In vitro studies of ligand affinity and activity

In vitro ligand screening and characterization is a compulsory part of drug discovery and development, and requires assays for description of ligand activity (for example differentiation between agonist, antagonist or PAM, NAM, or SAM) assays for studying different signaling pathways for the receptor, and assays measuring affinity and potency. As previously described, GPCRs can activate multiple signal pathways, and concepts such as biased signaling needs to be taken into consideration when selecting or developing functional assays to avoid rejecting potential valuable drug candidates (Zhang and Xie, 2012).

Ligand-binding assays using radioactive ligands are commonly applied, alone or in combination with other ligands, in order to resolve which binding site the ligand occupies (orthosteric, allosteric), the binding affinity and kinetics among other purposes (Hulme and Trevethick, 2010; Miyano et al., 2014). A disadvantage of such assays is their hazardous nature and in addition the custom production and labeling of ligands with a radioisotope is time- and

cost expensive (Sykes et al., 2019). Fluorescence-based methods are emerging as an alternative to radioligand-based methods as they are not hazardous (Sykes et al., 2019). These types of binding studies require a fluorophore to be attached via a linker to the ligand(s) and the signal from the probe can then be detected upon ligand binding. A challenge with fluorescence labeling is that the molecular weight is increasing and can influence the physicochemical- and pharmacological properties of the ligands (Sykes et al., 2019). Time-resolved fluorescence resonance energy transfer (TR-FRET) assay is an example of a relatively new fluorescence based method that can be applied in a HTS where a distance-dependent transfer of energy from a donor (e.g. a tagged receptor) to an acceptor (e.g. a tagged ligand) results in a traceable signal (Zhang and Xie, 2012). This technology can be applied for multiple types of studies from kinetic measurements to protein-protein interaction, dynamics and trafficking (Vernall et al., 2014). There are multiple variants ligand binding assays, and they are important tools for identification of compounds targeting different GPCR classes, but to determine the functional properties and biological responses of ligands, functional assays are necessary (Zhang and Xie, 2012).

Ligand affinity can also be measured by biophysical techniques such as isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and fluorescence polarization (FP) (Du et al., 2016). ITC measures heat exchange during the binding process, and provides characteristics such as the affinity, enthalpy and entropy of a reaction. In general, the macromolecule under investigation is placed in a chamber in the calorimeter before the ligand is titrated into the chamber. The heat released, if the reaction is exothermic, or absorbed during the binding is measured and the data is used for calculating binding characteristics (Du et al., 2016). SPR can measure kinetics, affinity and specificity in real time without using labels. The optical-based method measures changes in the refraction index upon binding to proteins immobilized on a sensor surface made up of a thin film of gold on a glass support. As ligands bind to the protein, an increase in the refraction index can be measured and after a desired association time, the solution without ligands is injected to dissociate the ligand binding complexes. This causes an decrease in the refraction index, and the refraction index curves can be used to calculate the rate constants (Du et al., 2016). FP measures kinetics based on the principle that polarized light becomes unpolarized over time, and a decrease in molecular weight caused by disassociation of the ligand-receptor complex causes the emitted light to depolarize (Lea and Simeonov, 2011). The method can also be applied for competition binding assays using fluorescence labeled ligands and unlabeled ligands, where the FP signal can be correlated to the

concentration of the unlabeled ligand necessary to displace the labelled ligands (IC50 value) (Du et al., 2016).

Functional assays can be applied to detect activated G-proteins, G-protein mediated events or G-protein independent events (Zhang and Xie, 2012). A GTPgS binding assay can be used to determine if a ligand initiates receptor-G-protein coupling and for identifying intrinsic activity.

In addition, the GTPgS assay can be applied independent of which of the four main G-protein families the receptor is interacting with. As activation of a G-protein causes exchange of Ga-bound GDP to GTP, the radioactive GTPgS is added and binds the Ga subunit and radioactivity can be counted (Zhang and Xie, 2012).

The four main families of G-proteins initiate different intracellular responses upon activation and the choice of assay is therefore dependent on which family of G-protein the receptor recruits. Many G-protein dependent assays are based on detecting the second messenger after ligand binding and receptor activation, and thereby require the receptor coupling mechanism to be known. cAMP-based assays are frequently used when the GPCR is coupled with Gi/o and/or Gs that causes negative or positive stimulation of adenylyl cyclase and thereby affects the cellular levels of cAMP which can be detected by the assay (Fig. 4). Labeled cAMP can be introduced in the assay to compete with endogenous cAMP, and later be detected by anti-cAMP antibody (Zhang and Xie, 2012). There are multiple variants of the cAMP assay both radiolabeled and radio-free approaches (Zhang and Xie, 2012). Please see the methods section for further description of the cAMP assay applied in this thesis.

GRKs phosphorylate specific intracellular sites of GPCRs and cause recruitment of arrestins that promote receptor internalization (Hilger et al., 2018). This processes can be investigated both by receptor internalization- and b-arrestin recruitment assays. However, receptor internalization can be studied in several ways, but very often specific antibodies binding to an extracellular part of the receptor is used. The antibody is co-internalized with the receptor upon activation and may be detected by a fluorophore-labeled secondary antibody or by tagging the receptor with fluorescent proteins (Zhang and Xie, 2012). b-arrestins are also involved in G-protein independent signaling and can act as scaffolds that interact with various G-proteins such as the signaling protein Extracellular signal-regulated kinase 1/2 (ERK 1/2), nonreceptor tyrosine kinases like Src, and trafficking proteins (Lefkowitz, 2005). Assays targeting b-arrestins can be used to study biased GPCR signaling (Zhang and Xie, 2012). The first

commercial recruitment assay for studying the effects of b-arrestin recruitment upon receptor activation used b-arrestin tagged with green fluorescence protein (GFP) that emits green fluorescence upon light exposure which is monitored by an imaging system (Zhang and Xie, 2012).