Homogeneous Non Radioactive Binding Assays

Fluorescent assays offer the advantage of eliminating the use of radioactivity. Peptide and protein ligands are most suitable for fluorescent labeling, whereas maintaining a functional fluorescently labeled small molecule can prove quite a challenge. Nonetheless, certain fluorescent small molecules have been synthesized (e.g., muscarinic receptor ligands are available from Invitrogen). A wide range of fluorophores is available for labeling, for example, BODIPY, Alexa and Cy dyes. BODIPY and Cy dyes are hydrophobic in nature, whereas Alexa dyes tend to be more hydrophilic. The physicochemical properties of these dyes can be important for maintaining functional activities of ligands.

Fluorescent applications for receptor binding involve fluorescence intensity, fluorescence polarization, and time-resolved fluorescence resonance energy transfer (TR-FRET). In some HTS labs, FMAT® (Applied Biosystems, Foster City, California) is also utilized for measuring receptor binding in 96-, 384-, or 1536-well plates.19 FMAT is a fluorescence macroconfocal, biological binding technology that enables mix-and-read assays with live cells and beads.

The most common nonradioactive format for HTS receptor binding assays is fluorescence polarization (FP) — a homogeneous format in which the ligand is labeled with a fluorescent tag. For each FP data point, two separate intensities (parallel and perpendicular) are measured and the polarization signal is calculated.20 All polarization values are expressed in millipolarization units (mP). The mP values of the FP assay are calculated based on the following equation:

where IS is the parallel emission intensity measurement and IP is the perpendicular emission intensity measurement. FP assays have a major advantage in that they eliminate the need for radioactivity. Another advantage of FP assays is that they involve no need to immobilize or capture receptors to a solid surface as in the case for SPA and flash plate formats.

However, one major disadvantage of the FP format for receptor binding assays is the difficulty in maintaining ligand affinity after labeling with fluorophores; specifically, the labeling can induce unwanted steric hindrance in receptor-ligand pairing. As mentioned earlier, not all ligands can be fluorescently tagged, particularly the small molecular weight antagonists and agonists commonly used in binding assays.

The receptor density should be relatively high (>1 pmol/mg) for a successful FP assay. Thus, nonrecombinant membrane receptor preparations are not feasible in this format. Also, in the case of green fluorescent tags, the cellular components present in the membrane preparation significantly increase the background, and colored or fluorescent compounds in the screening collection can contribute to the noise in the assay and/or false positives and negatives.

Labeling the ligand with red-shifted fluorophores such as rhodamine and red Alexa dyes can potentially alleviate this problem.21 Far-red dyes with excitation and emission spectra near the infrared region have been introduced recently and may offer several advantages compared to green fluorescence for HTS.22 Appropriate filters, dichroic mirrors and instrument settings should be checked before running any fluorescent assay; these are one of the most common factors for the failure in fluorescent assays and can be easily circumvented.

TABLE 9.1

Receptor Binding Assays in High Throughput Screening

Receptor Binding Assay

Radioactive Homogeneous

Compound Cells or

Interference Throughput Membranes?

TABLE 9.1

Receptor Binding Assays in High Throughput Screening

Receptor Binding Assay

Radioactive Homogeneous

Compound Cells or

Interference Throughput Membranes?

Filtration

Y

N

Low

Low

C + M

SPA beads

Y

Y

Medium

Medium

M

FlashPlate

Y

Y

Medium

Medium

M

FlashBlue beads

Y

Y

Medium

Medium

M

LEADseeker beads

Y

Y

Low-medium

High

M

Imageplate

Y

Y

Low-medium

High

M

Fluorescence

N

Y

Low-high

High

C + M

polarization

FMAT

N

Y

Low-high

Low

C + M

TRF

N

N

Low

Low

M

TR-FRET

N

Y

Low-medium

High

M

Finally, TR-FRET can be employed to measure receptor binding. TR-FRET utilizes time resolution to reduce background interference. It is based on long-lived fluorescence from lanthanides such as terbium (Tb) and europium (Eu) that can be measured after the short-lived background fluorescence has disappeared. The lanthanide-labeled ligand serves as the donor whereas a fluorophore-tagged WGA derivative binds to the membrane and serves as an acceptor for the emission of the lanthanide.

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