Complement component C3a is a small serum protein implicated in many immune and inflammatory diseases. It binds to the membrane-spanning G protein coupled receptor C3aR and promotes both adaptive and innate immune cell activation. Very few small molecule agonists or antagonists for C3aR are known and all have limitations.
We sought to optimise known C3aR ligands through structure-activity relationships (SAR), and to create novel fluorescent ligands for assessing affinity for C3aR in competitive ligand-binding assays that do not require use of the expensive protein C3a. We report: (i) a novel competitive ligand-binding assay for the C3aR; (ii) an investigation into the effectiveness of downsizing C3a to peptide, helical and peptidomimetic agonists with high affinity for C3aR; (iii) structure-activity studies on ligands with moderate to potent agonist activity, as measured via intracellular Ca2+ release in a fluorescence assay.
A fluorescent ligand for C3aR competitive binding assays was established by labelling established C3aR agonists FLTLAR with a fluorescent europium(III) complex. Combining activating components from different ligand series into new compounds gave mixed results, some leading to greater agonist potency but most reducing activity. To mimic the α-helical C-terminal region of C3a, helix-inducing constraints were incorporated into longer peptide agonists. While these modifications did induce helicity, they did not increase C3aR activation potency. On the other hand, incorporating heterocycles into peptides, particularly short sequences, gave small peptidomimetics with greater agonist potency and binding affinity than any compound previously reported. It was found that aromaticity in the left-hand side of the compound greatly increased activity, and rotational stabilisation of heterocyclic aromatic systems was also beneficial.
In summary, the newly identified fluorescent agonist is predicted to be an important tool for future discovery of novel C3aR ligands. Some structure-activity insights are described herein for 10-20 residue peptides, hexapeptides, helix-constrained peptides, and smaller peptidomimetics containing heterocycles to provide a basis for further research on peptide and peptidomimetic drug design for C3aR.