DUB Technology Platform

DUBs are 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]. Inhibition (or, in some cases, activation) of DUBs represents a novel, underexploited form of therapeutic intervention, and efforts are underway to discover or design inhibitors and activators of these enzymes that can be developed as medicines [Ubiquitin Drug Discovery and Diagnostics 2009, 51st ASH Annual Meeting and Exposition, AACR-NCI-EORTC International Conference Molecular Targets and Cancer Therapeutics].

The concept behind Progenra’s IsoPro Assay for DUBs and UBL isopeptidases is the requirement of a free N terminus on the reporter enzyme for catalytic activity. Upon conjugation to the C-terminus of ubiquitin/UBL, the reporter is rendered catalytically inactive. After cleavage of the Ub/Ubl-reporter system by isopeptidase activity, the activated, free reporter acts upon its substrate. Thus, in the coupled assay, the signal generated by cleavage of the reporter enzyme’s substrate is a quantitative measure of isopeptidase activity. Activity of the reporter is quantified by fluorescent or colorimetric readouts[Nicholson et al., 2008]. The IsoPro Assay platform is also called CHOP-Reporter that has been licensed to LifeSensors Inc. for distribution to consumer markets.

Benefits:
Rapid and robust readout for deubiquitylating activity within 30 minutes
Activity of the reporter is quantified by a fluorescent readout
Non-radioactive reporter substrates
Miniaturization to multiwell (96, 384, 1536) format
Fusion with PLA2 generates a larger, more relevant leaving group as compared to Ub-AMC
In contrast to Ub-AMC does not autofluoresce in the UV range
Availability of multiple reporter substrates to assess specificity and de-replication