Tremendous attention has shifted towards implementation of molecularly-targeted therapeutics in cancer treatment. In the case of multiple myeloma, the proteasome inhibitors Bortezomib and Carfilzomib have gained FDA approval; however, patients develop a resistance to these drugs. Recent studies have identified that inhibition of deubiquitinating enzymes (DUBs) have led to increased cancer cell death in bortezomib-resistance cell lines. However, it is still unclear how the inhibition of both DUBs and the proteasome can lead to increased patient survival. To address this question, we developed a reporter of DUB activity that is cell permeable, long-lived, fluorescent, and specific to DUBs. The reporter consists of an N-terminal β-hairpin motif that acts as both a ‘protectide’ to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The β-hairpin was conjugated onto the N-terminus of a substrate consisting of the last four amino acids in ubiquitin to facilitate DUB mediated cleavage and a fluorophore AFC. The reporter was characterized in both HeLa and OPM2 cell lysates and intact cells. Reporter performance was compared to an unprotected DUB reporter (Z-LRGG-AMC). The calculated kinetic rates in cell lysates were fitted to the Michaelis-Menten equation by non-linear regression to determine the kinetic rate constants. The Vmax/Km in OPM2 lysates was 0.602*10-3 for RW-LRGG-AFC and 1.378*10-3 for Z-LRGG-AMC. Control experiments with PR-619 DUB inhibitor demonstrated the specificity of the reporter to DUBs. The reporter was internalized by intact cells at concentrations from 200 to 1400 µM as assessed by fluorometry and microscopy. Results demonstrated a rapid uptake of the reporter in intact cells with the enzyme-substrate reaction rates becoming saturated after 5 hours using 1mM peptide. Intracellular peptide stability was assessed using a degradation assay in OPM2 lysates followed by RP-HPLC analysis. Although the kinetics of the Z-LRGG-AMC peptide were slightly better in cell lysates, it was demonstrated that this peptide is not cell permeable, and therefore not compatible with high-throughput screening methods. The novel DUB reporter presented here is cell permeable with rapid uptake kinetics, and superior in terms of intracellular stability and DUB sensitivity. These qualities make it ideal for performing dynamic high-throughput measurements of DUB activity in intact single cells.

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell permeable peptide-based reporter

255th National Meeting (2018)

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