Background:
The anaphylatoxin C5a plays a key role in Complement-mediated inflammation, inducing pro-inflammatory effects such as chemotaxis, leukocyte degranulation and cytokine/chemokine production through interaction with its endogenous receptor, C5aR1. In comparison to C5aR1, the understanding of the role of the second C5a receptor, C5aR2, is limited. Previous studies have relied predominantly on knockout mouse models to elucidate the role of C5aR2, however the impact of the receptor on trafficking and expression of C5aR1 and/or C3aR is still not fully understood. Ideally, these studies would be complemented with pharmacological experiments to further elucidate the immunomodulatory role of C5aR2 in both healthy and pathological conditions. In order to perform these studies, selective ligands for the receptor are needed. Previous studies have uncovered selective partial agonists, however selective full agonists have, to-date, remained elusive.
Methods:
Using FMOC-based solid phase peptide synthesis, a series of peptide analogues of C5aR1 agonist peptides were constructed and the ability of these analogues to modulate C5aR2-mediated β-arrestin 2 recruitment was assessed in HEK-293 cells transiently expressing C5aR2. Analogues that exhibited ≥100% efficacy of 100 nM C5a were then counter-screened for activity at C3aR and C5aR1 in ligand-induced ERK 1/2 phosphorylation and β-arrestin 2 recruitment assays.
Results:
This study identified 11 small peptide ligands that act as full agonists of C5aR2-mediated β-arrestin 2 recruitment. 8 of these analogues exhibited functional selectivity for the receptor, with no induction of C3aR/C5aR1 ERK1/2 activation or C5aR1-mediated β-arrestin 2 recruitment up to concentration of 100 µM.
Conclusions:
This study has identified the first reported full agonists of C5aR2-mediated β-arrestin 2 recruitment. These functionally selective ligands represent valuable tool compounds for modulation of C5a activity and further probing the role of C5aR2 within the complement-mediated immune response.