• Authors
  • Nuclear Medicine and PET Research, VU University Medical Centre, Amsterdam, The Netherlands
  • Neuroscience Department, Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium
  • Johnson & Johnson Pharmaceutical Research and Development, Beerse, Belgium
  • Nuclear Medicine and PET Research, VU University Medical Centre, Amsterdam, The Netherlands

Receptors: Binding Assays


Labeled ligand binding assays; Ligand binding assays; Radioligand binding assays


Receptor binding refers to a technique in which a labeled compound, a ligand, which binds to a receptor is used to detect that receptor. Usually, the ligand is labeled by means of a radioactive isotope, such as 3H, 125I, 35S, etc., but a fluorescent moiety is also possible. The receptor can be localized in a tissue that is homogenized or sliced or in cells in culture that either have an endogenous expression of the receptor or have been transfected with a cloned receptor gene. Tissue preparations are incubated with a labeled ligand that has a high binding affinity for the target receptor. The labeled ligand bound to tissue is then collected and detected using various techniques, such as filtration techniques combined with radioactivity counting, scintillation proximity analysis and autoradiography for radioactive ligands, and time-resolved fluorescence resonance energy transfer (TR-FRET) or amplified luminescent proximity homogeneous assay (AlphaScreen) for ligands labeled with a fluorescent or a chemiluminescent probe, respectively. These techniques are applied in vitro or ex vivo. In vivo receptor binding can be investigated using positron emission tomography (PET) or single-photon emission computerized tomography (SPECT) imaging.

Principles and Role in Psychopharmacology

Receptor binding techniques were first introduced in the 1970s (historic reviews: Lefkowitz 2004; Snyder and Pasternak 2003). They were the first tools enabling receptors to be demonstrated in tissues using biochemical methods. In earlier years, there were no clues about the nature or structure of receptors. Extensive research throughout the 1980s led to the cloning of receptor genes and the identification of their structure. Today, four major classes of receptors that are located in cell membranes and one class of cytosolic receptors have been identified:

  1. The G-protein-coupled receptors (GPCRs), also called "7 transmembrane (7 TM)" receptors, are integral membrane protein monomers. With 802 known and predicted human GPCRs, derived from the human genome, this is the largest receptor family and at least 50 of them have been identified as major drug targets (Lagerström and Schiöth 2008). Sequence comparison revealed five subfamilies, all of which have a central core domain in common, which consists of 7 TM helices connected by three intracellular and three extracellular loops. The protein has an extracellular N-terminal and an intracellular C-terminal, which can be of widely varying lengths. GPCRs have diverse natural ligands comprising of small molecules (amines, amino acids, nucleotides, nucleosides, prostaglandins, peptides, lipid-like molecules, etc.), light, Ca++, odorants and pheromones, and proteins (for review of the GPCR classes, structures, features, and natural ligands, see Lagerström and Schiöth 2008). Upon activation, a GPCR associates with a G-protein that further activates effectors in the cell. Various different proteins can interact with GPCRs so that receptor complexes can exist in multiple states leading to possible complex kinetics for ligand binding (Christopoulos and Kenakin 2002).

  2. Ligand-gated ion channels are multimeric, often pentameric, protein complexes that form a pore in the membrane. The members of this family are, e.g., the nicotinic acetylcholine, GABAA, 5-HT3, glycine, purinergic P2X, and the NMDA, AMPA, and kainate subtypes of glutamate receptors, each of itself has several subtypes.

  3. Receptors that are enzymes with a ligand binding domain that couples to an intracellular membrane-anchored enzyme such as tyrosine kinase, tyrosine phosphatase, serine/threonine kinase, and guanylyl cyclase. The members of this class are, e.g., growth factor receptors, neurotrophic factor receptors, and transforming growth factor β (TGFβ) receptors.

  4. Cytosolic receptors regulate transcription in the cell nucleus. The members are steroid, retinoid, and thyroid hormone receptors.

