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Caroline Cardonnel | Sr. Applications Scientist | Molecular Devices

Introduction

The Tag-lite HTRF platform from Cisbio enables efficient labeling of proteins of interest on targeted sites with HTRF fluorophores. A cell surface receptor can be inserted into a SNAP-tag plasmid. Cells are then transfected with this construct and express the tagged receptor. The receptor is then covalently labeled with terbium (Tb) cryptate by adding SNAP-lumi4-Tb-substrate. To do a binding assay, the receptor’s ligand is labeled with an HTRF acceptor fluorophore, e.g. d2. When d2 ligand binds to the terbiumlabeled receptor, time-resolved fluorescence resonance energy transfer (TR-FRET) from receptor to ligand can be measured using an HTRF-compatible microplate reader (Figure 1).

Figure 1. Example of a Tag-lite cell surface binding assay. First, a GPCR gene of interest is inserted into a SNAP-tag plasmid. Then cells are transfected and express the Tag-lite GPCR. Next, the GPCR is covalently labeled with a cryptate donor (SNAP-lumi4-Tb-substrate). Finally, a binding assay is performed using labeled ligand.

Cisbio offers a variety of plasmids encoding tags and proteins of interest, as well as frozen cells already transfected with these constructs. The constructs enable the expression of a tagged protein, e.g. receptor, that can be labeled with terbium cryptate, while the receptor ligand (agonist or antagonist) is labeled with the acceptor fluorophore. The Tag-lite platform is ideal for a wide range of applications, such as receptor dimerization, ligandbinding assays, and second messenger assessment. Binding kinetics allow one to study the rate of association and dissociation of a drug-protein complex and are a necessary step in optimizing the in vivo efficacy of drug candidates¹.

The glucagon-like peptide-1 receptor (GLP1R) is expressed in pancreatic beta cells, where its activation stimulates the adenylyl cyclase pathway, resulting in increased synthesis and release of insulin. GLP1R has therefore been proposed as a potential target for the treatment of diabetes2. GLP1R is also expressed in the brain, where it is involved in the control of appetite and is potentially important in mechanisms of memory and learning.

Here, we show how the SpectraMax® i3x and SpectraMax® iD5 Multi-Mode Microplate Readers are used to perform robust, no-wash saturation binding assays using HTRF. GLP1R ligand binding was evaluated on the HTRF-certified SpectraMax i3x and iD5 readers using Tag-lite technology.

Saturation binding assays

Ligand binding assays are important in estimating the affinities of particular ligands for a receptor of interest and are critical to understanding the mechanism of interaction between ligand and receptor. Binding studies are therefore part of the drug discovery process to help design drugs that bind their targets selectively and specifically. Binding affinity defines the binding interaction between a single biomolecule, e.g. a receptor, and its ligands (e.g. agonists). It is typically measured with saturation assays and is represented by the equilibrium dissociation constant (K_d). The K_d is used to evaluate and rank the strength of the interaction between the ligand and its target(s). The smaller the K_d value, the greater the binding affinity of the ligand for its target. Using the appropriate concentration of ligand is essential to accurately determine the IC₅₀ and the inhibition constant (K_i) in competition or inhibition studies. Typically, a concentration that is at, or slightly below, the K_d value is acceptable. Using a concentration of ligand that is higher than the K_d value can make the drugs seem less potent than they appear in vivo¹.

Saturation binding assays measure total and non-specific binding of increasing concentrations of ligand (Figure 2). A fluorescent ligand is titrated into a solution containing a fixed amount of labeled cells, and this is incubated to reach equilibrium. Upon binding of the fluorescent ligand to the receptor, TR-FRET occurs and is measured with an HTRF-certified microplate reader. The HTRF ratio obtained represents the total binding. Non-specific binding is measured as a negative control using unlabeled ligand, accounting for non-specific binding of the labeled ligand to the receptor, non-receptor molecules, or the microplate².

Figure 2. Tag-lite saturation binding assay. Total binding measurement: Upon binding of the fluorescent ligand to the receptor, TR-FRET occurs. Non-specific binding measurement: unlabeled (in excess) and fluorescently labeled ligand compete for binding to labeled GPCR; unlabeled ligand binds to the receptor and TR-FRET does not occur.

For this measurement, the fluorescently labeled ligand is titrated into a solution containing a fixed amount of labeled cells and a 100-fold molar concentration of unlabeled ligand. The labeled and unlabeled ligands compete for binding to labeled GPCR. As the non-specific ligand is in excess, it binds to the receptor and TR-FRET does not occur. The HTRF ratio obtained from this titration represents non-specific binding. Specific binding is calculated by subtracting the non-specific binding from the total binding at each fluorescently labeled ligand concentration.

Materials

• Tag-lite GLP1R-expressing, terbium-labeled, frozen ready-to-use cells (Cisbio cat. #C1TT1GLP1)
• Tag-lite buffer (Cisbio cat. #LABMED)
• GLP1 receptor red agonist (Cisbio cat. #L0030red)
• Exendin 4 (Sigma-Aldrich cat.# 1269105)
• White, low-volume 384-well microplates (Greiner cat. #784075)
• SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices cat. #i3x) with HTRF Detection Cartridge (Molecular Devices cat. #0200-7011)
• SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices cat. #iD5) with HTRF Detection System (Molecular Devices cat. #6590-0144, includes Enhanced TRF module and HTRF filters)

Methods

Cells

Cells were prepared according to the product insert³. Frozen cells were thawed at 37°C and transferred to a tube containing 5 mL of 1X Tag-lite buffer (TLB). The tube was centrifuged for five minutes at 1200 x G at 4°C. Supernatant was then aspirated and the cell pellet was resuspended in 2.7 mL of 1X TLB.

