A high-content, in vitro cardiac fibrosis assay for high-throughput, phenotypic identification of compounds with anti-fibrotic activity.

A key feature in the pathogenesis of heart failure is cardiac fibrosis, but effective treatments that specifically target cardiac fibrosis are currently not available. A major impediment to progress has been the lack of reliable in vitro models with sufficient throughput to screen for activity against cardiac fibrosis. Here, we established cell culture conditions in micro-well format that support extracellular deposition of mature collagen from primary human cardiac fibroblasts - a hallmark of cardiac fibrosis. Based on robust biochemical characterization we developed a high-content phenotypic screening platform, that allows for high-throughput identification of compounds with activity against cardiac fibrosis. Our platform correctly identifies compounds acting on known cardiac fibrosis pathways. Moreover, it can detect anti-fibrotic activity for compounds acting on targets that have not previously been reported in in vitro cardiac fibrosis assays. Taken together, our experimental approach provides a powerful platform for high-throughput screening of anti-fibrotic compounds as well as discovery of novel targets to develop new therapeutic strategies for heart failure.

Fluorescent probe for high-throughput screening of membrane protein expression.

Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.