Nanobody-Based Quantification of GTP-Bound RHO Conformation Reveals RHOA and RHOC Activation Independent from Their Total Expression in Breast Cancer.

As key regulators of the actin cytoskeleton, RHO GTPase expression and/or activity are deregulated in tumorigenesis and metastatic progression. Nevertheless, the vast majority of experiments supporting this conclusion was conducted on cell lines but not on human tumor samples that were mostly studied at the expression level only. Up to now, the activity of RHO proteins remains poorly investigated in human tumors. In this article, we present the development of a robust nanobody-based ELISA assay, with a high selectivity that allows an accurate quantification of RHO protein GTP-bound state in the nanomolar range (1 nM; 20 µg/L), not only in cell lines after treatment but also in tumor samples. Of note, we present here a fine analysis of RHOA-like and RAC1 active state in tumor samples with the most comprehensive study of RHOA-GTP and RHOC-GTP levels performed on human breast tumor samples. We revealed increased GTP-bound RHOA and RHOC protein activities in tumors compared to normal tissue counterparts, and demonstrated that the RHO active state and RHO expression are two independent parameters among different breast cancer subtypes. Our results further highlight the regulation of RHO protein activation in tumor samples and the relevance of directly studying RHO GTPase activities involvement in molecular pathways.

The pharmacologic characterization of allosteric molecules: Gq protein activation.

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo. Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales. Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration-response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (ß cooperativity). Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model. Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

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

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 ß-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.

A Broad G Protein-Coupled Receptor Internalization Assay that Combines SNAP-Tag Labeling, Diffusion-Enhanced Resonance Energy Transfer, and a Highly Emissive Terbium Cryptate.

Although G protein-coupled receptor (GPCR) internalization has long been considered as a major aspect of the desensitization process that tunes ligand responsiveness, internalization is also involved in receptor resensitization and signaling, as well as the ligand scavenging function of some atypical receptors. Internalization thus contributes to the diversity of GPCR-dependent signaling, and its dynamics and quantification in living cells has generated considerable interest. We developed a robust and sensitive assay to follow and quantify ligand-induced and constitutive-induced GPCR internalization but also receptor recycling in living cells. This assay is based on diffusion-enhanced resonance energy transfer (DERET) between cell surface GPCRs labeled with a luminescent terbium cryptate donor and a fluorescein acceptor present in the culture medium. GPCR internalization results in a quantifiable reduction of energy transfer. This method yields a high signal-to-noise ratio due to time-resolved measurements. For various GPCRs belonging to different classes, we demonstrated that constitutive and ligand-induced internalization could be monitored as a function of time and ligand concentration, thus allowing accurate quantitative determination of kinetics of receptor internalization but also half-maximal effective or inhibitory concentrations of compounds. In addition to its selectivity and sensitivity, we provided evidence that DERET-based internalization assay is particularly suitable for characterizing biased ligands. Furthermore, the determination of a Z'-factor value of 0.45 indicates the quality and suitability of DERET-based internalization assay for high-throughput screening (HTS) of compounds that may modulate GPCRs internalization.

A fluorescent ligand-binding alternative using Tag-lite® technology.

G-protein-coupled receptors (GPCRs) are crucial cell surface receptors that transmit signals from a wide range of extracellular ligands. Indeed, 40% to 50% of all marketed drugs are thought to modulate GPCR activity, making them the major class of targets in the drug discovery process. Binding assays are widely used to identify high-affinity, selective, and potent GPCR drugs. In this field, the use of radiolabeled ligands has remained so far the gold-standard method. Here the authors report a less hazardous alternative for high-throughput screening (HTS) applications by the setup of a nonradioactive fluorescence-based technology named Tag-lite(®). Selective binding of various fluorescent ligands, either peptidic or not, covering a large panel of GPCRs from different classes is illustrated, particularly for chemokine (CXCR4), opioid (δ, µ, and κ), and cholecystokinin (CCK1 and CCK2) receptors. Affinity constants of well-known pharmacological agents of numerous GPCRs are in line with values published in the literature. The authors clearly demonstrate that the Tag-lite binding assay format can be successfully and reproducibly applied by using different cellular materials such as transient or stable recombinant cells lines expressing SNAP-tagged GPCR. Such fluorescent-based binding assays can be performed with adherent cells or cells in suspension, in 96- or 384-well plates. Altogether, this new technology offers great advantages in terms of flexibility, rapidity, and user-friendliness; allows easy miniaturization; and makes it completely suitable for HTS applications.