Study on homogeneous high-throughput method in viable cell GPCR functional and surface receptor binding assay

Study on homogeneous high-throughput method in viable cell GPCR functional and surface receptor binding assay

Introduction:

G protein-coupled receptors ( GPCRs ) are currently the largest branch of the cell surface receptor family, and 40% of current drug screenings are performed on GPCRs . In the screening process of lead compounds, the efficacy of these potential drugs (compounds) should be evaluated first (for example, in cardiovascular diseases and other fields). If you want to fully understand the mechanism of action of these drug candidates, such as receptor and ligand binding, A large number of experiments are required for body aggregation or receptor internalization. Therefore, we need to establish a highly automated and highly sensitive test method for high-throughput screening work. The Tag-lite cell screening platform was originally designed to increase the flexibility of cell surface receptor studies. This platform can be designed for a wide range of different protocols for pharmacological properties research and development of therapeutic antibodies, making it ideal for a wide range of primary and secondary screening operations. Here we show the results of using agonists and antagonists of GPCR research Tag-lite receptor ligand binding assay and internet microporous SpectraMax® Paradigm plate reader machine, including dopamine D3, glucagon GLP1 And Mu opioid test. The key indicators for measuring the test are the Z value and the window value, and we also give the results of the tests for cAMP , pERK and pAKT .

method:

Principle of TR-FRET detection

HTRF technology ( Homogeneous Time-Resolved Fluorescence ) is based on the TR-FRET technology (that is, combining TRF and FRET technologies). The HTRF donor fluorescent dye molecule uses a cryptate compound of Eu3+ cryptate or Lumi4®-铽( Tb2+ cryptate ), which is a recent collaboration with Lumiphore . A wide variety of acceptor molecules can be used as red or green fluorescent emitters. When a biomolecule interacts with two fluorescent dye molecules, a portion of the energy is captured by the acceptor fluorescent dye molecule after being excited by the donor molecule labeled with the cryptate compound. The figure below shows the standard test procedure and detection principle for TR-FRET in the use of Eu3+ cryptate as donor molecule and XL665 as acceptor molecule.

Results and discussion:

Tag-lite live cell GPCR binding assay

The Tag-lite detection platform uses HTRF dye molecules to target the proteins of your interest. The Cisbio Bioassay System provides a plasmid encoding the TAGs tag and the protein sequence of interest, or a cryopreserved cell after transfection of the constructed plasmid. When the ligand (agonist or antagonist) of the receptor binds to the receptor, the protein that constructs the expression tag can be labeled with the cryptate compound of the lanthanum element. Tag-lite detection technology is a widely used and ideal detection platform for mechanism research, receptor dimerization, ligand binding, and second messenger detection.

Figure 1: Tag-lite cell surface binding test principle

cAMP test

The cAMP assay kit uses HTRF technology to detect cAMP levels directly from suspended or adherent cells . We used a monoclonal antibody against cAMP labeled with Eu3+ cryptate as a tracer molecule, and an immunological competition method to analyze endogenously expressed cAMP and cAMP labeled with d2 fluorescent dye molecules in order to evaluate SpectraMax Paradigm.   with   The titer curve of the performance of the M5e multi-function plate reader in such tests is shown in the figure below.

Figure II:   SpectraMax Paradigm   And the SpectraMax M5e multi-function plate reader for cAMP concentration titer curve evaluation: W = 21.4, Z' = 0.91 . M5e: W = 15.5, Z' = 0.97

HTRF Cell Kinase Kit to detect pAKT and pERK levels

Cellul'erk is a HTRF- based assay reagent method for the detection of phosphorylated AKT ( Ser473 ), which detects activated ERK1/2 and AKT directly in intact cells . When the receptor is stimulated, the activation of the relevant kinase is triggered, and the amount of phosphorylated protein in the cell lysate can be detected by the kit. The phosphorylated protein was measured using a double antibody sandwich method, and both antibodies were monoclonal antibodies. One of them is an antibody against phosphorylated protein, labeled with Eu3+ ; the other is an antibody against other sites of the protein, labeled d2 .

