Discovery of novel selective C5a2 ligands that can modulate IL-6 release from macrophages — ASN Events
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  3. Discovery of novel selective C5a2 ligands that can modulate IL-6 release from macrophages

Discovery of novel selective C5a2 ligands that can modulate IL-6 release from macrophages (#32)

Daniel E Croker 1 , Reena Halai 1 , Geraldine Kaeslin 1 , Dimitrios Morikis 2 , Trent Woodruff 3 , Christodoulos A Floudas 4 , Peter N Monk 5 , Matthew A Cooper 1
  1. Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD, Australia
  2. Department of Bioengineering, University of California, Riverside, CA, USA
  3. School of Biomedical Sciences, The University of Queensland, St. Lucia, QLD, Australia
  4. Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA
  5. Department of Infection and Immunity, Sheffield University Medical School, Sheffield, UK

Background: The complement cascade is a highly sophisticated network of proteins that are well regulated and directed in response to invading pathogens or tissue injury and forms a major component of our innate immune response. Complement C5a binds to two G-protein coupled receptors (GPCR); namely the C5a receptor (C5a1) and C5a receptor like-2 receptor (C5a2). C5a2 is a non-signalling GPCR and its role has been difficult to validate due to the unavailability of selective ligands.

Aims: Discover novel C5a2 ligands that allow us to probe and explore the functional role of C5a2 in cytokine release from macrophages.

Methods: Radioligand binding assays were used to screen a small peptide based library of 61 ligands designed to act at C5a1 for the ability to displace 125I-C5a from C5a2 expressing membranes. A novel β-arrestin 2 recruitment via C5a2 BRET assay was developed to test for ligand activity at C5a2 and ligands were counter screened for β-arrestin 2 recruitment via C5a1. ELISA was used to measure cytokine release from human monocyte derived macrophages (HMDM).

Results: Radioligand binding testing of the 61 ligands revealed two ligands (P32 & P59) which showed ≥ 40% inhibition of 125I-C5a binding to C5a2 membranes at 100 µM. P32 and P59 were able to dose dependently recruit β-arrestin 2 via C5a2 but not C5a1 each with an EC50 of 7.6 µM.  Neither P32 nor P59 was able to recruit β-arrestin 2 to a similar level as C5a, with P32 able to recruit to ~55% and P59 ~30% of the level of C5a. Both P32 and P59 could dose dependently inhibit the release of IL-6 from HMDM when co-stimulated with LPS but had no effect on IL-10 release.

Conclusions: P32 and P59 are the first reported selective ligands for C5a2 and show novel pharmacology with the ability to modulate cytokine release from HMDM.