A certificate of analysis describes what is in a vial. A pharmacology paper describes what that molecule does once it reaches a receptor, in a different vocabulary: EC50, Emax, relative potency, biased agonism. For a triple receptor agonist like LY3437943, reported to engage the GLP-1, GIP and glucagon receptors with deliberately unequal strength, that vocabulary is the difference between reading a number and understanding it. This article covers how published LY3437943 and incretin-peptide studies generate those numbers, what each measurement captures, and where the limits of that data sit for a reader comparing a pharmacology table against a product label.
| Term | What it describes | What it does not tell a reader |
|---|---|---|
| cAMP accumulation assay | Downstream signal in cells expressing one receptor after peptide exposure | Whole-organism exposure or elimination |
| EC50 | Concentration for half-maximal response in that assay | An absolute, assay-independent strength value |
| Emax | Maximum signal the peptide produces, regardless of concentration | Whether it is a full or partial activator at every receptor engaged |
| Relative potency | EC50 expressed as a ratio against a native ligand in the same assay | How that ratio translates to a different tissue or format |
| Beta-arrestin recruitment | A pathway tracked alongside cAMP to detect pathway-selective (biased) signaling | Whether a bias in one cell line reproduces in native tissue |
| Receptor internalization | Surface receptor removal after activation, tracked by imaging or labeling | The functional significance of a given rate |
What is a cAMP accumulation assay, and why HEK293 cells expressing GLP-1R, GIPR or GCGR?
GLP-1R, GIPR and GCGR are class B1 G-protein-coupled receptors that couple predominantly to Gs proteins, so activation stimulates adenylate cyclase and raises intracellular cyclic AMP (cAMP). A cAMP accumulation assay exploits that: a cell line, often HEK293 because it transfects easily and has low background expression of the receptors under study, is engineered to express one human receptor, exposed to a range of peptide concentrations, and intracellular cAMP is measured after a short incubation with a commercial reporter kit. A single-receptor line is what makes the readout interpretable for a multi-receptor molecule like LY3437943; a line expressing only GIPR isolates that receptor's signal from any GLP-1R or GCGR contribution, a separation impossible in native tissue expressing all three. Coskun and colleagues' discovery paper on LY3437943 (development code LY3437943) used this approach, reporting cAMP-based potency and efficacy at each of the three human receptors expressed individually in recombinant cell lines (Coskun et al., 2022).
What do EC50 and Emax actually describe on a response curve?
Plotting cAMP signal against the log of peptide concentration produces a sigmoidal curve, and EC50 and Emax are the two values most often pulled from it. EC50 is the concentration producing half of the maximum response in that assay, a measure of how little peptide is needed for a given fraction of signal. Emax is the plateau itself, the largest cAMP response the peptide can produce in that system regardless of concentration. The two do not move together: a peptide can match a comparator's Emax while needing a higher concentration to get there (a right-shifted EC50, lower potency), or reach full potency quickly but plateau below the comparator's maximum (lower Emax, a partial agonist at that receptor). A paper reporting only an EC50 without Emax or the underlying curve has left out half of what the assay measured, which is why a single number lifted from an abstract is a weak substitute for the methods and figures.
What does "relative potency" mean when a paper compares LY3437943 to native GLP-1, GIP or glucagon?
Because absolute EC50 values can shift between laboratories, cell passage numbers, assay kits and even the specific clone of a receptor-expressing cell line, published pharmacology papers commonly normalize a test peptide's potency against a native ligand run in the same assay on the same day, reporting the result as a ratio or percentage. Coskun and colleagues used this framing to describe LY3437943's engineered imbalance across the three receptors: relatively strong potency at GIPR, intermediate potency at GLP-1R and comparatively weaker potency at GCGR relative to each receptor's own native ligand (Coskun et al., 2022). That internal comparison controls for the assay itself, but it is still specific to the format used to generate it; a ratio from a transfected cell line with overexpressed receptor is not the same quantity as one from a native tissue preparation, and the two will not necessarily agree. Background on why the three receptors were engineered with unequal, rather than matched, potency is covered in how LY3437943 engages GLP-1, GIP and glucagon receptors.
Why does albumin in the assay buffer shift apparent potency for an acylated peptide like LY3437943?
LY3437943, like several other long-acting incretin peptides, carries a fatty acid or diacid side chain that binds serum albumin, a modification associated with extended circulating exposure in animal studies. That same property complicates in vitro potency readouts. Albumin added to assay buffer, or already present in the serum used to culture the reporter cells, can bind a fraction of an acylated peptide and reduce the concentration actually free to engage the receptor, which shows up as a right-shifted, apparently weaker EC50 relative to an unmodified peptide tested under identical conditions. Bech and colleagues characterized how divalent, small-molecule albumin interactions shape the systemic behavior of fatty-acid-modified GLP-1 analogues, illustrating why binding engineered for extended circulating exposure is the same property that confounds a simple cell-based potency comparison (Bech et al., 2017). Papers typically address this by running parallel assays with and without added albumin, or by using albumin-free buffer for the primary readout and labeling the albumin-containing condition separately. A reader comparing EC50 values across two papers on the same peptide should check whether both used the same albumin condition first.
What do beta-arrestin recruitment and internalization assays add beyond a cAMP readout?