Labeled (fluorescent) ligand

GLP1 Receptor red agonist is an Exendin 4 derivative labeled with a red fluorescent HTRF probe. The 400 nM concentration of red agonist was prepared by diluting the stock concentration in 1X TLB (see kit insert for stock concentration). Ten additional 1:2 dilutions were then prepared using 1X TLB to obtain final concentrations ranging from 100 nM down to 0.097 nM.

Unlabeled ligand

A 40 μM final concentration of unlabeled Exendin 4 ligand was prepared by diluting the stock concentration in 1X TLB (see product insert for stock concentration). This corresponded to 100-fold molar excess of the maximum labeled ligand concentration of 400 nM.

Assay plate setup

Reagents were dispensed following Cisbio’s ligand binding protocol (Figure 3). 10 μL of GLP1 receptor cells were dispensed into wells of a 384-well white plate. 5 μL of TLB was added to the total binding wells while 5 μL of unlabeled ligand was added to the nonspecific binding wells. Finally, 5 μL of GLP1 receptor agonist exendin 4-red was then added to all wells. Assay plates were incubated for two hours at room temperature (RT) and read with optimized instrument settings (Table 1) on the SpectraMax i3x and iD5 readers. Both microplate optimization and read height adjustment were performed to ensure optimal assay sensitivity and dynamic range.

Table 1 Instrument settings used for HTRF with terbium donor and red acceptor.

Figure 3. Assay setup for a 384-well low-volume plate. The plate was covered and incubated for two hours at room temperature. TR-FRET was measured on the SpectraMax i3x and iD5 readers (see Table 1 for instrument settings).

Data Analysis

Analysis of HTRF assays involves Cisbio’s patented ratiometric reduction method based on the two emission wavelengths detected. Donor emission at 616 nm is used as an internal reference, while acceptor emission at 665 nm is used as an indicator of the biological reaction (binding) being assayed. This ratiometric measurement (ratio of acceptor to donor fluorescence) reduces well-towell variation and eliminates compound interference. Delta F, calculated in step 4 below, reflects signal to background of the assay and is useful for inter-assay comparisons. Results are calculated from the 665 nm / 616 nm ratio and expressed in Delta F as follows:

Data were generated and analyzed using SoftMax® Pro Software, which contains preconfigured HTRF protocols to simplify detection and analysis.

Results

The HTRF ratio was measured for each labeled ligand concentration for the total and non-specific binding wells. The specific binding was calculated by subtracting the non-specific binding from the total binding at each fluorescent ligand concentration. Data were analyzed as described above and graphed with SoftMax Pro Software using a bi-rectangular hyperbola curve fit (Figure 4). Best results were obtained with the reader settings indicated in Table 1. The SpectraMax i3x reader generated a saturation curve (not shown here) similar to that obtained with the SpectraMax iD5 reader. The bi-rectangular hyperbola curve fit allows calculation of the K_d as the curve fit parameter B on the specific binding saturation curve. A K_d value of 5 nM or lower was established by Cisbio to confirm the ability of the microplate reader to successfully measure Tag-lite assays with red acceptors. The Kdvalues were 0.816 nM for the SpectraMax i3x reader and 2.347 nM for the SpectraMax iD5 reader (Table 2), confirming the ability of both instruments to detect these Tag-lite assays.

Figure 4. Saturation binding curves measured on the SpectraMax iD5 reader. A saturation binding assay measures total and non-specific binding for increasing concentrations of ligand at equilibrium. The ratio of the specific binding (blue curve) is calculated as the ratio of the total binding (red curve) minus the ratio of the non-specific (green curve) binding at each concentration.

Table 2. Results summary for Tag-lite binding saturation curves.

Conclusion

SpectraMax i3x and iD5 readers can be equipped with an HTRF detection cartridge or Enhanced TRF module and filters, respectively. Both readers demonstrated their ability to detect the HTRF signal from the terbium donor and red acceptor combination, and they could be used for saturation studies using the Tag-lite technology. Data acquisition and analysis are simplified using SoftMax Pro 7.0.3 (or higher) Software with preconfigured HTRF protocols.

References

1. https://www.htrf.com/htrf-technology
2. http://learn.cisbio.com/hubfs/cisbio-ls/docs/application-notes/Determination%20of%20association%20(kon)%20and%20 dissociation%20(koff)%20rates%20constants%20using%20the%20Tag-lite%20platform.pdf?hsCtaTracking=10f4759a-f255-4f21-aa95- b03c24b839e9%7C736c51a4-4301-4256-ab76-a10df00e9593https://cisbio.wistia.com/medias/i2eup2dah5
3. https://fr.cisbio.eu/media/asset/c/i/cisbio_dd_pi_c1tt1glp1.pdf

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