TOLLREG

Fibrotic diseases (sometimes known as scarring) are the most important causes of mortality worldwide. They are the common consequence of multiple initiating events (trauma, infection, surgery …) and affect multiple organs, including the cardiovascular system, lungs, liver, skin and cornea. Fibrosis is a form of defective tissue repair, characterised by increased cell proliferation and deposition of extracellular matrix, notably fibrillar collagens. It is the result of a complex chain of events involving inflammation, tissue repair and remodelling. In view of this complexity, several strategies have been proposed to prevent or treat fibrosis, though most of these suffer from a general lack of specificity. Particularly promising targets are the procollagen C-proteinases (also known as tolloid proteinases), the extracellular enzymes that trigger collagen fibril formation by release of the C-propeptides from soluble procollagen precursors. Recent work has shown that tolloids are subject to complex regulation at the protein level, involving a number of endogenous activators and inhibitors. Among the activators, the most extensively studied are the procollagen C-proteinase enhancers (PCPEs) which have the unusual property of increasing tolloid proteinase activity, up to 20-fold, in a substrate specific manner, making them potential anti-fibrotic targets in their own right. As for inhibition, the first protein inhibitor of tolloid proteinases has recently been identified, called Sizzled, thus opening up new potential routes for therapeutic applications. Finally, a number of synthetic inhibitors of tolloid proteinases have been developed, though most are based on hydroxamate technology which has a number of drawbacks. The overall goals of the proposed project are to: (a) understand the mechanism of action of the major PCPE, by high resolution structure determination in complex with its protein target, the C-propeptide trimer of fibrillar procollagens, (b) understand the mechanisms of action of the endogenous inhibitor Sizzled, and related proteins, by interaction, mutagenesis and structural studies, and (c) develop specific phosphinic based inhibitors and activity-based probes for tolloid proteinases, by structure based design. The project is timely in that we have recently obtained crystals of the C-propeptide trimer and have isolated a stable complex with PCPE. This and the recent determination of the 3D structure of a tolloid catalytic domain make a structural approach feasible, with a high probability of success. With regard to Sizzled, since this protein was originally identified in Xenopus and has no human counterpart, understanding the mechanism of action could provide new avenues for the development of highly specific tolloid inhibitors. Concerning synthetic molecules, phosphinic peptide inhibitors, developed by one of the partners, have shown high potency against a related zinc metalloproteinase from crayfish which suggests that such inhibitors could also be developed for tolloids. Finally, a particularly novel part of the project is the development of activity-based probes that will permit for the first time detection of tolloid proteinase activity in complex biological tissues and extracts. The knowledge acquired during the course of the proposed project will form the basis for the development of new therapeutic strategies in the prevention and treatment of fibrotic disorders, where the world market is of the order of billions of euros. In the shorter term, it will provide new molecules and tools to gain further understanding of the mechanisms of tissue remodelling. The project is both innovative and multidisciplinary in that it will establish a new consortium, and bring together leaders in the field in the French scientific community (Moali, Aghajari, Dive), combining structural biology, chemical and biochemical approaches to tackle different mechanisms of tolloid proteinase regulation.