Opioid Receptor Binding Assay

Background and Assay Principle           

 The β-arrestin GPCR assay measures the activation of G-protein coupled receptors using a luciferase reporter system (19). The luminescent product of luciferase activity, in this assay, is used to measure ligand binding to the GPCR as a way to identify potent κ agonists. The assay uses “fragment complementation technology” which uses an enzyme split into fragments (donor and acceptor), that will join and produce an active form of the enzyme when brought into close proximity. The activated enzyme can then be used to produce a measurable signal.

Step 1: Ligand binding

In this assay, complementary fragments of β-galactosidase are fused to the G-protein coupled receptor and β-arrestin, two important players in opioid signaling. The cell line used in this experiment expresses opioid receptors that are fused to a peptide fragment of β-galactosidase. These cells also express β-arrestin that is fused to the second, complementary enzyme acceptor fragment. The assay design begins with ligand binding to the GPCR. The ligand is the molecule of interest, which for this purpose is the synthetic opioid or the peptide opioid being studied.

Step 2: β-arrestin recruitment

When a ligand binds and activates a G- protein coupled receptor, it recruits β-arrestin. β-arrestin is fused to the complementary enzyme acceptor (EA) fragment of β-galactosidase; and when it is recruited to the GPCR, the two enzyme fragments (enzyme donor and acceptor) are brought into close proximity.

Step 3: Fragment complementation results in active β-gal

The ProLink™ peptide fragment of β-gal fused to the GPCR joins with its enzyme acceptor counterpart that is fused to β-arrestin. This results in an active β-galactosidase enzyme. The chemistry done by this activated enzyme is important for detection and quantification.

Step 4: β-Galactosidase cleaves sugar to produce luciferin

Fragment complementation of β-Galactosidase is important because of the chemistry performed by the active enzyme. More specifically, active β-galactosidase cleaves the sugar from 6-O-galactopyranosyl-luciferin to produce luciferin. The starting molecule in this reaction is added to cells as a part of the detecting agent.

Step 5: Luciferase activity provides luminescent signal                                         

The product of step 4, Luciferin, is the substrate for Luciferase, which is an enzyme used in many biochemical assays. The result of luciferase activity is a luminescent signal, which allows for quantification of the opioid agonist binding (given that the production of the substrate for this reaction is the result of GPCR activation).

Measurements and readout 

The opioid receptor binding assay yields an EC₅₀ value for the molecules tested. This value is termed the “half-maximal effective concentration” of the molecule of interest. In other words, the concentration of ligand at which half of the maximum signal is observed. A lower EC₅₀ indicates better binding affinity. Typically, EC₅₀ values are determined by fitting a dose response curve to the data.

Applications for opioid peptides

This assay has been used to quantify the efficacy of different peptide opioids being explored as less addictive alternatives for traditional pain management drugs. For example, Hughes et.al. used this assay to determine the most potent peptides in their library of κ agonists. Using this assay, they identified their most interesting derivative, JT09 for further investigation which had an EC₅₀ of 29.9 nM(20, 21). We discuss this finding further under “Recent Research.”