Receptor binding assays have become increasingly popular since the introduction of multiple filtration platforms for automation. Recent advances have also enabled researchers to perform receptor binding assays in a homogeneous format so that no additional separation steps are required. The selection of an ideal radioligand for a binding assay depends on ligand stability, specific activity and selectivity; higher values for all of these properties are preferred. The affinity of the ligand for the receptor also dictates the choice of the radio label.
Suppliers such as Amersham Biosciences (Piscataway, New Jersey) and Perkin-Elmer (Boston, Massachusetts), sell iodinated and tritiated ligands for several GPCRs. If the receptor density is high and the ligand has high affinity for the receptor, either 125I- or 3H-labeled ligands can be used. If the receptor density is high and the ligand has low affinity, a 3H-labeled ligand is a better choice, especially for scintillation proximity assay (SPA). If the receptor density is low and a high affinity ligand is available, a 125I- labeled ligand is better for screening. Typical expression levels above 50,000 receptors per cell are required for 125I-labeled ligands and 10-fold higher receptor densities are required for 3H-labeled ligands.
Another important step is deciding whether to use an agonist or antagonist ligand for an HTS campaign. Indeed, it is generally accepted that GPCRs can exist in low-and high-affinity states, depending on whether they interact with G proteins.1516 While this has been a topic of discussion for many years, a favorable explanation may be that GPCRs are in a high affinity state when interacting with a guanosine nucleotide-free G protein. Agonists bind with a higher affinity to a high affinity state of a receptor, whereas antagonists bind with equal affinity to either state of a receptor.
There are exceptions to this generalization, for example, ergoline derivatives such as LSD and lisuride are serotonergic or dopaminergic agonists that do not readily distinguish between low- and high-affinity states of these receptors. In whole cell binding assays, agonists activate the receptors and can lead to homologous or heterologous desensitization of receptors. Also, agonists tend to label only portions of the receptors. As a result, the signal may be too low, especially when the receptor is expressed at a low level. Labeled antagonists are generally preferred in HTS receptor binding assays when the option is available unless the biology requires an agonist screen.
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