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    • An Ubl modification requires several steps that are catalyze

    2019-08-29

    An Ubl modification requires several steps that are catalyzed by three enzymes, referred to as E1 (activating enzyme), E2 (conjugation enzyme), and E3 (ligase). The SUMO E1 is a heterodimer of SAE1 and Uba2 (also known as SAE2). In brief, an Ubl is first activated by E1 through ATP hydrolysis and forms a thioester conjugate with E1. The Ubl is then transferred to E2, forming a thioester conjugate with E2. Finally, the Ubl is transferred to target proteins, a step usually catalyzed by an E3. Usually, Ubl modifications add new docking sites to target proteins. For example, SUMO modifications enables new protein-protein interactions through the SUMO-interacting motif in receptor proteins (Song et al., 2004, Song et al., 2005). At least three members of the SUMO family (SUMO1, 2, and 3) are ubiquitin-like proteins that can conjugate to other cellular proteins by a biochemical mechanism similar to ubiquitylation (Hay, 2005, Sarge and Park-Sarge, 2009, Yeh, 2009). Currently, the only known mechanism to inhibit the E1 Ivacaftor benzenesulfonate clinical targets their ATP-binding sites (Brownell et al., 2010, Soucy et al., 2009). Historically, covalent drugs have had great success (e.g., aspirin and penicillin), and covalent drugs have become a focus in anticancer and antiviral drug discovery (Kalgutkar and Dalvie, 2012, Singh et al., 2011). These compounds contain low reactivity warheads that allow covalent adducts to form only when a non-covalent complex forms first. Their duration of action depends on the turnover rate of their protein targets, independent of the drug\'s stability in the blood.
    Results
    Discussion In this study, we have discovered an allosteric inhibitor chemotype that inhibits SUMO E1 through a covalent mechanism. Our further studies demonstrated covalent adduct formation of COH000 to Cys30 of SUMO E1, a cysteine that is not involved in binding ATP or SUMO. This is consistent with the results that COH000 does not compete with ATP or SUMO1 binding (Figure 2A). Cys30 is fully buried in previously determined crystal structures of SUMO E1s (Lois and Lima, 2005, Olsen et al., 2010), and the analogous sites are buried in previously published Ubl E1 crystal structures (Lee and Schindelin, 2008, Noda et al., 2011, Walden et al., 2003). Crystal structure of COH000 in complex with SUMO E1, indeed, revealed a more extensive conformational flexibility than seen previously for this family of enzymes (Lv et al., 2018). Therefore, binding of COH000 to the SUMO E1 requires a conformational state of the enzyme not previously observed. Sequence comparison of E1 enzymes revealed that the covalent modification site and the amino acid sequence surrounding it is conserved among the human Uba1 (ubiquitin E1), Uba2 (SUMO E1), Uba3 (Nedd8 E1), Uba4 (Urm1 E1), Uba7 (ISG15 E1), and Atg7 (E1 for Atg8 and Atg12) (Figure S1C). Thus, the covalent allosteric inhibition mechanism exemplified by COH000 could be applicable to other E1 enzymes. Findings described here also raise a question of whether E1 enzymes can be allosterically regulated by small-molecule metabolites that bind to the same site as COH000. Covalent inhibitors are well suited for targeting the E1 enzymes of Ubl modifications. Because the E1 enzymes in Ubl modifications, such as the SUMO E1 and Atg7, have a slow turnover rate (Boggio et al., 2004), prolonged inhibition can be achieved without requiring compounds to be stable in circulation, reducing the pharmacokinetic hurdle associated with conventional drug discovery processes. In addition, cancers and viruses are less likely to develop resistance to covalent than to non-covalent inhibitors as shown by existing covalent drugs (Kalgutkar and Dalvie, 2012, Singh et al., 2011). Our finding of an allosteric, covalent inhibitory mechanism of the E1 enzymes is a major advancement in targeting the Ubl modifications. As we have previously discussed, the lack of drugs targeting Ubl modifications highlights the need to identify new approaches to inhibit this class of enzymes. Members of E1 enzymes are potential targets for inhibiting c-Myc and KRas oncogenes involved in the development of the majority of human cancers and contribute to cancer cell stemness and resistance (Bogachek et al., 2016, Du et al., 2016, He et al., 2017, Kessler et al., 2012, Luo et al., 2009, Yu et al., 2015). Therefore, identification of a mechanism to inhibit the E1 enzymes not only provides a complementary approach to overcome resistance developed through mutations for compounds that target the ATP-binding sites (Xu et al., 2014) but also provides new directions for developing potent and selective inhibitors to potentially treat cancers and other life-threatening diseases.