P.19.5 Development of an alphascreen-based substrate ubiquitination assay to identify inhibitors of the muscle ubiquitin ligase MuRF1

MuRF-1 (Muscle Ring Finger protein-1) is an E3 ubiquitin ligase that is thought to play key role in muscle atrophy. MuRF-1 levels are increased in multiple animal models of muscle atrophy and it has the ability to ubiquitinate myofibrillar proteins and target them for proteasomal degradation. Mice that lack MuRF-1 are protected from muscle wasting during starvation, sciatic nerve denervation and hindlimb suspension. Lack of MuRF-1 has also been shown to prevent the loss in muscle function induced by TNF-alpha treatment in mice. As such, inhibitors of MuRF-1 activity are expected to preserve muscle mass and function in muscle wasting situations. In order to identify inhibitors of MuRF1, we have developed an alphascreen-based substrate ubiquitination assay that is amenable for screening compounds in a high throughput mode. The assay utilizes flag-tagged Troponin I as a substrate for MuRF-1. In the presence of E1 and E2 ubiquitinating enzymes and biotinylated ubiquitin, MuRF-1 adds on ubiquitin moieties to Troponin I. The ubiquitinated Troponin I is then detected by the addition of streptavidin-coated donor beads and anti-flag antibody coated acceptor beads that are then brought in close proximity by ubiquitinated Troponin I. Stimulation with laser light facilitates energy transfer between the donor and acceptor beads that results in a fluorescent signal. Inhibitors of MuRF-1 activity result in a dose-dependent decrease in the alphascreen signal. Actives that come out from this screen can be confirmed using a Troponin I ubiquitination ELISA that uses an anti-ubiquitin antibody. The alphascreen assay was also modified in certain ways to eliminate compounds that act upstream of MuRF-1 (by assessing E2 auto-ubiquitination) and to weed out compounds that inhibit MuRF-2 and MuRF-3. Use of these assays is expected to facilitate the identification of selective inhibitors of MuRF-1 for doing pharmacological proof of concept studies in animal models of muscle wasting.