ethylcarboxamidoadenosine; PAM, positive allosteric modulator; SPA, [D-Arg1,D-Phe5,DTrp7,9,Leu11]-substance P. 992 Functional Assays for Gq-Protein–Activating Receptor Ligands 993 of calcium flux into the cells using permeant dyes, in which complemented culture medium at the desired concentration. Cells fluorescence emission increases upon binding to calcium were seeded into 96-well culture-treated plates for IP1 and calcium (Chambers et al., 2003). On the other hand, the inositol mono- experiments (Greiner Bio-One, Courtabœuf, France) at 100 ml/well phosphate (IP1) assay measures the accumulation of IP1, which is and incubated overnight at 37°C with 5% CO2 before being used in a metabolite of inositol triphosphate, through a competition assay functional assays. based on homogeneous time-resolved fluorescence technology (Trinquet et al., 2011). It will be seen that both IP1 and calcium Measurement of the Intracellular Accumulation of IP1 assays function well in screening mode but differ considerably in The homogeneous time-resolved fluorescence IP-One-Gq assay (IP1) lead optimization mode. This paper discusses whether these was used to measure intracellular IP1 accumulation according to the functional readouts deliver accurate data for ligand characteriza- manufacturer’s instructions (Cisbio Bioassays, Codolet, France). In tion or if they lead to dissimulating and misleading profiles. brief, on the day of the experiment, serial dilutions of compounds were It is well known that calcium transients emanate from prepared in the stimulation buffer of the kit (containing 50 mM LiCl to prevent IP1 degradation) at the desired working concentration. Cell hemi-equilibrium assays that do not furnish sustained agonist culture medium supernatant was removed from the plates. After a responses; therefore, pharmacological procedures normally washing step with phosphate-buffered saline, cells were treated by requiring these, such as detecting inverse, partial, and addition of 70 ml/well of the different compounds and incubated for the positive allosteric modulator (PAM) agonism, are precluded. indicated time at 37°C, 5% CO2. Then, IP1 detection was allowed by Downloaded from However, there are published procedures that have been used addition of the detection reagents previously diluted in the appropriate to furnish mass action pharmacological profiles for these types kit lysis and detection buffer (30 ml total). Plates were incubated for of ligands through coaddition formats, which apparently yield 1 hour at room temperature and the homogeneous time-resolved the correct profiles for some of these types of ligands. It can be fluorescence signal was recorded using a PHERAstar FS reader (BMG shown that while these procedures apparently provide correct LABTECH, Ortenberg, Germany) with flash lamp excitation. qualitative patterns of drug effect, they in fact can yield Assay parameters, such as the cellular density required to work within the assay dynamic range or the determination of the appro- molpharm.aspetjournals.org erroneous drug parameters that lead to misclassification of priate stimulation time for each compound (i.e., ACh, Alvameline, drug activity. The key to identifying such dissimulations is the Atropine), were properly optimized with the cellular models used application of internal checks within the mathematical models (CHO-M1 and HEK293-GHSR1a). of drug receptor pharmacodynamics. These concepts are illustrated in the present paper with examples of five general Measurement of Calcium types of ligands [agonists, antagonists, inverse agonists, PAM agonists, and PAMs increasing agonist efficacy (b-PAMs)]. After cell culture medium removal, cells were washed once with phosphate-buffered saline and incubated for 1 hour at 37°C, 5% CO Specifically, the results obtained suggest that the use of an 2 with 2 mM of Fluo-4 AM (ThermoFisher Scientific, Villebon sur Yvette, equilibrium assay based on the determination of the intracel- France) previously diluted in Hanks’ balanced salt solution (Thermo-