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  • br Results br Discussion The ubiquitin system has in recent


    Discussion The ubiquitin system has in recent years become an exciting area for drug discovery (Cohen and Tcherpakov, 2010), as multiple enzymatic steps within the ubiquitylation process are druggable. The potential of targeting the ubiquitin-proteasome pathway was first demonstrated in 2003 by the approval of the proteasome inhibitor bortezomib (Velcade; Millennium Pharmaceuticals) for use in multiple myeloma. While proteasome inhibition is a broad intervention affecting general survivability, E3 ubiquitin ligases and DUBs (Ritorto et al., 2014) represent the most specific points of intervention for therapeutic tools as they specifically regulate the ubiquitylation rate of specific substrates. For Estradiol valerate example, nutlins, cis-imidazoline analogs able to inhibit the interaction between MDM2 and tumor suppressor p53, have recently entered early clinical trials for the treatment of blood cancers (Burgess et al., 2016). The small number of drugs targeting E3 ligases currently on the market is partly due to the lack of suitable high-throughput assays for drug discovery screening. Traditionally, screening for inhibitors of ubiquitin ligases and DUBs has been performed using different fluorescence-based formats in high-throughput and ELISA, SDS-PAGE, and western blotting in low-throughput. These approaches show a number of limitations. ELISA- and SDS-PAGE-based approaches are time consuming and low-throughput by nature, and therefore mostly incompatible with HTS. The applicability of fluorescence-based techniques such as FRET is dependent on being able to get FRET donors and acceptors in the right distance, and the fluorescent label might affect inhibitor binding. To address these issues, we have developed a sensitive and fast assay to quantify in vitro E2/E3 enzyme activity using MALDI-TOF MS. It builds on our DUB MALDI-TOF assay (Ritorto et al., 2014), which has enabled us to screen successfully for a number of selective DUB inhibitors (Kategaya et al., 2017, Magiera et al., 2017, Weisberg et al., 2017), and adds to the increasing number of drug discovery assays utilizing label-free high-throughput MALDI-TOF MS. Apart from E2/E3 Estradiol valerate and DUBs (Ritorto et al., 2014), high-throughput MALDI-TOF MS has now successfully been used for drug discovery screening of protein kinases (Heap et al., 2017b), protein phosphatases (Winter et al., 2018), histone demethylases, and acetylcholinesterases (Haslam et al., 2016), as well as histone lysine methyltransferases (Guitot et al., 2014). 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 enzymes 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.