Introduction Ubiquitylation is a post
Introduction Ubiquitylation is a post-translational modification which impacts almost every biological process in the cell. Dysregulation of the ubiquitylation pathway is associated with several diseases, including cancer, neurodegenerative disorders, and immunological dysfunctions. Single ubiquitin moieties or polyubiquitin chains are added to the substrate by the combined action of three different Fluticasone propionate molecular of enzymes: the E1 activating enzymes, the E2s conjugating enzymes, and the E3 ligase enzymes (Pickart, 2001). In the first step, a single ubiquitin molecule is coupled to the active site of an E1 ubiquitin-activating enzyme in an ATP-dependent reaction. In the second step, the ubiquitin molecule is transferred from E1 to an E2 ubiquitin conjugating enzyme. In the final step, ubiquitin is transferred to the protein substrate in a process mediated by an E3 ubiquitin ligase, which provides a binding platform for ubiquitin-charged E2 and the substrate. Ubiquitin chain formation is highly specific and regulated by a plethora of different E2 conjugating enzymes and E3 ligases. The human genome encodes two ubiquitin-activating E1, >30 ubiquitin-specific E2, and 600–700 of E3 ligases (Kim et al., 2011). Thus, including about 100 deubiquitylating enzymes, approximately 800 ubiquitin enzymes regulate the dynamic ubiquitylation of a wide range of protein substrates (Kim et al., 2011). Within this complexity, E3 ligases are the most diverse class of enzymes in the ubiquitylation pathway as they play a central role in determining the selectivity of ubiquitin-mediated protein degradation and signaling. E3 ligases have been associated with a number of pathogenic mechanisms. Mutations in the E3 ligases MDM2, BRCA1, TRIMs, and Parkin have been linked to multiple cancers and neurodegenerative diseases (Fakharzadeh et al., 1991, Hatakeyama, 2011, Welcsh and King, 2001), and MDM2-p53 interaction inhibitors have already been developed as a potential anti-cancer treatment (Shangary and Wang, 2009). This highlights the potential of E2 enzymes and E3 ligases as drug targets. Although all E3 ligases are involved in the final step of covalent ubiquitylation of target proteins, they differ in both structure and mechanism and can be classified in three main families depending on the type of E3 ligases promoting ubiquitin-protein ligation and on the presence of characteristic domains. The RING ligases bring the ubiquitin-E2 complex into the molecular vicinity of the substrate and facilitate ubiquitin transfer directly from the E2 enzyme to the substrate protein. In contrast, homologous to the E6-AP C terminus family (HECTs) covalently bind the ubiquitin via a cysteine residue in their catalytic HECT domain before shuttling it onto the target molecule. RING between RINGs (RBRs) E3 ligases were shown to use both RING- and HECT-like mechanisms where ubiquitin is initially recruited on a RING domain (RING1) then transferred to the substrate through a conserved cysteine residue in a second RING domain. The vast majority of human E3 enzymes belong to the RING family, while only 28 belong to the HECT and 14 to the RBR family of E3 ligases (Chaugule and Walden, 2016). 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), antibodies (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.