RADIOLIGAND BINDING ASSAYS
2.4.2 Important Considerations for Developing Radioligand Binding Assays
RADIOLIGAND BINDING ASSAYS
2.4.1 Introduction
Human receptors have been shown to be clearly associated with a multitude of human diseases and disorders. Thus, these receptors have become molecular targets for drug discovery. There are several different structurally related superfamilies of human receptors including G-protein coupled receptors, cytokine receptors, receptor protein tyrosine kinases, ligand-gated ion channels, and steroid receptors and members from all of these superfamilies have been targeted in drug discovery programs. Screening for agonists and/or antagonists of various receptors has traditionally been accomplished by using radioligand binding assays. These assays are relatively simple to perform and are based upon the principles of a competitive binding assay in which the radiolabeled ligand competes with the unlabeled ligand for binding to a receptor in a cell or on the cell surface membrane. Thus, compounds or natural product extracts are evaluated for their ability to compete with a known ligand and this competition is measured by a decrease in the binding of the labeled known ligand. The focus of this section is to describe important aspects of developing and validating receptor radioligand binding assays used to screen combinatorial chemistry reaction products and natural product extracts.
2.4.2 Important Considerations for Developing Radioligand Binding Assays
There are certain assay parameters that need to be carefully evaluated for development of radioligand binding assays for natural product extract screening. These parameters are listed in Table 9.
Table 9. Specific Assay Parameters to Consider for Developing
• Receptor source and assay concetration • Selection of radioligand and assay concentration • Selection of assay incubation conditions • Selection of assay buffer
• Selection of assay volume
2.4.2.1 Receptor source and assay concentration
2.4.2.1.1 Nonrecombinant Human Cell Lines
Stable non-recombinant human cell lines can often be used as a source of receptor for developing screening assays. Advantages of using this source include: cells contain unperturbed receptors, normal receptor post-translational modifications are usually present, and the physiologically activated signal transduction system is present for development of receptor functional assays. Possible disadvantages may include: the presence of undesired receptor subtypes, agonist-mediated receptor desensitization and internalization, and receptors may be structurally altered in transformed cells.
2.4.2.1.2 Membrane Preparations from Nonrecombinant Cells and Tissues
Crude plasma membranes prepared from human cells expressing receptors of interest are widely used for radioligand binding assays. Advantages of using membrane preparations include: agonist-mediated desensitization and internalization do not occur, large preparations can be made easily and frozen until needed, normal receptor postranslational modifications are present, and guanine nucleotides can be washed away to increase high affinity agonist binding to G-protein coupled receptors. Possible disadvantages include: undesirable receptor subtypes may be present and receptor-associated signal transduction molecules may not be present for developing functional assays.
2.4.2.1.3 Recombinant Receptor Containing Cells or Membranes
The primary advantage of using recombinant cells or membranes from recombinant cells as a source of receptors for radioligand binding assays is that they can provide a cheap, consistent, and pure population of human receptors for screening large numbers of compounds or extracts. Receptor densities expressed in cells can often be increased over physiological levels allowing increased ratios of signal-to-noise and the use of lower amounts of membranes. Large quantities of recombinant cells can be scaled up using spinner flasks or bioreactors, membranes can be prepared, and stored until needed for assays. Possible disadvantages of using recombinant cells or recombinant cell membranes include: cloning and expressing receptors of interest can be difficult and time consuming, over-expression of receptors may lead to activation of non-physiological signal transduction pathways, and physiological post-translational modifications may not occur in certain expression systems.
Several different expression systems can be used for recombinant human receptors including several types of mammalian cells, insect cells, yeast or bacteria. Important considerations for choosing a receptor expression system would include whether endogenous receptors are present, whether the appropriate receptor post-translational modifications will occur, whether appropriate signal transduction molecules are present for developing functional receptor assays, and which expression system will provide the largest quantity of receptor at the lowest cost.
2.4.2.1.4 Selection of Raioligand and Assay Concentration
For any given receptor, there are often several different radioligands that are commercially available for use in developing a radioligand binding assay. There are several important considerations that should be made when selecting a suitable radioligand and these considerations are listed in Table 10.
The radioligand chosen should possess high affinity for the receptor so that the assay is sensitive, low concentrations of radioligand can be used to minimize assay costs, and levels of nonspecific binding can be minimized. Similarly, the chosen radioligand should have a high specific activity for similar reasons. High specific activity ligands are also essential when membrane preparations used contain low densities of receptor. The radioligand should also possess low nonspecific binding allowing maximization of signal-to-noise ratio. The chosen radioligand should also be selective for the subtype of receptor of interest, especially if related receptor subtypes are present in the receptor preparation used.