Figure 3: After stimulation and unstimulated cell lysate as the internal quality control conditions of the test to detect the quality of the obtained results, the window shows two quality control

Dopamine D-3 binding assay

Dopamine receptors are members of the G- protein coupled receptor family and are mainly distributed in the central nervous system of vertebrates. Dopamine receptors are involved in many nerve conduction processes, including excitability, cognition, memory, and small muscle development. Abnormal dopamine receptor signaling can cause nervous system disorders, so dopamine receptors are the target of common neurological drugs. Antipsychotic drugs are antagonists of dopamine receptors, while psychostimulants are usually dopamine receptor agonists. Dopamine D3 is based on the cell Tag-lite binding assay, which facilitates the detection of agonists and antagonists and the development of new antipsychotics.

Figure 4: Binding assay: HEB293 cells express D3 receptor labeled Tb cryptate, then the receptor is incubated with the ligand for 90 minutes, and different delays and detection times are set on the two instruments for evaluation. The shorter the detection time. Will increase the W and Z' values.

Delay – 20s; Int – 200s
Delay – 20s; Int – 500s

We evaluated the performance of the dopamine D3 Tag-lite binding test after optimizing the settings using SpectraMax Paradigm and SpectraMax M5e , respectively . The performance of the two instruments in the competition inhibition test is as follows. Paradigm's ideal results also unmatched convenience, allowing users to upgrade their systems to meet future needs at any time by purchasing optimized cartridges.

Figure 5: Competition inhibition assay, which detects several unknown agonists and antagonists of dopamine receptors and derives IC50s values. The cells were incubated in a 6 nM receptor ligand binding system and a receptor agonist PPHT and bromocriptine or antagonist NAPS, and the two instruments yielded similar IC50s values.

Optimize SpectraMax Paradigm   with   SpectraMax M5e two instrument settings

By optimizing the delay time and detection time for optimization, we found that when both instruments shorten the delay time and detection time, a larger window value is obtained.

Figure 6: Using the SpectraMax Paradigm instrument to set different detection times and delay times yields different titer curves (left) and test window values W (right), which are optimized for a 20us delay time and 200us detection time.

Mu   Opioid receptor binding assay:

The μ - opioid receptor has a higher affinity for endorphins (μ = morphine) and the μ receptor agonist is the opium alkaloid morphine. Activation of μ receptors by receptor agonists, such as morphine can relieve pain, calm, lower blood pressure, refreshed and reduce respiratory rate, long-term or high-dose opioids can lead to decreased tolerance, if the opioid intake is excessive Causes death due to lack of oxygen due to difficulty in breathing. Opiate antagonists (such as naloxone and naltrexone) can be used to counteract the effects of excessive opioid intake.

Here we present the results of the live cell receptor binding assay. Mu- opioid receptors are labeled with the Tb/ Red Receptor ( T-type ) and the SpectraMax Paradigm multi-function reader is used to evaluate the match. In vivo binding and inhibition constants for reaction with different compounds.

Figure 7: Results of the Mu- opioid ligand binding and competition inhibition assay using the SpectraMax Paradigm multi-function reader system (right), the test window and Z values ​​were 5.3X and 0.89 , respectively .

Glucagon   GLP-1 binding test:

The GLP1 receptor is thought to be widely expressed in islet β cells. Activation of the GLP1 receptor stimulates the adenylyl cyclase pathway, which ultimately leads to increased insulin release and release. Therefore, the GLP1 receptor is a potential target for the treatment of diabetes, and the GLP1 receptor can also Expressed in the brain, involved in controlling appetite, regulating memory and learning. The glucagon GLP1 receptor ligand binding and competition inhibition assays were evaluated by the SpectraMax Paradigm Multi-Purpose Reader System .

Figure 8: SpectraMax Paradigm Multi-Purpose Reader for test results of GLP1 peptide receptor agonists with test window values ​​and Z values ​​of 19X and 0.78 , respectively .

in conclusion:

• The SpectraMax Paradigm and M5e Multi-Purpose Readers perform exceptionally well in several Cisbio assays: cAMP assay, pERK and PAKT kinase assays, and Tag-lite ligand binding and competition assays.
• By optimizing the settings of the two instruments, the Tag-lite test (using a 384- well plate) was evaluated for their performance by the test window and Z value, and the results were very good.
• Shorter delays and inspection times for the SpectraMax Paradigm multi-function reader will give better results.
• The combination of the SpectraMax Paradigm Multi-Purpose Reader and Tag-lite technology will be a powerful, high-throughput platform for studying cell surface binding and functionality.

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