Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Dec 12;283(50):34643-9.
doi: 10.1074/jbc.M805181200. Epub 2008 Oct 9.

Discovery and validation of novel peptide agonists for G-protein-coupled receptors

Ronen Shemesh  1 , Amir Toporik, Zurit Levine, Iris Hecht, Galit Rotman, Assaf Wool, Dvir Dahary, Eyal Gofer, Yossef Kliger, Michal Ayalon Soffer, Avi Rosenberg, Dani Eshel, Yossi Cohen
Affiliations

Discovery and validation of novel peptide agonists for G-protein-coupled receptors

Ronen Shemesh et al. J Biol Chem. .

Abstract

G-protein-coupled receptors (GPCRs) represent an important group of targets for pharmaceutical therapeutics. The completion of the human genome revealed a large number of putative GPCRs. However, the identification of their natural ligands, and especially peptides, suffers from low discovery rates, thus impeding development of therapeutics based on these potential drug targets. We describe the discovery of novel GPCR ligands encrypted in the human proteome. Hundreds of potential peptide ligands were predicted by machine learning algorithms. In vitro screening of selected 33 peptides on a set of 152 GPCRs, including a group of designated orphan receptors, was conducted by intracellular calcium measurements and cAMP assays. The screening revealed eight novel peptides as potential agonists that specifically activated six different receptors in a dose-dependent manner. Most of the peptides showed distinct stimulatory patterns targeted at designated and orphan GPCRs. Further analysis demonstrated a significant in vivo effect for one of the peptides in a mouse inflammation model.

PubMed Disclaimer

Figures

FIGURE 1.
FIGURE 1.
Dose-response activation measured for Ca2+ flux-activating peptides. Peptides that showed significant results in the screening assay were tested for dose-dependent activation on the activated receptors (MRGX1, MRGX2, FPRL1, and MAS1, counterclockwise). Dose-response activation for positive control peptides are indicated with a thick line and solid circles. Dose-response curves for the tested peptides are indicated by thin lines and open squares. The best fit curves and EC50 were calculated as described under “Experimental Procedures” and are presented in Table 1. For the MAS1 receptor, no activation was observed by the suggested positive control (Ang 1–7).
FIGURE 2.
FIGURE 2.
Inhibition of cAMP in forskolin-stimulated cells by activation of LGR7 and LGR8. cAMP was stimulated by incubation for 10 min in the presence of 10 μm forskolin, as described above. The peptide was added at a final concentration of 1μm, and the sample was incubated for 20 min following the readout of the end point cAMP concentration (measured in arbitrary fluorescence units (AFU)). NT represents addition of the buffer only as a negative control. Forskolin with no added peptide serves as a positive control.
FIGURE 3.
FIGURE 3.
Effects of P58 and its derivatives on zymosan-induced PMN infiltration into murine air pouch. Mice were treated intravenously with the peptides (20 or 80 nmol per mouse) or vehicle, immediately followed by an intra-pouch challenge with zymosan A at time 0. Four hours after the zymosan A injection, pouch cavities were washed, and the leukocytes recovered in the lavage fluids were stained with an anti-GR-1 antibody and analyzed by fluorescence-activated cell sorter. Data are shown as GR-1+ cells (×105) per mouse (mean ± S.E.). *, p < 0.05 versus vehicle group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Imming, P., Sinning, C., and Meyer, A. (2006) Nat. Rev. Drug Discov. 5 821–834 - PubMed
    1. Hill, S. J. (2006) Br. J. Pharmacol. 147 Suppl. 1, 27–37 - PMC - PubMed
    1. Lin, S. H., and Civelli, O. (2004) Ann. Med. 36 204–214 - PubMed
    1. Leifert, W. R., Aloia, A. L., Bucco, O., Glatz, R. V., and McMurchie, E. J. (2005) J. Biomol. Screen. 10 765–779 - PubMed
    1. Kutzleb, C., Busmann, A., Wendland, M., and Maronde, E. (2005) Curr. Protein Pept. Sci. 6 265–278 - PubMed

LinkOut - more resources