Molecular mechanisms of the pathological stabilization of the complement system C3 convertase and its therapeutic control – COMPC3

The complement system is a master innate immune defense cascade, playing a key role in inflammation, pathogen elimination and mounting of an adaptive immune response. The central enzyme of the complement cascade is the C3 convertase C3bBb. Therefore the C3 convertase needs to be tightly regulated to avoid overactivation and subsequent cell and tissue damage, leading to severe inflammatory, thrombotic and age-related diseases, affecting primarily the kidneys. A large number of negative regulators of C3bBb exist, but only one protein –properdin, is able to stabilize the complex, to increase its half-life and to render it resistant to regulation. Properdin binds with greater affinity to cell bound C3bBb than to cell-bound C3b and is implicated in the conversion switch between C3 to C5 convertase. The exact binding site of properdin to C3b and to the C3 convertase is poorly studied and its mode of action and its influence in tissue injury is not well understood. In pathological conditions, the stabilization of the C3 convertase is achieved also by a particular class of autoantibodies, called C3 nephritic factors (C3Nef), which bind to a neo-epitope(s), present on the C3bBb complex. The location(s) of this neo-epitope(s) is not known and there was no evidence about the position or number of relevant epitopes and about the overlay binding site with the others regulatory proteins of the C3 convertase, in particular the Factor H. C3Nef is associated with the C3 glomerulopathy, which defines a disease spectrum caused by abnormal control of the complement activation that results in predominant glomerular C3 fragment deposition along or within the basal membrane. We already analyzed a French multicentric cohort of 150 patients with C3G and a C3NeF was found in 60% of patients. The epitope mapping of the C3Nefs was always considered as a very complex issue due to the fact that they bind to a neo-epitope, exposed on a very short leaved complex. The objective of this project is to map the area on the C3 convertase, to which the stabilizing factors bind (i.e, properdin and C3Nef). The identification of the binding sites will bring novel basic knowledge and will allow to understand their mode of action. We have the ambition to provide an accurate characterization of the binding sites, which are very difficult to be addressed by structural biology approaches (crystallography or NMR) due to the complex heterogenic oligomeric nature of properdin and the size of the C3bBb complexes. We propose an original approach to resolve this problem, using a specific functional assay. We already isolated three subtypes of C3NeF according to the capacity of patients ‘IgG to stabilize the C3 convertases C3bBb and C3bBb-P. We demonstrated that Properdin have an additive or competitive effect on the C3 convertase stabilisation according to the subtype of the C3NeF. Mapping of the key residues for the binding will allow original design of inhibitory molecules with therapeutic potentials. Another objectives of the project is to generate rapid diagnostic tests and novel bio-markers to follow complement activation with the implementation of novel sensitive and specific detection systems (e.g. diagnostic antibodies, specific ELISAs and ‘signature arrays’). We will design a novel method for direct screening of C3NeF using immunoreactivity analysis against already identified inhibiting peptides.
We aim at developing several optimized peptides and for some of them, we will attempt to design small drug-like chemicals that mimic these peptides. We expect to identify several inhibitory peptides and drug-like compounds that will undergo several rounds of optimization and that could be used as a proof of concepts to start a full drug discovery project using the functional assays developed during this project. Such molecules could be very valuable for many complement-mediated diseases, where the activity of the C3 convertase has to be controlled.