The ubiquitin-proteasome pathway degrades ~80-90% of cellular proteins and is of great interest as a source of new therapeutics for multiple diseases. The pre-proteasomal phase of the pathway, catalyzed by the ubiquitin E1 activating enzyme, E2 conjugating enzyme, and E3 ligase, is predicted to afford drugs that act more selectively than proteasome inhibitors, the first class of drug developed from the ubiquitin proteasome pathway, and thus be less compromised than proteasome inhibitors by side effects. Evidence implicates E3 enzymes in various physiological processes and disease states. For these reasons, major pharmaceutical companies have spent several years identifying novel, selective E3 inhibitors for drug development, but to date the results have been disappointing, as very few E3 inhibitors have entered clinical trials. Reasoning that improved assays were needed to discover high quality molecules in screening, Progenra developed, in Phase I of this grant project, facile, homogeneous assays for E3 ligases of choice, including an -screen based assay that can be employed for substrate ubiquitylation as well as auto-ubiquitylation. The E3 ligase whose physiological substrate (Troponin I) was configured for the substrate based assay is the simple RING finger E3 MuRF1, which is associated with muscle wasting, or myopathy, a pathological complication of numerous diseases, including cancer, HIV/AIDS, and diabetes, as well as a natural consequence of inactivity and aging. Muscle wasting is a result of increased rates of protein breakdown and decreased rates of protein synthesis, with a secondary net loss in muscle weight of 10-15%. MuRF1 is one of several genes overexpressed in myopathy and it is a well-validated target for therapeutic agents to treat muscle wasting. In the phase II 2-year project, it is proposed to utilize the MuRF1/Troponin I assay developed in Phase I to screen Progenra's 220,000 member small molecule library for inhibitors of this substrate ubiquitylation. These inhibitors will be characterized using secondary assays to establish selectivity and efficacy. Initial preclinical evaluation will be performed using in vitro and in vivo models of muscle wasting, and medicinal chemistry will be employed for lead optimization. The identification of specific inhibitors of MuRF1 will be an important first step in producing novel targeted therapies for muscle atrophy.
Progenra proposes to utilize a recently developed assay to find inhibitors of MuRF1, a ubiquitin pathway enzyme implicated in muscle wasting/cachexia, a severe complication of many diseases and aging. The assay was developed in Phase I of this SBIR project, and in Phase II it will be employed in high throughput mode to develop clinical candidate inhibitors to treat muscle wasting.