Structure-Guided Design of C5aR1-Selective Peptide Agonists

Selective peptide agonists for complement C5a receptor 1 (C5aR1) are valuable tools for dissecting receptor-specific inflammatory signalling, but their optimisation is complicated by overlap with closely related anaphylatoxin receptors and by pathway-dependent pharmacology. Here, we applied a structure-guided computational workflow to prioritise mutations within two C5a-derived peptide agonist scaffolds. FoldX-guided modelling identified position 5 as a candidate optimisation site, with hydrophobic substitutions predicted to improve C5aR1 engagement without corresponding gains at C3aR. Predicted BM1 and BM221 analogues were synthesised by solid-phase peptide synthesis and evaluated across C3aR, C5aR1 and C5aR2 using ERK1/2 phosphorylation and {beta}-arrestin recruitment assays. Position-5 substitutions enhanced C5aR1 functional preference in ERK assays, although additional replacement of Leu6 with Ala reduced target potency and revealed pathway-dependent receptor discrimination. BM1 P5M provided the clearest overall improvement across ERK and {beta}-arrestin readouts. In the BM221 series, A5Nle improved C5aR1 preference over C3aR, whereas A5Nle Abu6Ala produced the most favourable serum stability profile. These findings support position 5 as a transferable optimisation site and demonstrate that C5aR1 potency and receptor selectivity must be balanced during next-generation agonist design.