High-throughput compatible FRET based assay to identify small molecule inhibitors of AMSH deubiquitinase activity

摘要

Deubiquitinases (DUBs) play an important role in regulating the ubiquitin landscape of proteins. The DUB AMSH (Associated Molecule with the SH3 domain of STAM) has been shown to be involved in regulating the ubiquitin-dependent downregulation of activated cell surface receptors via the endo-lysosomal degradative pathway. Therefore small molecule AMSH inhibitors will be useful chemical probes to study the effect of AMSH DUB activity on cell surface receptor degradation. Currently there are no known selective inhibitors of AMSH or high-throughput compatible assays for their identification. We report the development and optimization of a novel FRET based add-and-read AMSH deubiquitinase assay in a 384-well format. In this format, the optimal temperature for a high throughput screen (HTS) was determined to be 30°C, the assay tolerates 5% DMSO and has a Z-score of 0.71 indicating HTS compatibility. The assay was used to show that AMSH selectively cleaves Lys63-linked diubiquitin over Lys48- and Lys11-linked diubiquitin. The IC50 value of the non-specific small molecule DUB inhibitor N-ethylmaleimide was 16.2 ± 3.2 μM and can be used as a qualitative positive control for the screen. We conclude that this assay is HTS compatible and can be used to identify novel small molecule inhibitors of AMSH.