The complement system is critical for rapid detection and clearance of pathogens and immune complexes. Distinction between self and nonself is conferred by regulator proteins, which downregulate complement activation on host cells. Complement dysregulation is involved in several inflammatory conditions, including age-related macular degeneration (AMD). Deficiencies or mutations in specific complement proteins are linked to increased risk of developing AMD, and complement-targeted therapeutics have shown promise in treating AMD. In addition, it is critical to be able to monitor the onset and progression of AMD, as aberrant complement activation occurs on the retinal pigmented epithelium (RPE) in AMD patients. Complement proteins and regulators are constituents of drusen, including the terminal product C3d that remains covalently attached on surfaces on which complement has been activated. Our goal is to develop low-molecular mass chemical compounds and peptides that have strong binding affinities for C3d and fluorescence properties. These molecules will have potential as quantifiable biomarkers of complement activation in the eye and as diagnostics for early stage AMD. Our methods are computational and experimental. We will use pharmacophore-based virtual high-throughput screening, and protein-ligand docking to discover small chemical compounds with predicted binding affinities for C3d and intrinsic fluorescence. We will also use peptide design principles to design peptides with attached clinically-approved fluorophores that have predicted binding affinities for C3d. Both approaches are based on the interactions of C3d with its receptor, complement receptor 2 (CR2), and complement regulator Factor H (FH), using crystallographic structures and molecular dynamics simulations. We will test our computationally predicted C3d ligands, using an in vitro direct binding, biochemical and functional assays, and we will examine their photophysical properties using fluorescence spectroscopy. We will then evaluate the efficacies of the C3d ligands with highest potency and optimal fluorescence properties in human retinal pigmented epithelial cell-based assays. Finally, we will test the most efficacious ligands in histological sections of drusen-laden retinal tissues from non-human primate models of AMD, as well as using retinal sections from human AMD eyes. The proposed studies will generate data for a more focused study to evaluate a lead compound in animal models. !
This work will develop chemical compounds and peptides with fluorescent properties and high-affinity for the complement system protein C3d, a biomarker for age-related macular degeneration. These molecules will subsequently be explored as noninvasive in vivo diagnostics of early-stage age-related macular degeneration. Furthermore, this work paves the way for targeting therapeutics to sites of retinal inflammation.
