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

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.

Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons.

Complement activation products are major components of the inflammatory response induced by cardiac surgery and cardiopulmonary bypass which contribute to postoperative organ dysfunction, fluid accumulation, and morbidity. Activation of the complement system occurs during extracorporeal circulation, during reperfusion of ischemic tissue, and after the formation of heparin-protamine complexes. In this study we examine the efficacy of Compstatin, a recently discovered peptide inhibitor of complement, in preventing heparin/protamine-induced complement activation in baboons. The study was performed in baboons because Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans); Compstatin inhibits only the activation of primates' complement system. After testing various doses and administration regimens, Compstatin produced complete inhibition at a total dose of 21 mg/kg when given as a combination of bolus injection and infusion. Compstatin completely inhibited in vivo heparin/protamine-induced complement activation without adverse effects on heart rate or systemic arterial, central venous, and pulmonary arterial pressures. This study indicates that Compstatin is a safe and effective complement inhibitor that has the potential to prevent complement activation during and after clinical cardiac surgery. Furthermore, Compstatin can serve as the prototype for designing an orally administrated drug.

Binding kinetics, structure-activity relationship, and biotransformation of the complement inhibitor compstatin.

We have previously identified a 13-residue cyclic peptide, Compstatin, that binds to complement component C3 and inhibits complement activation. Herein, we describe the binding kinetics, structure-activity relationship, and biotransformation of Compstatin. Biomolecular interaction analysis using surface-plasmon resonance showed that Compstatin bound to native C3 and its fragments C3b and C3c, but not C3d. While binding of Compstatin to native C3 was biphasic, binding to C3b and C3c followed the 1:1 Langmuir binding model; the affinities of Compstatin for C3b and C3c were 22- and 74-fold lower, respectively, than that of native C3. Analysis of Compstatin analogs synthesized for structure-function studies indicated that 1) the 11-membered ring between disulfide-linked Cys2-Cys12 constitutes a minimal structure required for optimal activity; 2) retro-inverso isomerization results in loss of inhibitory activity; and 3) some residues of the type I beta-turn segment also interact with C3. In vitro studies of Compstatin in human blood indicated that a major pathway of biotransformation was the removal of Ile1, which could be blocked by N-acetylation of the peptide. These findings indicate that acetylated Compstatin is stable against enzymatic degradation and that the type I beta-turn segment is not only critical for preservation of the conformational stability, but also involved in intermolecular recognition.

Herpes simplex virus type 1 glycoprotein gC mediates immune evasion in vivo.

Many microorganisms encode proteins that interact with molecules involved in host immunity; however, few of these molecules have been proven to promote immune evasion in vivo. Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) binds complement component C3 and inhibits complement-mediated virus neutralization and lysis of infected cells in vitro. To investigate the importance of the interaction between gC and C3 in vivo, we studied the virulence of a gC-null strain in complement-intact and C3-deficient animals. Using a vaginal infection model in complement-intact guinea pigs, we showed that gC-null virus grows to lower titers and produces less severe vaginitis than wild-type or gC rescued virus, indicating a role for gC in virulence. To determine the importance of complement, studies were performed with C3-deficient guinea pigs; the results demonstrated significant increases in vaginal titers of gC-null virus, while wild-type and gC rescued viruses showed nonsignificant changes in titers. Similar findings were observed for mice where gC null virus produced significantly less disease than gC rescued virus at the skin inoculation site. Proof that C3 is important was provided by studies of C3 knockout mice, where disease scores of gC-null virus were significantly higher than in complement-intact mice. The results indicate that gC-null virus is approximately 100-fold (2 log10) less virulent that wild-type virus in animals and that gC-C3 interactions are involved in pathogenesis.

Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3b1 inactivates the alternative complement pathway.

Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.

Structure, functions, and evolution of the third complement component and viral molecular mimicry.

The third component of the complement system, C3, is a common denominator in the activation of the classical, alternative, and lectin pathways. The ability of C3 molecule to interact with at least 20 different proteins makes it the most versatile component of this system. Since these interactions are important for phagocytic, immunoregulatory, and immune evasion mechanisms, the analysis of its structure and functions has been a subject of intense research. Here we review our current work on the C3-ligand interactions, C3-related viral molecular mimicry, evolution of the complement system, and identification of C3-based complement inhibitors.

Glycoprotein D of herpes simplex virus (HSV) binds directly to HVEM, a member of the tumor necrosis factor receptor superfamily and a mediator of HSV entry.

Glycoprotein D (gD) is a structural component of the herpes simplex virus (HSV) envelope which is essential for virus entry into host cells. Chinese hamster ovary (CHO-K1) cells are one of the few cell types which are nonpermissive for the entry of many HSV strains. However, when these cells are transformed with the gene for the herpesvirus entry mediator (HVEM), the resulting cells, CHO-HVEM12, are permissive for many HSV strains, such as HSV-1(KOS). By virtue of its four cysteine-rich pseudorepeats, HVEM is a member of the tumor necrosis factor receptor superfamily of proteins. Recombinant forms of gD and HVEM, gD-1(306t) and HVEM(200t), respectively, were used to demonstrate a specific physical interaction between these two proteins. This interaction was dependent on native gD conformation but independent of its N-linked oligosaccharides, as expected from previous structure-function studies. Recombinant forms of gD derived from HSV-1(KOS)rid1 and HSV-1(ANG) did not bind to HVEM(200t), explaining the inability of these viruses to infect CHO-HVEM12 cells. A variant gD protein, gD-1(delta290-299t), showed enhanced binding to HVEM(200t) relative to the binding of gD-1(306t). Competition studies showed that gD-1(delta290-299t) and gD-1(306t) bound to the same region of HVEM(200t), suggesting that the differences in binding to HVEM are due to differences in affinity. These differences were also reflected in the ability of gD-1(delta290-299t) but not gD-1(306t) to block HSV type 1 infection of CHO-HVEM12 cells. By gel filtration chromatography, the complex between gD-1(delta290-299t) and HVEM(200t) had a molecular mass of 113 kDa and a molar ratio of 1:2. We conclude that HVEM interacts directly with gD, suggesting that HVEM is a receptor for virion gD and that the interaction between these proteins is a step in HSV entry into HVEM-expressing cells.

Complement activation by a B cell superantigen.

Staphylococcal protein A (SpA), acting as a B cell superantigen, binds to the Fab region of human VH3+ Igs. Using SpA abrogated of its IgG Fc binding activity (Mod SpA) as a model B cell superantigen, we determined whether such an interaction causes complement activation. Addition of Mod SpA to human serum led to complement consumption and the generation of C3a. To determine whether this complement activation 1) was due to an interaction between VH3+ Igs and the Fab binding site of SpA and 2) proceeded via the classical complement pathway, we tested a panel of monoclonal IgM proteins for the ability to hind C1q following interaction with SpA. C1q binding was restricted to SpA-reactive, VH3+ IgM proteins. To formally determine whether the binding of SpA to the reactive VH3+ IgM proteins led to complement activation, we reconstituted the serum from a hypogammaglobulinemic patient with monoclonal IgM proteins and measured complement consumption and C3a generation following the addition of Mod SpA. We observed complement consumption and C3a production only in Mod SpA-treated serum reconstituted with a VH3+, SpA-binding, IgM protein. Taken together, these results provide compelling evidence that the interaction of the Fab binding site of SpA and VH3+ Igs can lead to complement activation via the classical pathway. This novel interaction may have significant implications for the in vivo properties of a B cell superantigen.