Large numbers of members of each of these receptor classes as well as (neurotransmitter) transporters have been studied with receptor binding techniques and receptor binding assays, and hundreds of different molecular targets have been described. In this chapter, in vitro and ex vivo methods used for investigating membrane-bound receptors will be discussed.


Analytical Rules

Receptor binding assays require working according to analytical rules and procedures. Water must be of freshly double-distilled or milliQ quality and all chemicals (salts, metal ions, organic solvents, etc.) of analytical grade. Labeled ligands must have a purity of >98 % and purity must be checked regularly (radioligands, in particular with high specific activity, suffer from radiolysis). Stability of compounds must be checked and purified where necessary (e.g., catecholamines and indoleamines are very unstable and solutions need to be purified just prior to use). (Micro)balances and pipettes (manual and automated) must be regularly gauged. Compounds must be solubilized and diluted according to their physicochemical properties. Lipophilic compounds are best solubilized and diluted in pure dimethyl sulfoxide (DMSO) and buffer should be used only for the last dilution step, or the organic solvent solution should be added directly into the incubation mixture. However, the organic solvent concentration in the assay mixture should not exceed 0.5 %. Dilution in the organic solvent is indicated to avoid the loss of lipophilic compound due to adsorption to glass or plastic ware. Plastic tubing must be avoided when using robotic devices. Hydrophilic compounds must be solubilized and diluted in buffer or water. Sometimes protecting agents such as antioxidants must be added. Biological materials must be kept on ice in buffer of neutral pH until the start of the incubation. Buffer pH must be adjusted to the temperature of the incubation.

Law of Mass Action

Ligand-receptor binding experiments are usually analyzed according to the simple model of law of mass action:

where [R], [L], and [RL] represent the free concentration of receptor, ligand, and receptor-ligand complex, respectively, and where it is assumed that the reaction components can freely diffuse within the medium. kon and koff are the rate constants for association and dissociation of the ligand-receptor complex. It is assumed that after dissociation, receptor and ligand are not altered.

Association and dissociation rates are temperature dependent. The reaction is driven by the concentration of the reacting agents. Equilibrium is reached when the rates at which ligand-receptor complexes are formed and dissociate are equal. At equilibrium, the following applies:

The equilibrium dissociation constant, Kd, a measure for binding affinity, is defined as

The constants have the following units: kon: M−1∙s−1; koff: s−1; Kd: M, where M stands for Molar or mol∙L−1. When receptor binding experiments are performed, [R] usually is unknown (needs to be determined), [RL] is measured in the assay, and [L] usually is assumed to be equal to the applied ligand concentration. This implies, however, that only a minor fraction (<5 %) of the total applied ligand should become bound (either by specific binding, nonspecific binding, or adsorption to tissue and assay container) so that the free concentration is not significantly altered.

Criteria Required by the Law of Mass Action: Can They Be Met?

It is important to realize that the reaction conditions never meet the required criteria. The reaction model itself is largely simplified, as receptors can occur in different states and ligands may bind to orthosteric and allosteric sites (see Christopoulos and Kenakin 2002). "Free in solution" and "freely moving in solution" do almost never apply to biological tissue, either as an homogenate or a membrane suspension, and certainly not when tissue slices, whole cells, or tissue fixed on a support are used. Membrane preparations consist of membrane vesicles of varying dimension, possibly with strips of membrane suspended in aqueous medium. Surface phenomena will inevitably take place when ligands (hydrophobic or hydrophilic) approach the membrane vesicle. Examples of surface phenomena are electrostatic interactions of ionized compounds or surface excess of lipophilic compounds. Surface phenomena, which are difficult to measure or estimate, have received virtually no attention in receptor binding research. Yet, they are a matter of fact and, for haloperidol, a 1,000× surface excess was calculated to exist in the monolayer around a membrane vesicle as compared to the concentration in aqueous medium (Leysen and Gommeren 1981).

