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 (Mattern et al and Edelmann et al).
E3 ligases conjugate ubiquitin to specific target proteins, signaling changes such as 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 (Mattern et al, 2014, in press, Mattern et al and Edelmann et al).
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 termed the UbiPro™ Drug Discovery Platform for studying DUBs and E3 Ligases which we use to screen our diverse collection of >200,000 drug-like small molecules. There are three components to the UbiPro™ platform:
Traditionally, these assays are considered to be low throughput assays that are not amenable to HTS. However, Progenra scientists have successfully screened a number of therapeutically relevant DUB and E3 ligase targets against our compound collection in a HTS format using a biophysical assay. These assays also serve as ideal orthogonal assays for confirming hit compounds identified using alternative approaches.
The identification of small molecules that antagonize the interaction between a E3 ligase or a DUB and its substrate is a rational approach for the identification of potential therapeutic agents. Progenra has developed HTS compliant binding assays for identifying and evaluating ubiquitin pathway enzyme inhibitors.
Progenra scientists have developed a range of different HTS ready DUB and E3 ligase activity assays. Progenra's DUB technologies utilize physiological substrates 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. Furthermore, our team has 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 assays 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).