by Catherine Shaffer
Radioactive labels for ligands have long been a part of the biological laboratory repertoire. Radioactivity gives a clear, unmistakable signal, and its use is fairly straightforward; however, radioactive ligands are relatively expensive, problematic to dispose of, and the short half-lives of some isotopes can be inconvenient. Scientists are looking for alternatives to radioactive ligand binding assays, turning to fluorescent and chemiluminescent labels, and in response, many manufacturers are discontinuing their radioactive ligand products. In most cases, the new non-radioactive ligands can yield equal or better results than a radioactive ligand.
One such technology is the Tag-lite system from Cisbio. Tag-lite uses homogeneous, time-resolved fluorescence (HTRF) to assay GPCR ligand binding. A suicide enzyme is fused to the N-terminus of the GPCR 7™ fragment, and the fluorophore-labeled substrate is included in the Tag-lite medium. This system allows the expression of a tagged GPCR at the cell surface without affecting its activity. The assay can be used for ligand binding studies, receptor activity assays, and GPCR dimerization studies.
Says François Degorce, head of marketing for Cisbio Bioassays, “Tag-lite offers the same results as using regular radioactivity, but with better quality and without its disadvantages. We are seeing a strong push from the marketplace to eliminate the use of radioactivity in drug discovery, as
Pharma companies incur heavy costs for the license to use it and to manage the waste. Many of our customers have been very excited to see that Tag-lite and radioactivity are similar in output; we have compared results from the two for a number of different peptidic and non-peptidic ligands and the Kd's are comparable. This enables us to provide researchers with a reliable, cost-effective alternative.”
Another choice for a fluorescent ligand binding assay is the LanthaScreen® system from Invitrogen, part of Life Technologies Corporation. LanthaScreen® is a time-resolved fluorescence resonance energy transfer (TR-FRET) based technology. TR-FRET has the advantage of less susceptibility to compound interference and can be used with many types of targets. The system uses a long-lifetime terbium or europium chelate as the FRET donor and a variety of acceptor fluorophores, including fluorescein, green fluorescent protein (GFP), and Alexa Fluor® 647. Bonnie Hanson, PhD, a principal scientist in the cell systems division at Life Technologies, says, “The time-resolved aspect gets away from a lot of the background fluorescence coming from conventional fluorophores, which emit very quickly after excitation. With TR-FRET, the emission is measured after a delay so only the TR-FRET from long-lifetime species is detected."
Invitrogen also offers a fluorescence polarization assay kit for the hERG ion channel, branded the Predictor™ assay. This non-radioactive ligand binding assay uses a fluorescently labeled tracer that competes with other ligands. The hERG-tracer complex is very large and tumbles slowly in solution, giving a highly polarized signal under polarized light. When a ligand binds, it displaces the tracer, the tracer rotates out of the plane of polarized light, and the polarized light signal is lost. Says Hanson, “The Predictor assay is the only non-radioactive binding assay that is out there right now for the hERG channel.”
In the category of GPCR binding studies, one recurring problem is that binding assays are generally limited to Gi-coupled receptors, since they are more abundant in cells and have a faster GDP/GTP exchange rate. This makes it difficult to study orphan GPCRs—those for which no ligand has been identified. The PathHunter product line from DiscoveRx is a non-radioactive, functional, cell-based assay that can be used to study ligand binding independent of G-protein coupling status. It is based on the beta-galactosidase enzyme. The two portions of the split beta-gal enzyme are fused to the C-terminus of beta-arrestin and the GPCR of interest. When beta-arrestin binds to the GPCR, the two parts of the beta-gal enzyme are reunited, making the enzyme active. The system is independent of G-protein coupling status, so it is a universal system for studying any GPCR, especially orphan GPCRs.
In addition to binding, PathHunter can be used as a functional assay. Elizabeth Quinn, PhD, senior product manager for the PathHunter product line, explains: “Really we're the only technology in the world that can give you both binding information and functional readout. What our technology allows you to do is to differentiate mode of action and uncover multiple pharmacologies from a single compound. We can differentiate agonist, antagonist, inverse agonist allosteric modulators, and arrestin-biased ligands.”
Ligand binding is one of the functions of the In Cell Western™ from LI-COR Biosciences. All of LI-COR's technology is based on near-infrared fluorescence—wavelengths of 650 nm and above. The In Cell Western™ (ICW) is an immunocytochemistry assay, which is a type of assay that has been around in one form or another for quite a few years. In the ICW assay, ligands are fluorescently labeled with IRDye 800CW to detect ligand binding to cellular proteins, especially cell surface receptors. Unlike using microscopy to look at the assay result in the cells, the ICW assay uses direct detection to measure total well fluorescence in one channel, while the addition of a DNA stain in a second channel allows the data to be normalized for cell number.
Says Harry Osterman, PhD, principal scientist at LI-COR Biosciences, “What you get is very nice data showing ligand binding. You can do competitive assays as well.” Examples of ligand binding applications for the In Cell Western include assaying for ligand binding in the Ras/Raf/MEK/ERK pathway by epidermal growth factor (EGF) to epidermal growth factor receptor (EGRF). Adds Osterman, “Another example is 2-deoxyglucose, which we believe binds to one of the glucose transporters. All of these were developed and assessed using the In Cell Western assay.”
A non-radioactive ligand binding assay is a viable alternative to the use of radioactive labels. Advances in detection technology for fluorescence and chemiluminescence make direct detection of non-radioactive ligands comparable to radioactive ligands, without the mess and expense. GPCRs and other cell surface receptor proteins are ideal subjects for non-radioactive ligand binding assays, which can be done in vitro or in vivo in most cases without disrupting the biological processes under investigation. Although radioactivity is undoubtedly still advantageous in many situations, new options for non-radioactive ligand binding are worth considering.