The purpose of this proposal is to establish high-throughput assays that measure fibronectin deposition in fibroblast cell monolayers to be used for screening for small molecule compounds that inhibit fibronectin assembly. The assembly of fibronectin controls the deposition of other extracellular matrix (ECM) molecules that contribute to the pathology of fibrotic diseases, which lack effective therapies. Fibronectin is also involved in tumorigenic neo-vessel formation and is the target of anti-angiogenesis therapies. Despite past difficulties in identifying small molecules that disrupt protein-protein interactions, there are an increasing number of examples where this approach has been successful. During initiation of assembly, a defined region at the N-terminus of fibronectin binds specifically and in a saturable manner to activatable receptor-like molecules. Display of these assembly sites is tightly controlled by signaling pathways that can be modulated by various pharmacological agents. During assembly progression, fibronectin binds integrins, transmembrane receptors shown to be amenable to peptidic and small molecule modulation. Thus, there may be multiple points of potential disruption of fibrillogenesis by small molecule compounds. We have developed a primary cell-based assay that measures binding and incorporation of fluorescently- labeled fibronectin into the ECM using a fluorescence microplate reader. The assay shows dose-dependence of both, binding of fluorophore-labeled fibronectin, and inhibition with a 49 amino acid peptide (FUD) that has high affinity for the N-terminal region of fibronectin, as well as inhibition with forskolin, a small molecule activator of protein kinase A. A secondary assay using high-content imaging is also in place using dual fluorescence to ascertain fibronectin fibrils and the integrity of the cytoskeleton. Modulation of fibrillogenesis will be quantified using a segmentation method developed by the imaging facility (UWCCC). These assays now require optimization into robust quantitative assays that will give a precise measure of fibronectin fibrillogenesis. In consultation with the Small Molecule Screening Facility on campus (SMSF), the primary assay will be optimized to a 384-well format. Once the assay is determined to be robust, as measured by a Z'-factor >0.4, it will be transferred to the SMSF where it will be optimized again for automated liquid handling in preparation for high throughput screening. A preliminary screen for modulation of fibrillogenic activity will be performed and potential non-cytotoxic hits (ascertained by a luminescence assay measuring cell viability) will be re-screened using the aforementioned high-content imaging secondary assay. Compounds that inhibit fibrillogenesis by disrupting initial binding to cell surfaces without affecting cytoskeletal integrity will be prioritized for further testing in select screens measuring modulation of cellular fibronectin and collagen incorporation into ECM. Future plans include submission of the fibrillogenesis assays for evaluation by MLPCN for subsequent screening of large libraries with the long-term goal of identifying compounds that will generate therapeutics useful in the treatment of fibrotic diseases and cancer.
Fibrotic diseases, primarily of the lung and kidney are characterized by an excessive accumulation of connective tissue proteins that ultimately damages organ function. In order to obtain nutrients, tumors promote the formation of new blood vessels in their vicinity. Current therapies for both fibrotic diseases and some cancers are not entirely effective and produce undesirable side effects. Fibronectin is a protein that forms fibrils in connective tissue and is an important regulator of connective tissue protein deposition and of the development of new blood vessels. We are interested in developing assays that will measure fibronectin fibril formation, with the idea that compounds that modify fibronectin fibril formation may eventually be useful in therapies for fibrotic diseases and cancer.
|Tomasini-Johansson, Bianca R; Johnson, Ian A; Hoffmann, F Michael et al. (2012) Quantitative microtiter fibronectin fibrillogenesis assay: use in high throughput screening for identification of inhibitor compounds. Matrix Biol 31:360-7|