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Understanding the structure of GPCRs is important in the design of novel therapeutics, but it is also fundamental to understand the pharmacological action of a prospective drug or lead compound. A brief overview of the pharmacological terms and assays relevant for the work presented in this thesis is provided in this section.
1.3.1. Binding Affinity
Drugs bind to receptors by forming favorable interactions with residues in the binding pocket. It is essential in the drug discovery process to be able to quantify how strongly a ligand binds at a receptor.60 This pharmacological property, called "binding affinity," is typically reported as the Kd, Ki, or IC50 of a compound. In the work presented in this thesis, binding affinity was the first property assessed by our collaborators when testing novel compounds at the apelin receptor.
Binding affinity can be reported as the dissociation equilibrium constant (Kd). This value can be derived from the equation for the binding of a genergic ligand 'L' to a receptor 'R' (Equation 1.1).61,62
This equation means that, at equilibrium, the rates of ligand association and dissociation at the receptor are equal.63 This is given by the association (k+1) and dissociation (k-1) rate constants.62 From Equation 1.1 at equilibrium, the value for Kd can be written as:62,63
It is from Equation 1.2 that the conceptual meaning of Kd can be understood. If we take the concentration of the receptor, notated as [R], to be equal to the concentration of the ligand-receptor complex [RL], this infers that half of the receptors are complexed with the ligand and half have empty binding sites. When this holds true, Kd is the same as the ligand concentration. Therefore, the Kd for a given compound can be thought of as the concentration of the ligand when half of the receptors are occupied and half are ligand-free. Medicinal chemists typically strive to design compounds that have high affinity for a receptor, so compounds that exhibit small Kd values (nanomolar affinity) are often desired.
1.3.2. Binding Assays
The affinity a natural ligand has for its cognate receptor can be determined through radioligand saturation binding experiments. Here, the ligand is labeled with a radioactive atom and the resulting radioligand is tested for binding at the receptor at varying concentrations.63 The amount of radioligand bound to the receptor is plotted against the concentration of the radioligand (Figure 1.10).60 However, it is important to note that, in addition to binding at the primary site, ligands may also bind at other sites on the receptor.60 Therefore, an additional experiment is conducted in saturation binding assays to determine the amount of nonspecific binding at each concentration of radioligand. For this assay, a high concentration of the unlabelled ligand is first administered to the receptors, after which it can be assumed that all of the orthosteric receptor binding sites are occupied and only nonspecific sites remain available for binding.60 The radioligand is then tested for affinity at each concentration, giving the nonspecific binding. The specific binding is plotted as the total binding subtracted by the nonspecific binding at each concentration (Figure 1.10).
Figure 1.10. Saturation binding curve for a radioligand at increasing concentrations. Values for Bmax, the receptor density, and Kd, the equilibrium dissociation constant, can be determined from the plot. Figure adapted from Hein et al.63
Two important values can be determined from saturation radioligand binding plots. The first is Bmax, the receptor density for a particular assay.63 This value can be estimated from the plateau of the saturation curve (Figure 1.10), because after the value of bound ligand fails to increase in response to high radioligand concentrations the maximum receptor occupancy has been reached. Half the value of Bmax on the saturation curve gives the concentration of radioligand when 50% of the receptors are occupied, which we can infer from Equation 1.2 is the affinity value Kd.63
Saturation binding experiments are useful for characterizing the pharmacological properties of endogenous ligands, but are unsuitable for the rapid screening of novel compounds. HTS, for instance, aims to rapidly determine the binding affinity of thousands to millions of compounds against a drug target and hit rates of only 0.1-1% are typical.64 Therefore, generating saturation binding curves for each compound would be highly inefficient. Instead, initial screening assays are usually competition binding assays that measure how well a test compound can compete with a high-affinity radioligand for binding. Competition binding assays are initially conducted with a fixed concentration of both the radioligand and test compound. The affinity of the compound from a fixed concentration competition binding assay is reported as the percent inhibition of radioligand binding. If a compound shows remarkably high radioligand displacement, additional points can be determined for a range of test compound concentrations (Figure 1.11). The amount of bound radioligand is plotted against the logarithm of the test compound concentration, and the concentration at which the ligand is able to displace 50% of specific radioligand binding is the inhibitory concentration (IC50).