Furthermore, reaction temperature needs to be carefully controlled. A binding reaction is temperature dependent and tissue is temperature sensitive; the transition temperature of the lipid cell membrane (usually around 15 °C) must be considered. Tissue and compounds may also behave differently according to pH, in particular when they have ionizable groups with an acid association constant, pKa, between 5 and 8. Therefore, it is advised never to consider experimentally measured values for, e.g., Kd or Ki as "absolute" but refer to them as "apparent" under the conditions of a particular assay. Those conditions should always be fully reported.

Types of Binding

Labeled ligands show different types of binding, depending on their physicochemical and pharmacological properties. There is specific binding to the target receptor, which occurs in a concentration range of 2 log units around the Kd value. This binding is saturable, as the number of receptors present is limited. Binding to the target receptor should be reversible and can be inhibited by a compound (competitor) that has the same pharmacological property, related to the target receptor, as the labeled ligand. Full inhibition of receptor binding is obtained at 100× Ki of the competitor (Ki, inhibition constant, see below), provided that the labeled ligand is used at concentrations around its Kd. A particular labeled ligand (or competitor) may bind to several different receptors within a narrow concentration range. In particular agonists or antagonists for various subtypes of dopamine, noradrenaline, serotonin, histamine, and acetylcholine GPCRs can show, what is called, a "broad receptor profile" (see Leysen 2002, 2004). Binding of a labeled ligand to several different receptors can be a problem, in particular when natural tissues are investigated (this is less of an issue when cultured cells transfected with a particular receptor gene are used and high receptor expression is obtained). If required, occluding agents can be added to prevent the labeling of nontargeted receptors. Labeled ligands also show "nonspecific" binding due to adsorption to tissue, this binding is linearly proportional with the labeled ligand concentration and non-saturable, i.e., non-displaceable. Nonspecific binding can be measured in an experiment where an appropriate competitor (with high affinity for the target receptor but preferably of a different chemical structure than the labeled ligand) is added at a concentration 100-1,000 times its Ki and incubated together with the labeled ligand and the tissue. Resulting binding represents nonspecific binding. Labeled ligands may show an additional type of binding, apparently of high affinity and saturable, hence displaceable, but related to a particular moiety in its structure and apparently unrelated to binding to a known biological target (e.g., [3H]spiperone, the 5-HT2A and dopamine D2 receptor ligand, labels "spirodecanone sites"); this binding can be occluded by adding a structural analogue of the labeled ligand, but which does not bind to the target receptor. When studying receptor binding in natural tissues, the various possible types of binding should be carefully investigated (Leysen 1984).

Types of Experiments

Saturation Binding Experiments or "Labeled Ligand Concentration Binding Isotherms"

Aim: To determine Kd and Bmax (maximum number of receptor binding sites in the tissue preparation) values

These assays are performed using a constant amount of tissue at a particular temperature (preferably 37 °C but sometimes lower temperatures are used) and pH (around 7.4) with increasing concentrations of labeled ligand. The specific binding isotherm, according to the simple law of mass action, follows the course of a hyperbola when the labeled ligand's free concentration on the abscissa and specific binding on the ordinate are plotted using linear scales. Half-maximal binding is reached at a concentration that equals the Kd value, and maximal binding is approached at four times the Kd value. Applied labeled ligand concentrations should span a range from 0.2 to 8 times Kd in a series of at least 12 points covering the rising part and plateau of the hyperbola (e.g., for a labeled ligand with Kd = 1 nM, appropriate test concentrations are 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0 nM). The "total binding" is measured in assays with the labeled ligand at its various concentrations, the tissue preparation, and a solvent sample; the "nonspecific binding" is measured in assays with the labeled ligand (at each of its concentrations), the tissue preparation, and a (non-labeled) competitor at a concentration of 100 times its Ki value. Specific binding is calculated by subtracting nonspecific binding from total binding at each labeled ligand concentration. To date, binding isotherms are calculated by nonlinear regression analysis and computerized curve fitting, which provide the Kd and Bmax values. In case a "one-binding-site model" does not fit the data, a "two-binding-site model" can be attempted. Curve-fitting programs for labeled ligand binding are available commercially, e.g., GraphPad Prism. The programs usually provide background on calculations with radioactivity, e.g., on converting bound radioactivity, measured in counts, into pmoles of bound labeled ligand. In the past, when computing power was not readily available, the hyperbola was transformed into a linear equation, e.g., a Scatchard plot.

