Meso-Scale Diagnostics (MSD) aims to discover compounds effective at inhibiting the ubiquitin E3 ligase activity of RNF28 (Murfl), a protein involved in muscle wasting. These compounds will be used to evaluate the importance of the ubiquitin E3 ligase activity in muscle wasting. We will screen our in house library of 120,000 compounds against a E3 ligase assay developed at MSD for measuring the activity of RNF28. Phase I of this project will include optimization of the assay for screening followed by screening of our compound library. This initial primary screen will be followed by selection of inhibitors from this screen for retesting and specificity testing vs other E3 ligases. Specific inhibitors will also be titrated to determine their potency. The product from the phase I study will be a selection of specific inhibitors validated via an In vitro biochemical screen and data on the IC50s for these compounds. Phase II of this project will involve the analysis of these compounds for activity in cell based models of muscle wasting. This will involve detailed investigation of protein turnover and degradation via assays for a number of key muscle proteins and associated pathways involved in muscle development such as MyoD and Akt.
Specific Aims : 1) Optimization of RNF28 (Murfl) E3 ligase assay 2) Screen of 120,000 compounds 3) Selection and retesting of inhibitors from primary screen 4) Determination of inhibitors specificity using other E3 ligase assays 5) Titration of confirmed inhibitors to establish IC50 and potency