Figure 1.11. Semi-logarithmic competition binding curve for percent of radioligand binding versus the logarithm of test compound concentration. Adapted from Hein et al.63
Although IC50 values are frequently reported in the literature, they are dependent on the experimental conditions of the assay and may make comparisons with the results of other experiments difficult. The Cheng-Prusoff equation65 provides a solution to this problem. Here, an absolute inhibition constant value (Ki) is calculated from the IC50 of the ligand, Kd of the radioligand, and the radioligand concentration [LR] (Equation 1.5).63
Competition binding assays were used throughout this thesis to assess the binding affinity of novel compounds designed through computational methods (see Chapters 4-7).
1.3.3. Potency and Efficacy
In GPCR signaling, binding of a cognate ligand is the first step in a signaling cascade that initiates with receptor activation and G-protein-coupling. As discussed in Section 1.2.1, activation of receptors which are coupled to the inhibitory G?-protein (G?i) causes inhibition of adenylate cyclase and results in attenuated intracellular cAMP production. Stimulatory G?-protein (G?s), on the other hand, causes an increase in cAMP production. It is critical in the drug discovery process to understand the effect that a prospective drug has on receptor response, as this is the property that will modulate receptor signaling.
Agonists are defined as compounds that activate the receptor and elicit a response, usually acting in a similar fashion as the endogenous ligand. Partial agonists also activate the receptor, but have a considerably smaller maximal effect compared to a full agonist.33 Competitive antagonists are ligands which occupy the orthosteric binding site of the receptor (i.e. compete with the natural ligand), but do not elicit a response.33 Inverse agonists comprise the final category and are compounds that cause the opposite response of an agonist.33
While the saturation binding and competition binding assays described in section 1.3.2 are useful for determining the affinity of a novel compound for a GPCR target, they fail to provide information about the response caused by binding. A second messenger assay can be used to determine if a compound is an agonist or partial agonist. This is typically plotted as a dose-response curve where the response, or amount of second messenger produced, is plotted against the logarithm of compound concentration (Figure 1.12A). The potency and efficacy are two pharmacological properties that can be calculated from such data. The efficacy of a ligand is the maximum response that an agonist can produce (Emax). The cognate ligand for a GPCR is taken as the reference for a full agonist (Emax = 100%), and the maximum response of test compounds can be calculated based on this value. Ligands that have a higher Emax than the endogenous ligand are referred to as super-agonists and those with a signficiantly smaller Emax are partial agonists. The potency of a compound is the concentration that can produce 50% of the maximum response and is reported as the EC50 (half maximal effective concentration).
Figure 1.12. (A) Dose-response curve for a test compound showing possible curves for either an agonist or partial agonist. The Emax (efficacy) and EC50 (potency) can be determined from this data. (B) The endogenous ligand for a GPCR is normally taken as the reference full agonist. A test compound with a similar Emax would also be a full agonist.
Competitive antagonists occupy the receptor binding site, but do not activate the receptor and subsequently fail to elicit a functional response. This makes it impossible to directly characterize antagonists through second messenger assays. Instead, dose-response curves are generated using an agonist in the presence of fixed concentrations of the antagonist. This causes the curves to shift rightward, indicating that higher concentrations of the agonist are required to achieve the maximal response in the presence of increasing concentrations of the antagonist (Figure 1.13). The extent to which the curves are shifted rightward is used to estimate the potency and efficacy of the antagonist.
Figure 1.13. Dose-response curve of second messenger response plotted against the logarithm of agonist concentration in the presence of different fixed concentrations of antagonist. The rightward shift indicates that as the functional assay is performed at increasing concentrations of antagonist, higher concentrations of agonist are necessary to elicit a second messenger response.
In this thesis, compounds which showed high binding affinity were also tested for function by our collaborators (see Chapters 4, 5, and 7).