Kinetic Binding Experiments

Aim: To determine the association and dissociation rate constants

Rate constants are relatively easy to measure for a labeled ligand.

To measure the association rate, a labeled ligand, at a given concentration (e.g., 3-4 × Kd), together with the tissue preparation, is incubated for various periods of time, and for each time, bound labeled ligand is measured. Association of ligands with nM affinity is fast and incubation times should be in the range from seconds up to 10 min. The maximal bound radioligand at equilibrium is usually reached within 2-4 min. Ligand-receptor association is a second-order reaction, involving two reaction partners: the free ligand and the free receptor. However, measured binding is the result of association and dissociation, and usually the free receptor concentration is small, and consequently the free ligand concentration drives the association. Therefore, the reaction proceeds according to the pseudo first-order kinetics. The plot of bound labeled ligand versus time is hyperbolic and can be transformed according to a pseudo first-order rate equation, in a linear plot:

Beq and bt are bound labeled ligand at equilibrium and at a given time. The pseudo first-order rate constant, kobs (s−1), is given by the slope of the line. For calculation of the association rate constant, kon (M−1∙s−1), the dissociation rate constant, koff (s−1), is needed.

The dissociation rate constant can be measured by incubating the tissue preparation with the labeled ligand at a given concentration (preferably not exceeding the Kd value) until equilibrium is reached and then either strongly diluting the reaction medium (more than 10 times) to favor dissociation and minimize reassociation or, more common, adding an excess of strong competitor to the reaction medium, which will induce displacement and hence dissociation of the labeled ligand. From that point onward, a series of incubation samples is taken to measure the remaining bound labeled ligand. The dissociation reaction is a first-order reaction, a semilogarithmic plot of log[bound labeled ligand] versus time is linear and the dissociation rate constant, koff, is given by the slope of the line × 2.3 (transformation of log10 into ln). The half-life of dissociation is given by t1/2 = 0.693/koff. Measurement of the dissociation rate of an unlabeled competitor requires a more elaborate procedure. The approximate half-life of dissociation of an unlabeled competitor can be assessed by an indirect method. Tissue with competitor at a given concentration (not exceeding 2 × Ki) is incubated until equilibrium and filtered over a glass fiber filter. The tissue (with bound competitor) adsorbed on the filter is rinsed for various periods of time, followed by short incubation of the tissue on the filter with a labeled ligand. The amount of labeled ligand that becomes bound is then an indication of the amount of competitor that has dissociated (Leysen and Gommeren 1986). Knowledge of the receptor dissociation rate of compounds has gained interest, in light of deriving information on factors that contribute to the duration of action of drugs and on the ease with which a drug bound to the receptor in vivo can be displaced, e.g., by the endogenous ligand for the receptor.

Competitive Binding Experiments

Aim: To determine the IC50 value (concentration producing 50 % inhibition) of a competitor Competitive binding experiments are used to compare different ligands acting at similar sites. The sigmoid curve obtained with a competitor should asymptotically approach the level of nonspecific binding, without surpassing it. Nonlinear curve-fitting programs can be used to estimate IC50 values. Curve fitting also yields a value for the slope (n) of the curve; n = 1, for competition at one binding site; n > 1 or n < 1 for multiple binding sites, binding of the competitor with different affinity to multiple states of the receptor, or involvement of cooperative binding. Curve-fitting programs allow for the analysis of data for multiple-binding-site models. IC50 values are dependent on applied concentration and Kd of the labeled ligand. The Cheng-Prusoff equation provides for the calculation of the equilibrium inhibition constant, Ki (equaling the equilibrium dissociation constant), which is independent of the applied labeled ligand:

The Cheng-Prusoff equation is derived from a simple model of competition at one and the same binding site; the same warnings of caution for the interpretation of Ki values as described earlier apply.

