OBESITY AND DIABETES PRODUCT SOLUTIONS

Sensitive cAMP Detection Assay Solutions for Peptides and Small Molecules

The cAMP assay is widely favored for therapeutic potency assessment as it reflects Gαs-mediated signaling triggered by receptor agonists, as reflected in numerous publications and regulatory filings.

Eurofins DiscoverX offers MOA-reflective, thaw-and-use cAMP Hunter^™ bioassays that are phase-appropriate for potency testing for GLP-1R activation and are rigorously qualified with semaglutide and tirzepatide to establish fit-for-purpose assays for potency and stability testing studies.

Potency Testing Using the cAMP Hunter Assay for Semaglutide

Semaglutide has been demonstrated to be a full agonist of GLP-1 receptor (GLP-1R) with minimal bias. This assay was developed and optimized following systematic qualification studies as per ICH guidelines to meet regulatory expectations for accuracy, precision, linearity, range, specificity, and robustness across varying potencies ranging from 150% to 50%.

cAMP Hunter semaglutide bioassay shows precision, accuracy, and linearity. A. Samples were tested at varying nominal relative potencies (RP; ranging from 50% to 150%) relative to a reference sample of 100% potency. Each nominal RP was tested in at least three independent experiments each by two analysts (a total of 6 assessments per potency level) over multiple days. The assay was found to be extremely accurate (97%), repeatable (8.7%) and precise (intermediate precision ≤8.5%). B. To demonstrate the dilutional linearity of the assay, observed values for each nominal RP were plotted against each expected RP, and the slope and R² value of the regression line were calculated, resulting in an R² value of 0.9990. C. Qualification study results demonstrated good accuracy, repeatability, intermediate precision, and dilutional linearity for the semaglutide bioassay kit.

Potency Testing Using the cAMP Hunter Assay for Tirzepatide

Tirzepatide is a unimolecular dual agonist of GLP-1 and GIP receptors. It activates the GIP receptor with a potency comparable to native GIP, while demonstrating significantly weaker potency in activating the GLP-1 receptor.

GLP-1 Receptor Agonist Target

The suitability of this assay for potency testing was tested for accuracy and precision across varying potency ranging from 150% to 50% according to ICH guidelines. This ready-to-use tirzepatide qualified-bioassay is fit-for-purpose for potency testing in the implementation in downstream development phases and lot release of the therapeutic drugs.

cAMP Hunter tirzepatide bioassay shows precision, accuracy, and linearity. A. Samples were tested at varying nominal relative potencies (RP, ranging from 50% to 150%) relative to a reference sample of 100% potency. Each nominal RP was tested in at least three independent experiments each by two analysts (a total of 6 assessments per potency level) over multiple days. The assay was found to be extremely accurate (95.2%), repeatable (4.5%) and precise (intermediate precision ≤11.8%). B. To demonstrate the dilutional linearity of the assay, observed values for each RP were plotted (against each “expected” RP, and the slope and R2 value of the regression line were calculated, resulting in an R² value of 0.9467. C. Qualification study results demonstrated good accuracy, repeatability, intermediate precision, and dilutional linearity for the tirzepatide bioassay kit.

GIP Receptor Agonist Target

Thaw-and-use cAMP Hunter GIP receptor agonist (RA) assay demonstrating robust response of tirzepatide at the GIP receptor.

cAMP Hunter Tirzepatide Bioassay (GIP RA) was used to quantify cAMP accumulation in response to tirzepatide. The assay demonstrated a robust and sensitive dose-dependent increase in cAMP levels with signal-to-background (S/B) of 20.8 and EC₅₀ of 196.3 pg/ml. 

Potency Testing Using the cAMP Hunter Assay for Retatrutide

Retatrutide is a novel triple receptor agonist peptide that targets GCGR, GIPR, and GLP-1R. Compared with the endogenous receptor ligands, retatrutide is reported to be less potent at the GCG and GLP-1 receptors but more potent at the GIP receptor.

GLP-1 Receptor Agonist Target

Thaw-and-use cAMP Hunter GLP-1 receptor agonist (RA) assay demonstrates a robust retatrutide response at the GLP-1 receptor.

cAMP Hunter Retatrutide Bioassay (GLP-1 RA) was used to quantify cAMP accumulation response with exendin, tirzepatide, and retatrutide. The assay demonstrated a robust, sensitive, and differential response in cAMP levels reflective of the accurate pharmacology of these drugs.

GIP Receptor Agonist Target

Thaw-and-use cAMP Hunter GIP receptor agonist (RA) assay demonstrates a robust retatrutide response at the GIP receptor.

cAMP Hunter Retatrutide Bioassay (GIP RA) was used to quantify the cAMP accumulation response with GIP, retatrutide, and tirzepatide. The assay demonstrated a robust, sensitive, and differential response in cAMP levels reflective of the accurate pharmacology of these drugs.

