A Novel Target for Antifibrotic Drugs and A High Throughput Assay to Screen for Such Drugs Keywords: Antifibrotic Drugs
B. Stefanovic
Department of Biomedical Science
College of Medicine Florida State University Tallahassee
FL 32306
United States
Abstract:
Fibrosis is a major health problem affecting 30% of world population. It is characterized by excessive synthesis of type I collagen in parenchymal organs. There is no cure for fibrosis and organ failure is the direct consequence of excessive collagen deposition. The development of antifibrotic drugs targeting collagen has been hampered by the lack of knowledge of the regulatory steps specific for type I collagen. We have discovered that synthesis of type I collagen critically depends on the interaction between the two molecules; the 5’ stem-loop structure (5’SL) present in collagen mRNAs and the protein LARP6. 5’SL is a unique sequence element found only in collagen mRNAs which encode for type I and type III collagens. It is not found in any other mRNA. LARP6 binds 5’SL with high specificity and affinity and this binding is critical for high expression of type I collagen. Knock down of LARP6 decreased collagen expression from human lung and scleroderma fibroblasts. Fibroblasts from knock-in mice in which the 5’SL was mutated in the context of endogenous collagen α1(I) gene produced <50% of type I collagen. Hepatic stellate cells (HSCs) are responsible for development of liver fibrosis. When HSCs were isolated from the 5’ SL mutant animals, they failed to upregulate type I collagen upon activation. 5’SL mutant animals also developed only a small degree of liver fibrosis compared to wt animals. These findings indicate that high level of collagen synthesis depends on the interaction between 5’ SL and LARP6 and validate this interaction as the prime target for development of specific antifibrotic drugs. We have developed a high throughput screen for antifibrotic drugs targeting LARP6/5’SL interaction based on fluorescence polarization. The details of this assay, its performance, applications for drug screening and potential to discover true antifibrotic drugs will be presented.
