Because of its involvement in a broad range of cellular signal transduction and other mechanisms, the ubiquitin pathway is now recognized as a new opportunity for discovering drugs to treat a variety of diseases. Initial success was achieved with the approval of the proteasome inhibitor VELCADE in 2003 for refractory multiple myeloma. It is believed that targeting a less general mechanism than proteasome inhibition would yield drugs with fewer side effects than those seen with VELCADE. E3 ligases, the enzymes that add ubiquitins to specific target proteins or groups of proteins, have been studied extensively in the last five years and are considered promising targets for selective drug discovery. Several of them have been linked genetically or biochemically with specific diseases, and assays have been employed to find inhibitors via high throughput screening. Despite this effort, no promising lead compounds have emerged, however, because the assays either lacked sufficient sensitivity or did not recapitulate correctly the physiological state of E3-mediated ubiquitylation. It is recognized that for the field to advance there is an urgent need for novel in vitro E3 assays with improved sensitivity and faithful representation of the native conditions of the ubiquitylation pathway. This proposal addresses both of these shortcomings by developing a homogeneous, cost effective, sensitive, and robust assay that quantifies true isopeptide bonds between ubiquitin and its substrate (a target protein or another ubiquitin). Moreover, the assay can be configured for high throughput screening. Poly-ubiquitylation will be detected by novel labeled Tandem Ubiquitin Binding Entities (TUBEs) and quantified by TR-FRET. Such an assay offers uniquely high sensitivity and physiological relevance, making it superior to those now in use, and increasing the likelihood of discovering selective, therapeutically useful inhibitors. The ultimate commercial goal is to develop an assay format that can be applied to various E3 enzymes and substrates, facilitating drug discovery for known and emerging E3-mediated disease processes.

Public Health Relevance

Enzymes acting act at early steps of the ubiquitin proteasome pathway should be more selective targets than the proteasome itself. E3 ligases act early to conjugate ubiquitin to certain target proteins, and Progenra proposes to develop a cell-based screening assay to discover inhibitors of ligases that are associated with particular diseases.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZRG1-IMST-G (10))
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Gerratana, Barbara
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Progenra, Inc.
United States
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Marblestone, Jeffrey G; Larocque, James P; Mattern, Michael R et al. (2012) Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins. Biochim Biophys Acta 1823:2094-7