The design and development of a potent and selective C5aR1 agonist (#146)
Unchecked inflammatory responses are responsible for a vast number of disease states such as Motor Neuron Disease. This chronic inflammatory response is both initiated and propagated by the host’s innate immune system, including the complement system. Anaphalatoxin C5a is a major factor in the complement system, and has been implicated in this characteristic chronic inflammation. Due to the large size of native C5a chemical synthesis is difficult, meaning that smaller peptide agonists of C5a’s endogenous receptor, C5aR1, are required to effectively study C5a mediated inflammatory pathways. Previous research has uncovered several full and selective agonists for C3aR (endogenous receptor for C3a), however a full and selective agonist of C5aR1 has remained elusive. The most promising published peptide agonist of C5aR1 is EP54 (YSFKPMPLaR), however this peptide functionally binds to both C3aR and C5aR1 with equimolar potency. In this study we generated a library of EP54 analogues using Fmoc-solid phase peptide synthesis with the aim of understanding the structure/activity relationships of this lead peptide and developing a selective full agonist of C5aR1. pERK signalling activity was measured in CHO cells stably transfected with either C5aR1 or C3aR to compare relative potencies for each analogue. The stabilities of the most promising candidates were then assessed by liquid chromatography mass spectrometry (LC-MS) analysis following proteolytic challenge. Finally, we compared the functional similarity between the candidate peptides and native C5a on LPS-induced cytokine release from human macrophages. This study ultimately resulted in the development of a selective, and stable, full peptide agonist for C5aR1.