For further reading on different types of binding experiments and illustrations and details on the analyses of receptor binding data, see Keen and MacDermot (1993).

Antagonist Binding

For GPCRs and ligand-gated ion channels, the majority of studied labeled ligands and competitors are antagonists or inverse agonists. Antagonist binding to GPCRs has appeared to be insensitive to the occurrence of the receptor in various states (e.g., G-protein coupled or uncoupled). Antagonist binding curves (labeled antagonist saturation binding and antagonist-antagonist competition) have often been found to obey apparent single-site binding kinetics.

Agonist Binding

Agonist binding to GPCRs is sensitive to the state in which the receptor occurs. Agonists have substantially higher affinity for the G-protein-coupled than for the G-protein-uncoupled receptor. As a consequence, competition between a labeled antagonist and an unlabeled agonist often yields a shallow inhibition curve, pointing to multiple binding sites or binding to the receptor in multiple states. Inhibition curves of agonists can be influenced by additives in the incubation medium, in particular by agents that affect the coupling of the receptor to the G-protein. Guanosine triphosphate (GTP), or a stable analogue thereof, uncouples the receptor-G-protein complex. The addition of GTP to the medium will shift a shallow or biphasic agonist inhibition curve to the right, make it monophasic, and decrease the slope to approach 1 (see Lefkowitz 2004). Agonist binding to GPCRs is also affected by divalent cations, in particular Mg++ or Mn++, which may favor receptor-G-protein coupling and increase the agonist binding affinity. The effect of divalent cations on agonist binding should be experimentally investigated. A good example is the investigation of the effects of bivalent cations on the affinity states of the M1 muscarinic acetylcholine receptor (Potter et al. 1988). Labeled agonist concentration binding isotherms will similarly be influenced by additives in the incubation medium and affect the measured apparent Kd and Bmax values. When labeled agonist saturation curves are measured in a low concentration range, usually only the high-affinity binding constant of the agonist, KH, will be detected. The Bmax value measured with a labeled agonist may only represent the G-protein-coupled portion of the receptor and, for the same tissue sample, usually will be lower than the Bmax value measured with a labeled antagonist.

GPCRs for which labeled agonist binding has been amply studied and for several of which agonists are used as therapeutic agents are μ − opiate, 5-HT1A, 5-HT1B, 5-HT4, and various peptide receptors (e.g., NK1, NK2, NK3, CRF1). Examples of GPCRs that have been studied with both labeled agonists and antagonists are dopamine D2, 5-HT2A, 5-HT2C, 5-HT4, 5-HT6 and muscarine M1ACh receptors, and β1- and β2-adrenoceptors.

Allosteric Competitors

The natural ligand or a synthetic agonist that binds to the orthosteric site and a compound that attaches to the allosteric binding site can concomitantly occupy the same receptor without mutual inhibition of binding. Positive allosteric modulators will enhance and negative allosteric modulators will reduce the activity/affinity of an orthosteric agonist. The allosteric receptor interaction of compounds is usually studied in functional, signal transduction assays ("Receptors: Functional Assays"). Labeled ligand binding to an allosteric site may be used in competition binding assays to detect competitors that bind to the same allosteric site.

Ligand-gated ion channel receptors are known to have several different allosteric binding sites. The GABAA receptor was the first receptor for which the phenomenon was discovered with drugs that were in use as therapeutic agents, namely, the benzodiazepines. GPCRs of family 3, the GABAB receptor, and the metabotropic glutamate receptor subtypes are amply studied for positive and negative allosteric modulators.