For many receptors, both radioactive agonists and antagonists may be commercially available for use in binding assays and in some cases, antagonists are preferred over agonists. For example, antagonists are generally preferred for developing binding assays for G-protein coupled receptors. The reason for this preference is due to the selective influence of endogenous guanine nucleotides on agonists binding. Only low affinity equilibrium agonist binding can be detected in whole cells due the presence of endogenous guanine nucleotides which promote conversion of high affinity to low affinity agonist binding sites (Thomsen et al, 1988). In contrast, antagonist binding is not effected by guanine nucleotides and high affinity binding is detected. Therefore, antagonists are required for labeling binding studies using whole cells. Similarly, agonist binding to membranes containing significant levels of endogenous guanine nucleotides will also be reduced to low affinity. Due to this potential membrane contamination, during preparation antagonists are generally preferred for radioligand binding studies to G-protein coupled receptors. Antagonist radioligands would also be preferred for cytokine receptor binding assays because agonists promote rapid receptor internalization. However, no cytokine receptor antagonists are available for this application. Antagonist
Table 10. Considerations for Selecting a Radioligand
• Receptor affinity • Specific activity • Nonspecific binding • Receptor selectivity • Agonist or antagonist • Type of radioisotope
radioligands are also preferred for ligand-gated ion channel receptor binding assays because agonists can promote rapid receptor desensitization.
The last consideration in choosing a suitable radioligand is the choice of radioisotope. Tritiated or radioiodinated ligands are most commonly used in screening assays. It is important to note that radioiodinated ligands have a relatively short half-life and radioactive decay must be taken into account during use of the radioligand. Significant decay is often manifested in a binding assay by an increase in nonspecific binding. Table 11 lists some important characteristics of commonly used radioisotopes.
The final assay concentration of radioligand used in an assay is also an important variable in developing a radioligand binding assay. Typically, a Kd concentration of radioligand (1/2 of a saturating concentration) is used. It is possible that lower concentrations can be added if a high specifie activity radioligand is available or if high receptor densities are present in the receptor preparation. Minimization of the radioligand concentration will reduce assay costs, minimize nonspecific binding, and reduce amounts of radioactive waste.
2.4.2.1.5 Selection of Assay Incubation Conditions
For radioligand saturation and competition experiments, the theoretical model used is one of equilibrium. Thus, the time of incubation needs to be sufficient to ensure that equilibrium or steady state radioligand binding is reached during the assay incubation (Bylund et al., 1993). The period of time required to reach equilibrium depends upon radioligand affinity and concentration and the assay incubation temperature. For most radioligands with Kd values in the low nanomolar range, equilibrium is usually reached in 20–60 minutes at room temperature in the presence of a Kdconcentration of radioligand.
Table 10. Characteristics of Radioisotopes Used for Radioligand Binding Studies
Isotope Specific (Ci/mmol ) Radioactivity Half- Life (years) Other Considerations [3H] 40 12.3 years
Bioactivity of ligand usually unchanged with tritiation. Stable for long periods.
[125I] 2125 60.2 days Requires tyrosine or unsaturated cyclic system, especially used when there is low receptor density. No scintillation fluid is required. Generally only stable for a month. Biological activity can be altered with iodination.
[32P] 9760 14.3 days Short half life is a technical problem [14C] 0.06 5730
years
Specific activity too low
Most receptor binding assays can be conveniently performed at room temperature. However, there are exceptions. For example, cytokine binding assays are generally performed at 0–4°C to reduce rapid agonist-mediated receptor internalization which occurs at higher temperatures. In addition, binding assays utilizing peptide radioligands are typically performed at room temperature or lower to minimize potential proteolysis that can occur at physiological temperatures. Inclusion of protease inhibitors is generally advisable when using peptide radioligands.
2.4.2.1.6 Selection of Assay Buffer
In general, radioligand binding assays are performed at physiological pH. There are several buffers commercially available that have good buffering capacity at physiological pH. Different buffers should be evaluated to determine which gives an optimal ratio of specific to nonspecific binding.
2.4.2.1.7 Selection of Assay Volume
The total assay volume for a radioligand binding assay should be minimized to conserve expensive reagents. Assay volumes ranging from 50 to 200 µl are typically used and volumes are generally limited by the precision of liquid handling devices used. In some cases where the receptor density is low, larger assay volumes up to 500–1000 µl may be required. New technologies are now being developed to enable dispensing of nanoliter quantities of reagents and it is likely that these technologies will be applied to reducing volumes of radioligand binding assays.
2.4.2.1.8 Selection of a Methodology for Separation of Free and Bound Radioligand
Subsequent to incubations, free and bound radioligand need to be resolved so that only bound ligand is measured. There are primarily three methods used, equilibrium dialysis, centrifugation, and vacuum filtration over glass fiber filters. For high volume screening, vacuum filtration is the only method of choice for resolving free and bound radioligand. The principle behind filtration is that filters will retain radioligand bound to membrane fragments or cells while unbound radioligand passes through the filter. Residual radioligand retained by filters is removed by subsequent washing of filters with ice cold buffer. One requirement for this washing procedure is that the dissociation of radioligand needs to be minimal during the washing step. Therefore, only radioligands with high affinity and slow dissociation rates can be used in filtration assays. Radioligands with affinities of less than 100 nM cannot generally be subjected to filtration assays because dissociation will occur in a fraction of a second (Yammamura et al, 1985). Even after washing, considerable amounts of free radioligand can still be retained on filters resulting in increased levels of nonspecific binding. Most glass fiber filters are available with different pore sizes and it is advisable to test several sizes to determine which gives the lowest nonspecific filter binding. In certain cases, non-specific binding can also be reduced by chemical treatments of filters. For example, filter retention of positively
charged radioligands to glass fiber filters can be reduced by precoating filters with polyethyleneimine (Bruns et al, 1983).