G-protein-coupled receptor (GPCR) activation is an essential step of signal transduction that leads to a myriad of secondary messenger systems cascading down to distal endpoints like changes in calcium and cAMP concentrations, or transcription activation. While many commercial assay systems target these surrogate readouts of activation, the evolving complexity and texture of GPCR signaling continues to present challenges for target validation & drug discovery.
These challenges include:
- Solutions for intractable targets
- Identifying & validating allosteric modulators
- Accessing new chemical spaces
- Deconvoluting receptor signaling through multiple G-proteins
- Identifying inverse agonists
- Improving the signal window
- Finding ways to assay G12 and G13 coupling
Improving our understanding of GPCR signaling and solving these challenges demands fresh approaches and new tools that do not relegate GPCRs to simple on-off switches. Whether you are looking for agonists, antagonists, inverse agonists, or allosteric modulators, Caden’s technology provides a new solution to this growing GPCR signaling complexity.
Caden’s Solution
Caden takes an entirely new approach to measuring GPCR signaling by focusing directly on GPCR:G-protein coupling, the initial event of receptor activation. Caden’s breakthrough is based on a series of proprietary peptides that mimic the C‑terminal tail of the G-alpha subunit. This critical interaction initiates binding of the heterotrimeric G-protein to the GPCR, resultant receptor activation, and all downstream signaling.
Caden’s proprietary peptides share high homology with the sequences of native, C-terminal peptides, but they exhibit specificity for an individual GPCR and have affinities 100–1000 fold greater than the corresponding native C-terminal G-alpha peptide. Their nanomolar affinities make the Caden peptides ideal tools for a number of applications, including screening and target validation.
Identifying Caden Peptides
Caden peptides are selected by mixing agonist-activated receptor insect cell membrane preparations expressing a single human recombinant GPCR with one of five different proprietary Caden peptide libraries that are each biased to, respectively, the sequence of one of the five primary human G-alpha subunits (Gs, Gi, Gq, G12, or G13). Caden’s patented process identifies unique high-affinity peptides that bind the GPCR at the G-protein binding site. Caden peptides are therefore sensitive tools for interrogating GPCRs by focusing on the GPCR:G-protein face.
High through-put screening
A compound’s ability to modulate the binding of a labeled Caden high-affinity peptide to a receptor is a direct measure of that compound’s ability to change the conformation of the receptor at the G-protein interface. Test compounds may cause the Caden peptide to bind more tightly or less tightly to the receptor, and they may alter peptide binding differently in the presence or absence of the natural agonist.
In this example, a known drug was able to reduce in a dose dependent manner the binding of a Caden peptide to the CCR1 receptor using either a fluorescentlylabeled peptide in a fluorescence polarization assay (left) or a biotin-labeled peptide in an ELISA (right).
Pathway validation
Caden high affinity peptides can also be used in a variety of ways to investigate the role of a given GPCR:G-protein pair in activating down-stream pathways. The peptide sequence, introduced to live cells as a minigene vector, can modulate the activity of individual GPCRs without affecting other GPCRs coupling to the same G-protein or other G-protein pathways through the target receptor.
In the panels to the left, endothelial cells transfected with minigenes encoding either the Caden native Gq C-terminal peptide sequence or a Caden high-affinity peptide are identified by the co-expression of GFP (yellow arrows). The high affinity peptide (Gt2-14) was identified from a Caden Gq peptide library screened against the thrombin-activated PAR‑1 receptor. In both cases, the cells expressing the peptides did not form actin filaments (red Alexa568 phalloidin staining), a process driven by PAR-1 coupling to Gq.
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