Radioligand Binding Assays

Pharmacological analysis in the simplest form for receptors, ion channels, and transporters is determination of the affinity of a drug for a target and this is determined by performing in vitro radioligand-binding assays. These assays are designed to measure the ability of a drug to compete with a radiolabeled compound that is selective for a particular target. Concentration-response curves are constructed and activity quantified as IC50: the molar concentration of drug that inhibits the binding of a radioligand by 50% (Figure 6.2).16 If the binding is competitive this can be converted into a Ki value using the Cheng-Prusoff equation.17 Ki is the inhibition constant for a drug: the concentration of competing ligand in a competition assay that would occupy 50% of the receptors if no radioligand was present. The IC50 value for a drug may vary between experiments depending on radioligand concentration used, whereas Ki is an absolute affinity value.

There are many advantages when applying the technique of radioligand-binding assays to pharmacological profiling; they are technically easy to perform, only small quantities of drug are required, and if validated correctly they are extremely robust, reproducible, and suitable for high-throughput screening (HTS) formats. Importantly, the drug is incubated

Log concentration (|jM)

figure 6.2

An example of a concentration-response curve for a drug in a radioligand-binding assay. Increasing concentration of drug (x-axis) causes an increase in the percentage inhibition of binding of the radiolabeled compound. The IC50 is defined as the concentration of drug that displaces 50% of the specific binding of the radioligand. In this example, the IC50 for the drug is 1 |M.

Log concentration (|jM)

figure 6.2

An example of a concentration-response curve for a drug in a radioligand-binding assay. Increasing concentration of drug (x-axis) causes an increase in the percentage inhibition of binding of the radiolabeled compound. The IC50 is defined as the concentration of drug that displaces 50% of the specific binding of the radioligand. In this example, the IC50 for the drug is 1 |M.

to reach equilibrium so quantitative determinations of affinity are attainable. Where the target has been cloned, the drug can be profiled against the human isoform of the target by screening using membranes from cells that express the recombinant form of the human receptor. Where in-house resources are not available, there are contract research organizations that specialize in the technique of radioligand binding for pharmacological profiling. However, there are some limitations to this technique. The main restriction is that binding assays do not provide any information on the pharmacological mechanism of action of the drug, i.e., they cannot distinguish between agonists, antagonists, partial agonists, inverse agonists, or allosteric modulators. In addition, radioligand-binding assays may be useful, but are not optimal for assessment of drug activity at all targets. For example, they may be inferior to functional electrophysiology assays for ion-channel targets, and the use of recombinantly expressed proteins means that assumptions are made that the binding affinity will be equivalent at the native human target. Overall, the interpretation and prediction of the functional consequences of a drug having affinity for a target in a radio-ligand-binding assay is a challenge, and the relevance of radioligand-binding assay data may only be revealed when a drug is subsequently tested in functional in vitro and in vivo assays and most importantly in humans. This will be discussed below. Many of the points discussed apply to enzymes where the most straightforward approach is to perform simple in vitro enzyme activity assays. Instead of measuring the ability of the drug to displace a radiolabeled compound, the ability of the drug to compete with the turnover of a substrate into a product is measured. Potency is determined in the same way as for radioli-gand-binding assays, i.e., expressed as IC50 or Ki. The substrate may be radiolabeled or in the form of a colorless substrate that is cleaved to form a colored product, in a reaction that can be easily quantified.

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