Receptor binding assay is used to measure the binding affinity of a drug or compound to the receptor binding sites of a particular receptor type. The cells that are usually used in this assay are cell membrane homogenates that are known to contain high population of the receptor needed. For opioids, the commonly used cells are brain cell membranes (eg: guinea pigs) or cell lines transfected with cloned receptors (eg: Chinese hamster ovarian (CHO) transfected cells) (Toll et al., 1998). The receptor binding assay can measure the binding affinity of a compound regardless of its pharmacological activity (agonist or antagonist nature). For a compound with antagonist activity, the isolated tissue preparation also can be used to estimate the compound binding affinity, which will be explained in the next subchapter (Schild analysis and Schild equation).
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Principle and mechanism of receptor binding assay
There are three ways of conducting binding assays which are through saturation, displacement and kinetic binding. The main principles behind these three binding assays are the same where the fraction bound of the measured ligand (specific binding) is different from the fraction unbound (non-specific binding, nsb) (specific binding = total binding – nsb). The nsb refers to the fraction of ligand that is bound to the sites other than the receptors, which also include the test tube and the cell membranes. The differences between saturation, displacement and kinetic binding techniques are how the tracer ligand (ligand labelled with radioactive isotope / fluorescence species) is measured (Kenakin, 2009). Saturation binding directly measures the binding of tracer ligand to the receptors. The ligand used has to be traceable which only can be done to the radioactive or fluorescence molecules. The tracer ligand in this case is the test compound. The second method, displacement binding, measures the interruption or reduction of radioactive signals through competitive binding (displacement) by a nontraceable ligand. The reduction of radioactive signal of the tracer ligand caused by the competitive activity of the nontraceable ligand at the receptor is used to measure the binding affinity of the nontraceable ligand. In this case, the tracer ligand was a standard drug while the nontraceable ligand was the test compound. The last technique, kinetic binding, measures directly the decay of radioactivity of tracer ligand with time (Kenakin, 2009).
The method used by John Traynor’s lab that is presented in this thesis is the displacement binding technique. The detailed method used will be discussed in
Chapter 3. Principally, the cells transfected with the specific receptor are pre-incubated with a constant (fixed) concentration of a tracer ligand (radiolabelled
ligand), together with a high concentration of nonlabelled ligand to measure the nsb, in the presence and absence of varied concentration of another nonradiolabelled ligand (the test compound) (Kenakin, 2009). Theoretically, the test compound will compete with and displace the tracer ligand which will disrupt the radioactivity of the tracer ligand in a concentration dependent manner. The remaining radioactivity of the tracer ligand bound to the receptor is measured using a scintillation counter. The percentage of receptor displacement of the tracer ligand is plotted against the
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concentration of the test compound (displacing ligand) in a log scale to get the IC50
value (Traynor et al., 1995). The IC50 refers to the concentration of the test
compound that causes 50% decrease in the radioactivity of the tracer ligand from the original (basal) value. This value is derived from the non-linear regression graph. This IC50 value is then fitted into the Cheng-Prusoff equation to calculate the binding
affinity (Ki) value of the displacing ligand (test compound) (Kenakin, 2009).
1.7.2 Functional assays
1.7.2.1 [35S]GTPS binding
[35S]GTPS refers to a non-hydrolyzable analogue of GTP that is tagged with a radioactive isotope of 35S, which can be measured by a liquid scintillation counter (Harrison et al., 2003; Traynor et al., 1995). The [35S]GTPS binding is a functional assay, usually conducted in cell lines transfected with a homogenous (isolated) receptor type. It is commonly used nowadays as a functional bioassay alternative to the isolated tissue preparations. Although the word ‘binding’ is used to describe this assay, it is not similar to the receptor binding assay because this assay is not directly quantifying the receptor occupancy, as in the receptor binding assay. The [35S]GTPS is a measures of receptor activation activity by an agonist as a result of agonist-receptor interaction (Harrison et al., 2003). Therefore, the ‘binding’ in this assay actually refers to the binding of the [35S]GTPS with the -subunit of the activated G-proteins.
Principle and mechanism of [35S]GTPS binding assay
The [35S]GTPS assay measures the very first events of the G-protein’s activation by an agonist at the receptor level (Harrison et al., 2003). This event refers to the nucleotide exchange between the membrane bound -GDP subunit of the activated
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heterotrimer complex and the intracellular GTP (Figure 1.2, top (right)). In this assay system, the function of the intracellular GTP is replaced with a radiolabelled, non-hydrolyzable GTP analogue, [35S]GTPS (Harrison et al., 2003). Since the amount of [35S]GTPS added in the test tube is known, the percentage of the -[35S]GTPS complex bound to the cellular membrane during agonist occupation at the receptor can be measured (Figure 1.9) after filtering the membrane, to determine the efficacy of the agonist (expressed as % stimulation) and compared against standard agonist. The [35S]GTPS is a stable species and is not subject to hydrolysis by the intracellular GTPase activity (Traynor et al., 1995). Therefore once activated, it will accumulate in the cell membranes which enables the level of this membrane-bound species to be measured (Figure 1.9).
Figure 1.9: Principle of [35S]GTPS binding assay and measurements.
Since [35S]GTPS is an artificial assay system, the level of agonist expression (% stimulation) may vary between different labs, which depends on the experimental protocol. Besides the [35S]GTPS species, the important materials for this assay are the GDP, charged ions (Mg2+ and Na+), and membranes (which contains the protein receptor) (Harrison et al., 2003). The amount of these materials and the types of cell lines can be adjusted to achieve a bigger receptor stimulation, which explains why
activated -GDP + -[35S]GTPS [35S]GTPS GDP (membrane-bound species)
Accumulate in the cell membranes
(filtered)
Measured (Liquid scintillation counter)
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sometimes different labs have reported huge differences of % receptor stimulation by the same agonist (Alt et al., 2002; Bloms-Funke et al., 2000; Spagnolo et al., 2008).