Studies of structure-activity relations of complement inhibitor compstatin

Compstatin, a 13-mer cyclic peptide, is a novel and promising inhibitor of the activation of the complement system. In our search for a more active analog and better understanding of structure-functions relations, we designed a phage-displayed random peptide library based on previous knowledge of structure activity relations, in which seven amino acids deemed necessary for structure and activity were kept fixed while the remaining six were optimized. Screening of this library against C3 identified four binding clones. Synthetic peptides corresponding to these clones revealed one analog, called acetylated Ile(1)Leu/His(9)Trp/Thr(13)Gly triple replacement analog of compstatin corresponding to clone 640 (Ac-I1L/H9W/T13G), which was more active than compstatin. This newly identified peptide had 4-fold higher activity when compared with the originally isolated form of compstatin and 1.6-fold higher activity when compared with acetylated compstatin (Ac-compstatin). The structures of Ac-I1L/H9W/T13G and Ac-compstatin were studied by nuclear magnetic resonance, compared with the structure of compstatin, and found to be very similar. The binding of Ac-I1L/H9W/T13G and the equally active acetylated analog with His(9)Ala replacement (Ac-H9A) to C3 was evaluated by surface plasmon resonance, which suggested similarity in their binding mechanism but difference when compared with Ac-compstatin. Compensatory effects of flexibility outside the beta-turn and tryptophan ring stacking may be responsible for the measured activity increase in Ac-I1L/H9W/T13G and acetylated analog with His(9)Ala replacement and the variability in binding mechanism compared with Ac-compstatin. These data demonstrate that tryptophan is a key amino acid for activity. Finally, the significance of the N-terminal acetylation was examined and it was found that the hydrophobic cluster at the linked termini of compstatin is essential for binding to C3 and for activity.