Methods for In Vitro Receptor Binding Using Tissue or Cell Homogenates or Membrane Preparations

Tissues and Preparation

Tissues used for receptor binding are, e.g., dissected brain regions or organs, cultured cells (cell lines or primary cultures) or blood cells that express a particular receptor endogenously, or cultured cells with (abundant) expression of a particular receptor following the transfection of the cells with that receptor's cloned gene from a specific species (often human). Although, when conditions are appropriate, receptor binding can be performed on intact cells, in most cases the tissue is homogenized, and either the whole homogenate or, more often, a membrane preparation is used for the assay. Used membrane preparations are the "total particulate fraction" (i.e., the total fraction of membranes spun down at high centrifugation speed following extensive homogenization of the tissue in buffer with a blender) or a membrane preparation from separated subcellular particles (e.g., heavy and light mitochondrial fractions, plasma membrane fraction) following careful homogenization of the tissue in 0.25 M sucrose followed by differential centrifugation (see Laduron et al. 1978). Membrane preparations are washed by resuspension in medium and recentrifugation. All tissue preparation steps are to be carried out at 0-4 °C. Tissue preparations can be stored below −20 °C.

Incubation in Tubes and Multiwell Plates

Incubations can be performed in test tubes (volumes 0.5-2 mL) or 96-multiwell plates (0.1-0.2 mL). Incubation mixtures are composed of a sample of cells or membrane preparation suspended in buffer, an aliquot of labeled ligand to give a desired final concentration and, depending on the type of experiment, an aliquot of a competitor to give a desired final concentration (see types of experiments). The buffer has a particular pH (usually around 7.4) and can contain certain additives such as metal ions. Depending on the type of experiment, the incubation is run at a given temperature for a given time period (see types of experiments). For competition binding experiments, the incubation time should be sufficiently long to reach binding equilibrium.

Filtration Methods

Labeled ligand, bound to the tissue preparation and free-labeled ligand, can be separated by filtration over glass fiber filters (filters are sometimes presoaked in polyethyleneimine to reduce absorption of the labeled ligand to the filters) under suction, followed by rapid rinsing with ice-cold buffer. For incubation in test tubes, a Brandel harvester (Brandel, Gaithersburg, USA) is used; for incubations in 96-multiwell plates, various harvesting devices are available (e.g., Micromate 196 and Mac II). The radioactivity of bound labeled ligand collected on the filters is counted; for β-ray emitting isotopes (e.g., 3H-labeled ligands), the filters are dried, liquid scintillation fluid is added, and radioactivity is counted in a liquid scintillation counter; for γ-ray emitting isotopes (e.g., 125I), radioactivity can be counted directly in a γ-counter. For competition binding assays, 96-multiwell incubation, filtration, and counting methods and devices are appropriate; for saturation binding experiments with the determination of Kd and Bmax values and the determination of ligand association and dissociation rates, incubation in test tubes and filtration over separate filters that are counted in vials in a calibrated liquid scintillation counter are advised.

Scintillation Proximity Assay (SPA)

SPA is a homogenous assay that avoids the filtration step. For SPA, either poly-vinyl-toluene beads filled with scintillant or 96-multiwell plates with scintillant coated on the inner surface of the wells are used. The beads or plates are coated with membrane preparation. Samples of coated beads added in 96-multiwell plates are incubated with labeled ligand with or without competitor at a desired concentration in buffer. Since the tissue is not "free in solution," several hours of incubation time are required to reach binding equilibrium. After incubation, the multiwell plates are counted directly in a TopCount NXT. The scintillant will only detect radioactivity in its immediate vicinity, i.e., the radioactive ligand that is bound to the tissue coated on the beads or on the plates. The mix-and-read format technology is useful for high-throughput screening, but long incubation times have to be taken into account.

Nonradioactive Proximity Assays

The burden on the environment and the inherent high cost for the removal of radioactive waste encouraged the development of nonradioactive techniques for receptor binding. TR-FRET, making use of fluorescent probes, and AlphaScreenTM, which is based on chemiluminescence, are examples that are mainly applied for high-throughput competition binding assays. The technologies are based on the excitation of the "donor" probe (e.g., attached to the ligand), which triggers an energy transfer to the acceptor probe (e.g., attached to the receptor preparation) if they are within a given proximity. The acceptor probe in turn emits light of a given wave length, which is detected. The application of these techniques is limited, since it requires the development of ligands labeled with relatively large probes, resulting in a compound with a chemical structure different from that of the original ligand. The receptor binding characteristics of such fluorescent or chemiluminescent ligands will be altered and need to be fully investigated. The development of apt fluorescent or chemiluminescent ligands is a matter of trial and error.

