Improvement of the anti-C3 activity of compstatin using rational and combinatorial approaches

Abstract

Compstatin is a 13-residue cyclic peptide that has the ability to inhibit the cleavage of C3 to C3a and C3b. The effects of targeting C3 cleavage are threefold, and result in hindrance of: (i) the generation of the pro-inflammatory peptide C3a, (ii) the generation of opsonin C3b (or its fragment C3d), and (iii) further complement activation of the common pathway (beyond C3) with the end result of the generation of the membrane attack complex. We will report on our progress on: (i) rational design of more active compstatin analogues based on the three-dimensional structure of compstatin, (ii) experimental combinatorial design based on the generation of a phage-displayed peptide library partially randomized with the implementation of structure-induced restraints, and (iii) theoretical combinatorial design based on a novel computational optimization method, structure-induced restraints and flexible structural templates. All three approaches have resulted in analogues with improved activities. Currently, the lead analogue has the sequence acetyl-I[CVYQDWGAHRC]T-NH(2) (where the brackets denote cyclization), and is 16-fold more active than the parent peptide. We will also report on our progress towards understanding the dynamic character of compstatin using molecular dynamics simulations. The identification of an ensemble of interconverting conformers of compstatin with variable populations is a first step towards the incorporation of dynamic elements in the design of new analogues using dynamics-activity relationships in addition to structure-activity relationships.

Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis

“…C3bi bound to zymosan at 37°C but not at 4°C. In contrast, in the presence of a C3-convertase inhibitor, compstatin, 25,26 binding of C3bi to zymosan was significantly suppressed but not in the presence of a control peptide ( Figure 1B). Using complement-opsonized zymosan, we assayed phagocytosis on macrophage-like HL60 cells after incubation with vitamin D 3 and TPA for 3 days (Day3-HL60).…”

“…24 A C3-convertase inhibitor, compstatin, is a 13-amino acid cyclic peptide that binds to and inhibits cleavage of C3. 25,26 The acetylated form of compstatin (Ac-ICVVQDWGHHRCT-NH2) and the control peptide (IAVVQDWGHHRAT-NH2) were purchased from Peptide Institute (Osaka, Japan) and Sigma-Aldrich (Tokyo, Japan), respectively.…”

Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

“…Nevertheless, it has been possible to improve the specific activity by structure-activity studies and experimental and theoretical combinatorial approaches (reviewed in Ref. 32). The structure of the complex between C3c and compstatin provides crucial information on the bound conformation of compstatin and the binding site on C3.…”

Characterization of Ehp, a Secreted Complement Inhibitory Protein from Staphylococcus aureus

“…We observed potent, dose-dependent inhibition of the alternative complement activation pathway by Ehp (IC 50 ϳ 120 nM Ehp). Interestingly, the inhibitory properties of Ehp were ϳ2-3-fold greater than those of Efb or compstatin, a complement inhibitory cyclic 13-mer peptide that has undergone several rounds of combinatorial optimization (21,(45)(46)(47). Furthermore, alternative pathway inhibition by Ehp correlated directly with C3d binding because the Ehp mutants described above had reduced inhibitory properties that reflected their individual affinities for C3d (Fig.…”