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).