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  • Due to the high attractiveness of


    Due to the high attractiveness of E2 and E3 ligases as drug targets, a number of drug discovery assays have been published, based on detection by fluorescence (Dudgeon et al., 2010, Krist et al., 2016, Zhang et al., 2004), BEZ 235 (Davydov et al., 2004, Huang et al., 2005, Kenten et al., 2005, Marblestone et al., 2010), tandem ubiquitin-binding entities (Heap et al., 2017a, Marblestone et al., 2012), surface plasmon resonance (Regnstrom et al., 2013), or cellular and bacterial two-hybrid (Levin-Kravets et al., 2016, Maculins et al., 2016). However, many of these tools are either too expensive for very high-throughput drug discovery or potentially result in false-positive and false-negative hits due to the use of non-physiological E2/E3 ligase substrates. We have addressed this gap by developing the first in vitro label-free MALDI-TOF mass spectrometry-based approach to screen the activity of E2 and E3 ligases that uses unmodified mono-ubiquitin as substrate. As a proof-of-concept, we screened a collection of 1,430 US Food and Drug Administration (FDA)-approved drugs for inhibitors of a subset of three E3 ligases that are clinically relevant and belong to three different E3 ligase families. The screen shows high reproducibility and robustness, and we were able to identify a subset of 15 molecules active against the E3 ligases tested. We validated the most powerful positive hits by determining the half maximal inhibitory concentration (IC50) values against their targets, confirming that bendamustine and candesartan cilexitel inhibit HOIP and MDM2, respectively, in in vitro conditions.
    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 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 enzymes 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).