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. Nov-Dec 2009;22(6):495-505.
doi: 10.1002/jmr.972.

Structure-kinetic Relationship Analysis of the Therapeutic Complement Inhibitor Compstatin

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Free PMC article

Structure-kinetic Relationship Analysis of the Therapeutic Complement Inhibitor Compstatin

Paola Magotti et al. J Mol Recognit. .
Free PMC article

Abstract

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 K(D) values for the panel of tested analogs ranged from 10(-6) to 10(-8) M. These differences in binding affinity could be attributed mainly to differences in dissociation rather than association rates, with a >4-fold range in k(on) values (2-10 x 10(5) M(-1) s(-1)) and a k(off) variation of >35-fold (1-37 x 10(-2) s(-1)) 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 beta-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.

Figures

Figure 1
Figure 1
Structure of compstatin and its analogs. (A) The core structure of compstatin is comprised of a cyclic tridecapeptide. (The structure shown represents peptide 4W in Table 1.) In the present study, changes were made to residues 4, 6, and 9 (Table 1) by introducing several natural (P, W, Y) and the following unnatural amino acids: (b) O-methyl-tyrosine (MeY), (c) O-ethyl-tyrosine (EtY), (d) O-allyl-tyrosine (AlY), (e) 5-methyl-tryptophan (5MeW), (f) 2-naphthyl-alanine (2Nal), (g) 1-methyl-tryptophan (1MeW), (h) 1-formyl-tryptophan (1ForW), (i) 4-pyrenyl-alanine (PyrA), (j) iso-aspartic acid (IsoD), (k) α-aminobutyric acid (Abu).
Figure 2
Figure 2
Kinetic ranking of compstatin analogs. A constant concentration (1 μM) of each peptide was passed over immobilized C3b. Binding signals were overlaid to visualize relative differences in their association and dissociation phases. Both the relative signal intensities and the observed dissociation correlated well with the measured affinities (Table 2). The weakest (4V9H; red) and strongest (4(1MeW); blue) analogs are highlighted in boldface type.
Figure 3
Figure 3
Detailed kinetic analysis of 13 compstatin analogs. Increasing concentrations of each compstatin analog were delivered to surface-bound C3b for 2 min, and the dissociation was monitored for another 5 min. All traces have been corrected for injection artifacts and non-specific binding by subtracting responses from an untreated reference surface and several buffer blanks. Processed SPR signals (black) were globally fitted to a Langmuir 1:1 binding isotherm (red simulation curves), and the kinetic rate constants kon and koff were extracted (Table 2). A schematic representation of the assay setup for the kinetic profiling is presented at the bottom.
Figure 4
Figure 4
Complement inhibition activity of compstatin analogs. The percentage of classical pathway complement inhibition is plotted against the peptide concentration and compared to that for the lead compound 4(1MeW). (A) 1ForW at position 4, (B) tyrosine analogs at position 4 (Y, MeY, EtY, and AlY), (C) PyrA at position 4, (D) IsoD at position 6, (E) proline at position 9. (F) Schematic representation of the ELISA assay to detect classical pathway activation, where the antigen-antibody complex was formed by an albumin-coated plate recognized by a polyclonal α-ovalbumin antibody. Inhibition assays for analogs 4(5MeW), 4(2Nal), and 9Abu have been reported previously (Katragadda et al., 2006; Mallik et al., 2005).
Figure 5
Figure 5
Correlation between measured parameters. (A) Inhibitory activities and binding affinities followed a linear trend. (B) Comparison between the KD values determined by ITC and SPR showed a good correlation between solution- and surface-based binding. (C) The on/off-rate plot revealed that structural modification had distinct effects on the rate constants. Dashed diagonal lines represent binding affinity (KD). Analogs with the weakest (4V9H) and strongest 4(1MeW) binding affinity are marked in bold, and minimum/maximum rate constants are indicated with dotted lines.
Figure 6
Figure 6
Structural base for the binding of compstatin. (A) Binding site of compstatin on C3c: The backbone of analog 4W is represented as a ribbon, with side chains as sticks in alternating colors (dark and pale blue). Positions 4, 6, and 9, which were modified in this study, are highlighted as bolded, red sticks. Contact amino acids for Trp-4 are marked with different colors on the surface of C3c (gray). (B) Structural model for the formation of hydrophobic contacts between C3c (brown) and analog 4(1MeW) (blue). The interatomic distances between the N-methyl group in compstatin analog 4(1MeW) and carbon atoms of residues 390–393 of C3c within a distance of 3.9 A are marked as dashed green lines. The binding site and the modification of Trp-4 were generated from PDB file 2QKI (Janssen et al., 2007) using PyMOL (www.pymol.org).

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