Labeled Ligand Autoradiography

Autoradiography is a general technique allowing the visualization of the distribution of a radioactive ligand, bound to a molecular target in a tissue section. The radioactive ligand can be injected (iv) in an animal, followed by sacrifice of the animal, tissue dissection, and sectioning. Otherwise, tissue sections can be incubated with radioactive ligand in vitro. Tissue (often brain) sections (20 μm thick) usually are cut with a cryostat and mounted on a coated microscope glass support. Incubation is performed by overlaying the tissue section with a drop of incubation medium containing the radioactive ligand. After incubation, the tissue sections are quickly rinsed and dried. To "visualize" radioactive ligand bound to the tissue, different techniques have been used. The highest resolution, but requiring the longest exposure time (weeks up to several months), is obtained by exposing the tissue sections to a photographic emulsion. Shorter exposure times (several days up to 1 week) can be achieved by using a phosphor imager. The most recently developed device, the β-imager (Biospace Lab, Paris, France), can produce an image of the bound radioactivity following over night counting, and up to 15 microscope glass slides can be counted simultaneously. The images are analyzed and quantified by counting the number of β-particles emerging from the delineated brain areas by using the β-vision program.

For technical details on receptor autoradiography protocols, see Wharton and Polak (1993).

Apart from neuroanatomical mapping, autoradiographic techniques can also be applied to measure receptor occupancy by non-labeled drugs (Schotte et al. 1993). A drug, at varying dosages, systemically is given to laboratory animals. The animals are sacrificed, tissue dissected and sectioned, and tissue sections are briefly incubated with a radioactive ligand for the target receptor. The difference in labeling in a defined area on matching tissue sections from drug-treated and nontreated animals is a measure for the occupancy of the receptor by the drug administered to the animal. This "ex vivo" incubation technique is only applicable for drugs with a sufficiently slow dissociation rate from the receptor. Alternatively, the radioactive ligand can be injected iv to the animal that is systemically treated with the investigational drug; in this way, the competition for receptor binding between the drug and the radioactive ligand will take place in vivo. Subsequently, the animal is sacrificed, tissue dissected and sliced, followed by the quantification of the image. With the introduction of the highly sensitive β-imager in the 1990s, allowing images to be obtained in a few hours, receptor occupancy assays ex vivo became a feasible and highly valuable drug screening technique (Langlois et al. 2001).

Application of Receptor Binding

Pharmacologists sometimes demean receptor binding data and may even view it scornfully because binding does not say anything about function. Yet, receptor binding has contributed substantially to pharmacology. It has demonstrated the existence of receptors at the molecular level. It has contributed to the isolation and purification of receptor proteins, leading to the cloning of the first receptor gene. It has allowed the study of the localization of receptors at anatomical and cellular levels and of receptor dynamics, such as receptor desensitization, receptor endo- and exocytosis, and transport of receptors along axons (see Lefkowitz 2004).

In terms of drug discovery and drug profiling, receptor binding has revolutionized the field. Drug screening with receptor binding started in the mid-1970s and soon led to high-throughput, automated assays and super high-throughput assays, allowing for the screening of thousands of compounds in 1 day at many different receptors with a minimum of personnel.

Receptor binding profiles of drugs have revealed that many of the drugs that were in clinical use, such as antidepressants and antipsychotics, bound to several different receptors within a narrow potency range, e.g., the antipsychotic clozapine was found to bind to more than 20 receptors with such potency, and that all of them could be hit at a therapeutic dose (Leysen 2002, 2004). Receptor binding is the method of choice to investigate and demonstrate the selectivity of action versus the multiplicity of action of drugs. Measurement of receptor occupancy in the brain by drugs that are administered to animals, using ex vivo autoradiography, has become a key component of CNS drug discovery programs. It allows the determination of the dose range within which central receptors become occupied and does provide information not only on the extent of receptor occupation but also on the brain penetration of the drug.



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