Since 2002 Progenra scientists have been working to understand the intricacies of the ubiquitin pathway to maximize discovery of new classes of molecules. The culmination of this work has led to the creation of a comprehensive toolbox falling under the UbiPro™ Drug Discovery Platform for studying DUBs and E3 Ligases.
DUB Technology Platform
DUBs constitute a class of isopeptidase that cleave ubiquitin from target proteins, including other ubiquitins. Other classes of isopeptidase perform the same function for ubiquitin-like proteins fused to their target proteins. Both conjugation and deconjugation of ubiquitin or ubiquitin-like proteins (UBLs) contribute to the maintenance of cellular homeostasis, the disruption of which can lead to disease. Accordingly, DUBs and UBL isopeptidases have been found to be overexpressed or dysregulated in a number of pathophysiologies[Nicholson et al, 2007].
Progenra’s DUB technologies utilize substrate specific assays to identify selective small molecule inhibitors of DUBs. Additional technologies, such as DUBSelect™ have been created to minimize the identification of reactive molecules in the first round of high-throughput screening.
· Rapid, robust readout for DUB activity within 30-60 minutes
· Reporter activity is quantified by fluorescence
· Miniaturization to multiwell (96, 384, 1536) formats
· Availability of multiple reporter substrates to assess specificity and de-replication
· Multiplexing capabilities
· Elimination of reactive molecules from screening hits
· Large panel of DUBs to determine selectivity
Ligase Technology Platform
E3 ligases conjugate ubiquitin to specific target proteins, directing them to the proteasome for degradation. In certain cases the E3 ligase receives ubiquitin from an E2 enzyme and transfers it to the target protein; in other cases it acts by interacting with both the E2 enzyme and the substrate but does not directly receive ubiquitin. Conjugation of ubiquitin to target proteins is essential to many key cell regulatory processes. There are approximately 600 different E3 ligases in the human proteome, many of which have been associated with various diseases.
Although pharmaceutical companies have struggled to identify specific inhibitors for E3 ligases of therapeutic interest, to date only a limited number of clinical candidates have emerged. Progenra scientists have developed a series of novel E3 ligase autoubiquitylation and substrate ubiquitylation assays which have been used to identify specific small molecule inhibitors of E3 ligases. These platforms take advantage of an ubiquitin binding domain's affinity for polyubiquitin chains, allowing for the analysis of ubiquitin chain formation; they have been used successfully with RING and HECT ligases [Marblestone et al, 2010]
· Rapid and robust readout
· Potential for miniaturization to multiwell (96, 384, 1536) formats
· Ability to detect auto or substrate ubiquitylation
· Modular nature; works for all E3 ligases tested
· Multiplexing capabilities
· E1/E2 assay to eliminate non-specific E3 inhibitors
· Large panel of E3 Ligases to determine selectivity