Receptor binding assays are essential for new drug development. Radioligand binding assays using radioactive isotopes like tritium and iodine were traditionally used but have drawbacks. Homogenous scintillation proximity assays avoid separation steps but are expensive. Fluorescent ligand binding assays provide safer alternatives with techniques like direct fluorescence measurement and fluorescence polarization. Label-free methods like surface plasmon resonance allow real-time kinetic analysis. NMR and X-ray crystallography provide high resolution structural data of receptor-ligand complexes.

1. Techniques for measuring receptor binding – Its uses
in new drug development

2. Binding assays…….
….the cornerstone of all pharmacological studies….. indispensable for
the development of new drugs targeting specific receptors……….
• Homogeneous assay refers to an assay format allowing to make an assay‐measurement by a simple mix and
read procedure without the necessity to process samples by separation or washing steps

3. Radioligand binding assay:
• Radioligand binding is initiated by the incubation of cells, cell homogenates or purified plasma membrane preparations with an
adequate radioactively labelled drug – “The radioligand”
• Tritium [3H] and iodine [125I] are the most frequently used isotopes
• Separation of free and receptor-bound drug represents the most delicate step.
Filtration: the free radioligand passes through the filter whereas the receptor-bound radioligand remains on the filter.
• Counting the radioactivity on the filter allows the amount of receptor-bound radioligand to be quantified.
• Able to handle a large number of samples with relative ease as well as the commercial availability of a variety of filtration devices.
• The filters are usually of glass fibre, but sometimes it is also necessary to coat them with
polyethyleneimine or to siliconize them to prevent radioligand absorption to the filter.
• For 'high throughput screening', the radioligand binding may be performed in
microtiter plates with 96 or 384 or even more wells.
• After the incubation, the contents of the wells are filtered simultaneously with a cell harvester.
• For modern high-throughput screening, robots are used to handle screen compounds and buffers
as well as to perform the filtration step.

5. • For this technique, small scintillant-containing beads are already present in the incubation tube/well.
• The principle of the technique: is based on the assumption that the overwhelming majority of the free radioligand molecules are too far from the beads for the
scintillant to be activated whereas the receptor-bound radioligand is in close proximity to the beads and, hence, capable of stimulating the scintillant. Therefore,
the measured scintillation will mainly arise from bound radioligand molecules.
• Tritium and iodine-125 are most suitable, but SPA has also been successfully applied using carbon-14, sulfur-35, and phosphorus-33.
• Characterized by its speed, sensitivity, reliability
• No separation step is required
• It has become an important technique in high-throughput screening (HTS) for new drugs, and for investigating their biological interactions
• The light output can be quantified by a photo multiplier tube (PMT)-based scintillation counter or by a charge coupled device (CCD)-based image reader.
Scintillation Proximity Assay (SPA)

6. Drawbacks….
• The use of radioactive ligands as tracers in binding assays presents several drawbacks, both technical and financial.
• the assay requires multiple washing steps
• scintillation proximity assays (SPA) which can be performed in homogeneous conditions have been developed, but are still expensive due to
the production cost of beads
• health reasons
• radioactive waste disposal,
• delimitation of working area and staff medical follow-up.
• As a result, and because of high costs resulting from these restrictions, other techniques have been introduced to replace the use of
radioactive binding assays, such as fluorescence techniques, without completely supplanting them.

7. • Fluorescence-based binding assays are promising alternatives to radioactive assays.
• The fluorescent probes needed for such tests are safe to use
• Bright and stable probes have been developed over the past two decades and are now commercially
available
Fluorescent Ligands to Perform Binding Assays

8. Direct Measurement of Fluorescent Intensity of Ligand Bound onto the Receptor
• Simplest one to perform and consists in the measurement of the fluorescent ligand bound fraction after
separation from the free fraction
• The sensitivity of this method is often questionable because of the auto-fluorescence of biological
preparations.

9. Fluorescence Polarization Assays
• Fluorescence polarization techniques can be used to measure more precisely the amount of bound fluorescent ligand.
• These techniques are based on the excitation of the biological sample with polarized light.
• Constrained fluorophores will emit highly polarized fluorescence.
• On the other hand, freely moving fluorophores which can easily rotate during the short period between their excitation and the emission of fluorescence will
scramble the polarized light.
• Therefore, when exciting the sample with a polarized light, a fluorescent ligand freely diffusing in the medium with an important molecular mobility will
emit a non polarized fluorescence, whereas upon binding a receptor, the molecular mobility of the fluorophore within the receptor-fluorescent ligand
complex will frequently decrease, and a polarized fluorescence emission will be observed leading to a high fluorescence anisotropy.
• Fluorescent ligands are simply added to the biological sample and no washing steps are required to measure fluorescence polarization, making it very simple
to adapt for high-throughput screening and allowing straightforward kinetics studies.
• However, the affinity of the fluorescent ligands should be high enough to bind to the receptor at low concentrations and therefore get a bound ligand-to-free
ligand ratio high enough to give a significant fluorescent polarization signal
• Fluorescent polarization assays which have been described as fast, sensitive and inexpensive have not been extensively used.

10. Label free ligand binding assays
Surface Plasmon resonance (SPR):
• Surface plasmon resonance (SPR) is a phenomenon occuring at metal surfaces (typically gold and silver) when an incident light beam strikes the surface at a
particular angle.
• Depending on the thickness of a molecular layer at the metal surface, the SPR phenomenon results in a graded reduction in intensity of the reflected light.
• Biomedical applications take advantage of the exquisite sensitivity of SPR to the refractive index of the medium next to the metal surface, which makes it
possible to measure accurately the adsorption of molecules on the metal surface and their eventual interactions with specific ligands
• Measurement in real-time of the kinetics of ligand-receptor interactions and to the screening of lead compounds in the pharmaceutical industry
• (SPP-Surface plasma polaritons: electromagnetic waves generated when light interacts with charges on gold surface)

11. Plasmon-Waveguide Resonance (PWR)
• Similar to SPR
• Receptors are immobilized onto SiO2 surface
• Ligand binding changes amplitude, position and width of reflected lights.
• PWR measures the conformational changes of receptors.

12. Structure based ligand binding assays
NMR:
(An NMR instrument allows the molecular structure of a material to be analyzed by observing and measuring the
interaction of nuclear spins when placed in a powerful magnetic field)
• Well known techniques to study receptor-ligand complexes
• Measures the shift in protein resonances, and therefore used to identify ligand binding sites.
X-ray crystallography
• Provides 3D structure of receptor-ligand complex.
• (experimental science determining the atomic and molecular structure of a crystal, in which the crystalline structure
causes a beam of incident X-rays to diffract into many specific directions.)
• Crystal structures of protein–ligand complexes provide a detailed view of their spatial arrangement and interactions.
• These high-resolution structures have greatly increased our understanding of ligand recognition and receptor activation.