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  • In the context of E ligase drug discovery it


    In the context of E3 ligase drug discovery, it is critical to identify the appropriate E2/E3 substrate pairing to ensure the development and use of the most physiologically relevant screening assay. There have been many reports of limited E2/E3 activity profiling with a small number of E2 and E3 RGDfK using ELISA-based assays, structural-based yeast two-hybrid assays, and western blot (Lechtenberg et al., 2016, Marblestone et al., 2013, Sheng et al., 2012). All of these approaches are time consuming, require large amounts of reagents, and are difficult to adapt for HTS. We have successfully used our E2/E3 MALDI-TOF assay to identify active E2/E3 pairings, which could then be further characterized using our HTS screen. The “E2 scan” was quickly and easily adapted, collecting data of three E3 enzymes against 29 E2 enzymes at 8 time points in one single experiment. Moreover, after identification of the right E2/E3 pairs, we applied the MALDI-TOF E2/E3 assay to determine inhibition rates and the IC50 values of small-molecule inhibitors. In a proof-of-concept study, we performed an HTS for inhibitors of three E3 ligases. The MALDI-TOF analysis speed of 1.3 s per sample (∼35 min per 1,536-well plate) and low sample volumes (reaction volume 5 μL/MALDI deposition 250 nL) make the E2/E3 MALDI-TOF assay comparable with other fluorescence/chemical probe-based technologies. Automatic sample preparation, MALDI-TOF plate spotting, and data collection allowed us to quickly analyze thousands of compounds through the use of 1,536 sample targets. The assay successfully identified bendamustine as a novel small-molecule inhibitor for HOIP, an attractive drug target for both inflammatory disease and cancer (Ikeda et al., 2011, MacKay et al., 2014, McGuire et al., 2016, Stieglitz et al., 2013). Bendamustine, a nitrogen mustard, shows likely very high reactivity against a range of targets in the cell including its intended target DNA. However, it is surprising that it shows a 12- and 18-fold higher activity against HOIP than against ITCH and MDM2, respectively, suggesting that there is possibly a structural effect and some selectivity can be reached between different E3 ligases. It also shows that E1 conjugating enzymes were not affected by bendamustine as the same enzyme was also used in the MDM and ITCH reaction. While this is just a proof-of-concept study characterizing E2/E3 activity and identifying inhibitors in an in vitro system, follow-up studies will need to verify results in cellular and ultimately in vivo models.
    Significance Our understanding of the ubiquitin biology has been rapidly expanding. The role of the ubiquitin system in the pathogenesis of numerous disease states has increased the interest in finding new strategies to pharmacologically interfere with the enzymes responsible of the ubiquitination process. However, the development of molecules targeting the ubiquitin cascade, especially the E2 conjugating enzymes and E3 ligases, has not being extensively sustained by the availability of robust and affordable technologies for extensive primary screening of compound libraries. Performing high-throughput screening in the ubiquitin field remains challenging and it usually requires engineered proteins or the synthesis of chemical probes. Here we show that the MALDI-TOF E2-E3 assay is a robust, scalable, label-free assay that can be employed for primary screening of compound libraries against E2 conjugating enzymes and E3 ligases belonging to different families and representative of all the currently known ubiquitylation mechanisms. The MALDI-TOF E2/E3 assay is a readily accessible addition to the drug discovery toolbox with the potential to accelerate drug discovery efforts in the ubiquitin pathway.
    Acknowledgments We would like to thank the DNA cloning, Protein Production, DNA sequencing facility, and mass spectrometry teams of the MRC Protein Phosphorylation and Ubiquitylation Unit for their support. We would like to thank Prof. Dario Alessi, Prof. Ronald Hay, Prof. Philip Cohen, Prof. Katrin Rittinger, Dr. Satpal Virdee, Dr. Sarah Buhrlage, Dr. Natalia Shpiro, Dr. Siddharth Bakshi, Dr. Andrea Testa, and Dr. Francesca Morreale for tools and helpful discussions; Bruker Daltonics, particularly Meike Hamester, Rainer Paape, and Anja Resemann for their technical support. We thank Dr. Anthony Hope, Alex Cookson, and Lorna Campbell for providing the FDA-approved compound library and support with the liquid handling. This work was funded by Medical Research Council UK (MC_UU_12016/5), and the pharmaceutical companies supporting the Division of Signal Transduction Therapy (DSTT) (Boehringer-Ingelheim, GlaxoSmithKline, and Merck KGaA).