Compstatin is a 13-residue peptide that inhibits activation of the complement system by binding to the central component C3 and its fragments C3b and C3c. A combination of theoretical and experimental approaches has previously allowed us to develop analogs of the original compstatin peptide with up to 264-fold higher activity; one of these analogs is now in clinical trials for the treatment of age-related macular degeneration (AMD). Here we used functional assays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) to assess the effect of modifications at three key residues (Trp-4, Asp-6, Ala-9) on the affinity and activity of compstatin and its analogs, and we correlated our findings to the recently reported co-crystal structure of compstatin and C3c. The KD values for the panel of tested analogs ranged from 10S6 to 10S8 M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4-fold range in kon values (2-10T105MS1 sS1) and a koff variation of >35-fold (1-37T10S2 sS1) being observed. The stability of the C3b-compstatin complex seemed to be highly dependent on hydrophobic effects at position 4, and even small changes at position 6 resulted in a loss of complex formation. Induction of a b-turn shift by an A9P modification resulted in a more favorable entropy but a loss of binding specificity and stability. The results obtained by the three methods utilized here were highly correlated with regard to the activity/affinity of the analogs. Thus, our analyses have identified essential structural features of compstatin and provided important information to support the development of analogs with improved efficacy.
- Isothermal titration calorimetry
- Surface plasmon resonance