HTS Assay for SUMO proteases based on AlphaScreen
The assay is based on developing a substrate whose cleavage can be monitored using AlphaScreen technology and employs the PHERAstar microplate reader from BMG LABTECH.
Small ubiquitin-like modifiers (SUMO 1-3) are a group of proteins which can be ligated to target proteins at lysine residues. SUMO conjugation/deconjugation is a highly dynamic process which plays a pivotal role in a number of pathological conditions such as diabetes and cancer. Therefore, it is of clinical significance to find compounds that interfere with the conjugation/deconjugation pathway. The current paper describes a specific HTS assay to discover SUMO protease inhibitors.
The technical note describes how the authors used a bacterial SUMOylation system to create a substrate that had SUMO3 modified with streptavidin conjugated to SUMO 2 which was His-tagged. In order to employ AlphaScreen this intact substrate is mixed with Strep-Tactin donor beads and Nickel-Chelate acceptor beads. Therefore in the absence of protease (or protease activity) the substrate will remain intact and excitation at 680 nm will lead to an emission signal at 520-620 nm; while if protease activity is present a decrease in emission signal will be detected. When run in a 384 well format and detected using the PHERAstar from BMG LABTECH they observed an average Z' = 0.83 indicating that this assay is indeed suitable for HTS applications.
The PHERAstar and CLARIOstar from BMG are both ideally suited to the performance of AlphaScreen as described in this application as either can be equipped with a red diode AlphaScreen laser.