Muscle atrophy (wasting), also known as myopathy, is a clinical complication of many diseases, e.g., cancer, AIDS, and diabetes, as well as a natural consequence of inactivity and aging;it significantly diminishes quality of life. The expression of a group of novel genes called atrogins (atrophy-specific genes) increases dramatically in skeletal muscles of fasting organisms and decreases rapidly upon resumption of feeding. One of these, MuRF-1 (Muscle- specific RING Finger-1), has been identified as an E3 ubiquitin ligase that is a component of atrophy-associated accelerated proteolysis. Ubiquitin pathway enzymes are considered excellent molecular targets for diverse indications, Thus, MuRF-1 is an attractive target for preventing or reversing muscle wasting associated with various pathologies. In a phase I grant application, it was proposed to establish an assay to screen MuRF-1 activity using a yeast based reporter assay in which human E3 ligase MuRF-1 was expressed along with a presumed substrate cardiac troponin. The yeast expression strain was generated, and it functioned as proposed, but numerous attempts at validation of this assay led to the conclusion that a robust screening assay could not be developed using the yeast system. The alternative approach -- an in vitro assay for MuRF-1ubiquitylation activity -- was subsequently undertaken and the in vitro assay was validated for high throughput screening (z'>0.6). In phase II it is proposed to utilize the in vitro E3 ligase assay to screen small molecule libraries (Maybridge HitFinder, Prestwick Chemical and Chembridge Premium Set libraries) to identify inhibitors of MuRF-1 (both self- ubiquitylation and substrate ubiquitylation). E3 RING finger ligases CARP2 and Praja-1 and the HECT domain ligase E6AP, which have been purified at Progenra, will be employed in selectivity screens and screening hits will be characterized in additional assays, including cell- based assays, to establish the specificity and efficacy of the inhibitors. Initial preclinical evaluation and chemical optimization will also be performed on selected lead compounds. The identification of specific inhibitors of MuRF-1 is the first step in the development of a class of novel targeted therapies for muscle wasting. These therapies should be efficacious in treating muscle wasting associated with aging, cancer, diabetes, and other chronic conditions.
Muscle atrophy (wasting), also known as myopathy, is a clinical complication of many diseases, e.g., cancer, AIDS, and diabetes, as well as a natural consequence of inactivity and aging;it significantly diminishes quality of life. A lack of effective therapies for muscle wasting has led to a search for new medicines based on molecular targets that have recently been identified. One of these targets is an enzyme called MuRF-1, which is linked to the degradation of muscle tissue;inhibitors of this enzyme may be effective therapies against muscle wasting. Progenra has developed a biochemical assay that can be used to identify chemical compounds that inhibit MuRF-1;in the second phase, it is proposed to use the assay to screen several compound collections and to place the most active and selective inhibitors found into development leading to clinical trial for muscle wasting.
|Goldenberg, Seth J; Marblestone, Jeffrey G; Mattern, Michael R et al. (2010) Strategies for the identification of ubiquitin ligase inhibitors. Biochem Soc Trans 38:132-6|