Atrogin-1 inhibitors for Muscle Wasting
DESCRIPTION (provided by applicant): Muscle atrophy (wasting) is a serious clinical complication of diabetes and other chronic pathoses, leading to increased morbidity and reduced life expectancy. While symptoms have been addressed by a number of interventions, no successful therapy has been developed. Recently, various proteins whose expression is enhanced under conditions of atrophy-inducing starvation have been identified in rats. In particular, the expression of a set of novel genes called atrogins (atrophy-specific genes) increases significantly in skeletal muscles of fasting organisms and decreases rapidly when feeding is resumed. The product of one of these genes - atrogin-1 - is an F-box protein ubiquitin E3 ligase, a critical enzyme of the ubiquitin-proteasomal degradation pathway. This pathway is implicated in muscle atrophy, since proteasome inhibitors protect against muscle wasting in model systems. Selective attenuation of proteasomal activity associated with muscle wasting may be achievable using inhibitors of atrogin-1. In Phase 1, E3 ubiquitination activity of atrogin-1 ectopically expressed in yeast will be demonstrated against a known substrate, calcineurin. Then, a yeast-based assay for atrogin-1 will be developed and validated, in preparation for high throughput screening of compound and natural products collections to discover inhibitors of atrogin-1 in Phase 2. For the assay, the following will be cloned and expressed in yeast S. cerevisiae: the substrate of atrogin-1, fused to p53 linked to a beta-galactosidase reporter; atrogin-1 E3 ligase complex; and an E3 ligase suitable as a selectivity control. Results of pilot experiments to validate this modular assay demonstrate that human E3 ligases, p-53 activated reporter plasmid, and p53-fused ligase substrate ectopically expressed in yeast give a null signal, but produce a robust reporter signal when E3 ligase is left out, consistent with ubiquitination/degradation of fused p53 by E3. Successful completion of Phase 1 should result in a validated screening assay that can be utilized in Phase 2. In Phase 2, active principles will be isolated from the best extract leads, with the goal of identifying novel, potent, and selective inhibitors of atrogin-1 for development as adjuvant therapy of muscle wasting associated with diabetes.
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