GCG Receptor Agonist Target

Thaw-and-use cAMP Hunter glucagon receptor agonist (RA) assay demonstrates a robust retatrutide response at the glucagon receptor (GCGR).

cAMP Hunter Retatrutide Bioassay (GCG RA) was used to quantify cAMP accumulation response with glucagon, tirzepatide, and retatrutide. The assay demonstrated a robust, sensitive, and differential response in cAMP levels reflective of the accurate pharmacology of these drugs.

Assessing Receptor Internalization and β-arrestin Recruitment for Pharmacological Characterization of GLP-1, GIP, and Glucagon Receptor Agonists

Achieving an optimal agonism ratio between receptors is crucial for designing multi-agonist T2D therapies, where targeting specific metabolic endpoints, such as GIPR agonism to alleviate GLP-1RA-induced nausea, necessitates finding the right balance of GLP-1R and GIPR activity for optimal insulinotropic outcomes.

The three key GPCR readouts, cAMP, β-arrestin, and receptor internalization, are studied to establish accurate pharmacology. Quantifying β-arrestin activity to the activated GPCR is a stoichiometric, sustained, and non-amplified readout offering a closer reflection of the in vivo response to receptor activation. Agonist-dependent GPCR internalization is commonly mediated by β-arrestin recruitment of endosomal trafficking and is typically implicated in biased pharmacology. β-arrestin recruitment assays are usually coupled with measurement of receptor internalization.

GPCR Biased Signaling Evaluation of Dual and Triple-receptor Agonist Against GLP-1, GIP, and Glucagon Receptors

Comparison of β-arrestin recruitment by tirzepatide and retratrutide at respective target receptors with endogenous ligands (exendin-4 for GLP-1, GIP for GIPR, and glucagon for GCGR).

cAMP Accumulation 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).

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.

β-arrestin Recruitment Assay Principle

The β-arrestin G protein-independent pathway and PathHunter GPCR β-arrestin assay principle.
–  A. Ligand-induced β-arrestin (types 1 or 2) recruitment activates signaling cascades independently of G-protein signaling to provide a stoichiometric, non-amplified signal. The ligand-activated GPCR is phosphorylated by a specific GPCR kinase (GKRs, not shown here), leading to β-arrestin recruitment. This β-arrestin binding blocks G protein-mediated signaling and results in the internalization (or endocytosis) of the GPCR and ending of the attenuation of GPCR signaling (known as desensitization). Subsequently, the GPCR is recycled back to the plasma membrane or degraded in the lysosome. This stoichiometric (1 receptor: 1 ligand), non-amplified system requires full ligand occupancy of the ligand bound to the receptor to give a full signal, which lowers sensitivity to agonists, but improves the ability to detect differences of efficacy between agonists and superior sensitivity for antagonists (compared to second messenger systems). By analyzing the β-arrestin pathway, you can fine tune GPCR biology when screening antagonists as well as deorphanize GPCRs and distinguish between full, super, and partial agonists. 

– B. PathHunter GPCR β-arrestin assays take advantage of DiscoverX’s proprietary Enzyme Fragment Complementation technology. The GPCR is fused in frame with a small enzyme donor fragment ProLink™ (PK) and co-expressed in cells stably expressing a fusion protein of β-arrestin and a larger, N-terminal deletion mutant of β-galactosidase (called enzyme acceptor or EA). Activation of the GPCR stimulates binding of β-arrestin to the PK-tagged GPCR and forces complementation of the two enzyme fragments, resulting in the formation of an active β-galactosidase enzyme. This interaction leads to an increase in enzyme activity that can be measured using chemiluminescent PathHunter Detection Reagents. Ultimately, β-arrestin recruitment assays offer an easy-to-use alternative to second messenger cAMP and calcium G-protein dependent pathways to enable enhanced profiles of compound pharmacology – a universal assay that expands opportunities for development of novel drugs.

Receptor Internalization Assay Principle

PathHunter total and activated GPCR internalization assay principles. In the total GPCR internalization assay format (left) cell lines are engineered to co-express two fragments of the β-galactosidase (β-gal) enzyme – a small EFC enzyme donor (ED) and a larger EFC enzyme acceptor (EA). The ED is tagged to the GPCR, and the EA is localized to the endosome. Small molecule or biologic (e.g. antibody) ligands that bind the GPCR-tagged fusion protein leads to internalization (endocytosis) of the receptor to the endosome, forcing the complementation of the two β-gal enzyme fragments (ED and EA). The resulting functional β-gal enzyme hydrolyzes a substrate to generate a dose-dependent chemiluminescent signal. In the activated GPCR internalization assay format (right), cell lines are engineered to co-express an untagged GPCR, an EA tagged β-arrestin, and an ED tag localized to the endosomes. Activation of the untagged GPCR induces β-arrestin recruitment, followed by internalization of the GPCR-β-arrestin-EA complex to the ED-tagged endosomes. Similar to the total assay format, this internalization forces the complementation of the two β-gal enzyme fragments, forming a functional enzyme that hydrolyzes substrate to generate a chemiluminescent signal.