Cyclic AMP is not the only signal a class B1 GPCR generates. After activation, these receptors are also phosphorylated and bound by beta-arrestin proteins, an event that terminates G-protein signaling at that receptor and initiates a separate set of downstream effects, including the receptor's eventual removal from the cell surface, known as internalization and typically tracked by fluorescent labeling and imaging or by loss of a surface-exposed tag over time. A peptide that activates cAMP signaling and beta-arrestin recruitment in the same proportion as the native ligand is a balanced agonist at that receptor, while one that favors one pathway is a biased agonist. Darbalaei and colleagues examined this for dual GLP-1 and glucagon receptor agonists, reporting that different peptide designs produced measurably different balances between cAMP signaling and beta-arrestin recruitment at the same two receptors, independent of raw cAMP potency (Darbalaei et al., 2020). Internalization rate matters in its own right because it affects how much receptor remains available at the cell surface during sustained or repeated exposure, separate from how potently a peptide activates the receptor on first contact: Wu and colleagues reported that biased agonists engineered for reduced beta-arrestin-mediated endocytosis produced prolonged downstream signaling relative to agonists with conventional internalization behavior (Wu et al., 2021), and Moo and colleagues described a GLP-1 receptor internalization pathway that proceeds independently of arrestin altogether, involving GRK, clathrin and caveolae-dependent mechanisms (Moo et al., 2025). A paper reporting cAMP EC50 values alone has characterized only one of these signaling arms, and a bias or internalization pattern measured in one reporter cell system is not guaranteed to appear the same way in a different cell background.
How should a researcher read these numbers across a paper and a certificate of analysis?
A certificate of analysis and a pharmacology paper answer two different questions and should not be conflated. A COA confirms identity and purity for a specific lot, covered in detail in reading a LY3437943 certificate of analysis, and says nothing about receptor potency, because potency is not a lot-release specification; it is a property of the molecule's sequence, established in the pharmacology literature and assumed to hold across correctly synthesized lots. A pharmacology paper's EC50 and Emax values, conversely, are specific to the assay system, cell line and albumin condition used to generate them, and do not vary by vial. James-Okoro and colleagues' characterization of glicentin as a low-potency GIPR agonist illustrates how a relative-potency classification depends on the comparator and assay chosen, since a peptide can read as low-potency relative to native GIP in one assay while still producing a measurable, reproducible signal (James-Okoro et al., 2026). Background on LY3437943's overall design is in what is LY3437943. Lot-specific identity and purity data for research-grade LY3437943 are posted on the LY3437943 product page.
Frequently asked questions
Is a lower EC50 always better?
A lower EC50 means less peptide was needed to reach half-maximal response in that specific assay. It does not by itself describe efficacy (Emax), selectivity across other receptors, or how the number would translate to a different assay format or tissue.
Can EC50 values be compared directly between two different published papers?
Only with caution. EC50 is sensitive to the cell line, receptor expression level, assay kit and incubation conditions used. Relative potency against a native ligand tested in the same assay on the same day is generally more comparable across studies than raw EC50 values are.
Does a cAMP assay capture everything a receptor does?
No. Class B1 GPCRs also signal through beta-arrestin recruitment and are subject to receptor internalization, both tracked by separate assays describing different parts of the signaling process.
Why does albumin matter for an acylated peptide's potency data specifically?
Fatty acid or diacid modifications on a peptide are designed to bind serum albumin for extended circulating exposure. That same binding can reduce the free peptide concentration available in an assay well, shifting the observed EC50 relative to an assay run without albumin.
References
- Coskun T, Urva S, Roell WC, et al. LY3437943, a novel triple glucagon, GIP, and GLP-1 receptor agonist for glycemic control and weight loss: From discovery to clinical proof of concept. Cell Metab. 2022;34(9):1234-1247.e9. PubMed 35985340.
- Darbalaei S, Yuliantie E, Dai A, et al. Evaluation of biased agonism mediated by dual agonists of the GLP-1 and glucagon receptors. Biochem Pharmacol. 2020;180:114150. PubMed 32682761.
- Bech EM, Martos-Maldonado MC, Wismann P, et al. Peptide half-life extension: divalent, small-molecule albumin interactions direct the systemic properties of glucagon-like peptide 1 (GLP-1) analogues. J Med Chem. 2017;60(17):7434-7446. PubMed 28771355.
- Wu Q, Chen S, Zhu H, et al. Biased agonists with less glucagon-like peptide-1 receptor-mediated endocytosis prolong hypoglycaemic effects. Eur J Pharmacol. 2021;907:174203. PubMed 34048741.
- Moo EV, Møller TC, Sørensen FA, Inoue A, Bräuner-Osborne H. Arrestin-independent internalization of the GLP-1 receptor is facilitated by a GRK, clathrin, and caveolae-dependent mechanism. FEBS J. 2025;292(7):1675-1695. PubMed 39756024.
- James-Okoro PP, Field J, Davies G, et al. Identification of glicentin as a low-potency glucose-dependent insulinotropic polypeptide receptor agonist. Peptides. 2026;199:171507. PubMed 42425317.
Research use only. Homegrown Peptides products are for laboratory research and are not for human or animal use. Nothing in this article is medical advice.
