Complement (C') is important in normal biology but it is also a pathological factor in a large number of inflammatory diseases. C'-mediated tissue injury has been reported in a wide variety of diseases including but not limited to autoimmune diseases, adult respiratory distress syndrome, Alzheimer's disease, stroke, heart attack, burn injuries, organ transplantation, and in extracorporeal blood oxygenation. There is a critical need for a therapeutically applicable C inhibitor. Several C' inhibitors have been described; however, the low-molecular weight inhibitors have shown low activity and high toxicity and are therefore pharmacologically undesirable. Recombinant forms of C' regulatory proteins such as CR1, DAF, MCP and CD59, and a monoclonal antibody to C5 have shown promise, as they have been effective in experimental diseases. All these inhibitors however, are large molecular weight protein, require intravenous administration and most of them have only a short half-life in vivo. Recent studies are focused on a second generation of smaller molecular weight derivatives with more desirable properties, but none of these has yet been adopted as a therapeutic agent. We have taken the alternative approach of screening a universe of random peptide information for C3-interactive peptides. Screening of a random peptide phage library yielded a novel small molecular weight cyclic peptide, Compstatin, (ICVVQDWGHHRCT) that binds specifically to human and primate C3 and inhibits the activation of C' by both the classical and alternative pathways. The peptide effectively inhibits C' in clinically relevant in vitro, ex vivo and most importantly in vivo models of C' activation. This application has three aims:
In Aim 1, the structure of Compstatin bound to a C3 fragment will be determined and the relationships between the structure and function of Compstatin will be studied by use of random peptide libraries, peptide synthesis, and NMR analyses.
In aim 2, photoaffinity probes, genetically engineered C3 molecules, and expressed C3 fragments will be used to identify the Compstatin binding Sites on C3, which may give important information on the C3 convertase recognition site on C3, a significant control point in the C3 architecture. In addition, we will also attempt to investigate the mechanism(s) by which Compstatin modulates C' activation.
Aim 3 will examine the C' inhibiting properties of Compstatin in clinical interventions. Half-life measurements, its biotransformation, and the ability of Compstatin to inhibit C' will be assessed in in vitro and in vivo experimental models of cardiopulmonary bypass (CPB) and in an ex vivo xenotransplantation model. In addition, the contribution of individual C' activation products in these clinical situations will be assessed using Compstatin, anti-C5 and C' receptor specific inhibitors.
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