GPCR cAMP Product Solutions

Advantages of cAMP Product Solutions

Optimized for a Variety of Applications

• Perform functional screens using small molecules or biologics (antibodies, peptides)
• Determine pharmacological profiles and rank order ligands
• Detect neutralizing antibodies (immunogenicity studies using serum and plasma)
• Characterize phosphodiesterase inhibitors

HitHunter cAMP Assays

• Eliminate optimization steps with an assay that has been designed and qualified for use with over 125 cAMP Hunter cell lines
• Achieve precise characterization of ligand pharmacology with large assay windows, sensitive detection, and wide dynamic range for ligand bias studies and many more applications
• Reproducible assay performance without fluorescence or serum interference and no need for specialized equipment

cAMP Hunter™ Cell Lines and Assay Ready Kits

• Over 125 stable cell lines with unlimited culture and overexpressed naturally coupled, wild-type GPCRs
• Ultra-convenient, complete eXpress kits for quick assessment without the need for cell culture
• Ready-to-use bioassay kits provide cryopreserved cells and optimized reagents with high lot-to-lot reproducibility to support the lifetime of the biologic drug

ChemiSCREEN Cell Lines

• Cell lines containing a promiscuous G protein, Gα15, to help funnel signaling to a common calcium readout, regardless of G protein coupling status
• High functional expression of receptors with the ability to assay both agonist and antagonist in a single well
• Stable cell lines for continuous culture and increased reliability and reproducibility assay results
• Perform orthogonal assay confirmation such as second messenger signaling, e.g. cAMP, and ERK phosphorylation, for most targets

ChemiBRITE Cell Lines

• Cell lines modified to flash with added brightness upon GPCR activation
• Exceptional high signal-to-background ratio with no assay interference with autofluorescent compounds
• Tool box capabilities allows generation of your own stable cell lines

ChemiSCREEN and ChemiBRITE Frozen Cells

• Ready-to-Assay GPCR frozen cells come with plating media and enough cells to run either 96- or 384-well plate assays in full or half plate formats
• Qualified for cAMP and calcium second messenger assays with results within 24 hours to accelerate your data generation

HitHunter cAMP Assay Principle

The cAMP G protein-dependent pathway involves a heterotrimeric (α/β/γ) G-protein containing a GDP molecule bound to the Gα subunit, which holds the trimer together. Upon activation, GDP is exchanged for GTP, leading to the dissociation of the Gβ/Gγ dimer from Gα. Both parts remain anchored to the membrane and become free to act upon their downstream effectors and initiate unique intracellular signaling responses. The activated Gα subunit interacts with and regulates many effector molecules such adenylyl cyclase that can ultimately lead to the accumulation of cAMP (a second messenger).

HitHunter cAMP assays are competitive immunoassays amenable for high-throughput and utilizes the Enzyme Fragment Complementation (EFC) technology where a fragment ß-galactosidase (ß-gal) enzyme donor (ED) is conjugated with cAMP. This ED-cAMP conjugate and cellular cAMP compete for binding to an anti-cAMP antibody (Ab). With low levels of cellular cAMP, most of the ED-cAMP binds to the cAMP Ab, making the ED-cAMP unable to complement with the enzyme acceptor (EA). With high levels of cellular cAMP, the anti-cAMP antibody becomes saturated allowing the ED-cAMP complex to complement with the ß-gal acceptor (EA) and form an active enzyme. The active enzyme then subsequently hydrolyzes a substrate to produce a chemiluminescent signal that is directly proportional to the amount of cAMP in the cells.

HitHunter cAMP Assays are cell-based functional, immunoassays with a chemiluminescent readout. The assays are available as complete kits that are robust, highly sensitive, and easy-to-use to study GPCR activity through cAMP production. The kits contain all the reagents needed for the detection of cAMP from whole cells expressing Gαi- and Gαs-coupled receptors induced with a biologic or small molecule ligand. The flexible assay system has been designed to work in agonist or antagonist mode for 96- and 384-well plate formats. After plating and stimulation of cells, the user simply adds the HitHunter cAMP Assay reagents to the cell following the homogeneous, simple protocol provided.

Accurately Rank Molecular Potency of Ligands

HitHunter cAMP assays provide powerful tools for drug discovery screening and revealing of the correct ligand rank order and pharmacological profile. Profiling experiment using a cAMP Hunter CHO-K1 adrenergic receptor β2 cell line to test five agonists. The data highlights the receptor’s sensitivity to the various agonists, ultimately revealing the correct rank order of the agonists and showing the agonist salbutamol (EC₅₀ of 160 nM) is less potent than the control ligand isoproterenol (EC₅₀ of 1.7 nM).

Easily Determine Pharmacological Profiles of Complex Assay Formats

Large assay windows and sensitive detection makes HitHunter cAMP assays ideal for studying complex assay formats like Gαi-coupled receptor antagonists. Comparative study of two antagonists of the Gαi-coupled metabotropic glutamate receptor 2 (GRM2) using a cAMP Hunter CHO-K1 GRM2 cell line. Results indicate that the Antagonist 2 (IC₅₀ of 8 nM) is a more potent inhibitor compared to Antagonist 1 (IC₅₀ of 24 nM).

Obtain Excellent Reproducibility and High Sensitivity for Testing Biologics

Perform quality control assays, including potency assays for lot release and stability testing in biologics. A. Evaluation of lot-to-lot consistency using a GLP agonist, GLP-1(7-36), and the cAMP Hunter Gαs-coupled GLP-1R bioassay that incorporates the HitHunter cAMP Assay for Biologics. Results indicate the assay’s excellent reproducibility for all three lots tested with S:B ratios over 10-fold and EC₅₀’s ranging from only 76 pM to 86 pM. B. Evaluation of lot-to-lot consistency using a biologic ligand, SDF1α, and the cAMP Hunter Gαi-coupled CXCR4 receptor (chemokine C-X-C motif receptor 4) cell line. Results show high sensitivity detection and excellent reproducibility with overlapping S:B ratios of ~6 and EC₅₀’s ranging from only 689 pM to 796 pM.

Identify Allosteric Modulators, Partial Agonists, Inverse Agonists, and Silent Agonists

HitHunter cAMP assays’ superior assay performance, with its large assay window and wide dynamic range, allows for easy identification of a diverse set of pharmacological ligands such as positive allosteric modulators (PAMs), negative allosteric modulators (NAMs), partial agonists, and more. Easily identify partial agonists. This experiment uses a cAMP Hunter Gαs-coupled glucagon receptor (GCGR) CHO-K1 cell line to analyze two agonists. Results reveal [des-His1, Glu9]- glucagon exhibits partial agonism (S:B of 9.2; EC₅₀ of 340 nM) compared to the full native agonist, glucagon (S:B of 14.8; EC₅₀ of 11 nM).