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  • The authors identified inhibitors of

    2019-10-12

    The authors identified inhibitors of SUMOylation using two assays to monitor the conjugation of SUMO1 to RanGAP1 by E1 and E2 enzymes: a fluorescence resonance energy transfer (FRET) primary screen that evaluated over 250,000 compounds and a chemiluminescence secondary screen. A counter screen was used to identify hits that selectively inhibited SUMOylation over ubiquitination. The sequence of triaging assays resulted in identification of one compound with high specificity to SUMOylation. A medicinal chemistry optimization campaign yielded an optimized inhibitor of SUMOylation (COH000) with an in vitro IC50 of 0.2 M. The inhibition of SUMO E1 by COH000 was long lasting and resistant to dialysis, and the authors used mass spectrometry and kinetic analysis to confirm that COH000 covalently labeled the enzyme. Unexpectedly, COH000 labeled a buried cysteine (Cys30) in SUMO E1 and not the catalytic cysteine (Cys173). Consistent with a non-competitive allosteric binding modality, the inhibition of SUMO E1 activity was not impacted by the concentration of ATP or SUMO. Cys30 is deeply buried in previously published crystal structures for SUMO E1 (Lois and Lima, 2005, Olsen et al., 2010). However, the SUMO E1 enzyme bound by COH000 has a different thermal denaturation profile and a higher melting temperature compared to the enzyme alone, suggesting the conformation of SUMO E1 is substantially different when bound by the inhibitor (Li et al., 2019). This was confirmed in a parallel publication of the crystal structure for SUMO E1 bound by COH000 (Lv et al., 2018). The structure revealed a dramatic rotation of the “second” catalytic cysteine half (SCCH)-domain, which contains the catalytic cysteine Cys173. This change reveals a cryptic pocket containing Cys30 that formed a covalent bond via a Michael addition reaction with COH000 and locked the enzyme in an inactive conformation (Lv et al., 2018). COH000 inhibited global SUMOylation in AZD3264 kinase and blocked the E1 catalyzed attachment of SUMO to the corresponding conjugating enzyme Ubc9, but not the attachment of ubiquitin or NEDD8 to their respective E2s, confirming inhibition and specificity in cellulo. Consistent with previous studies inactivating SUMO E1 using genetic knockdown or active site inhibitor, treatment of lymphoma and colorectal cancer cells with COH000 increased the miRNA miR-34b, decreased c-Myc protein expression and induced apoptosis. (He et al., 2017) Notably, analogs of COH000 lacking SUMO E1 inhibitory activity lacked the same phenotypes, supporting the conclusion that the observed effects are on-target (Li et al., 2019). Furthermore, COH000 exhibited anti-tumor activity in an in vivo colorectal xenograft model and ex vivo against primary colorectal PDX samples. Having two well-characterized inhibitors of SUMO E1 will allow researchers to probe inhibition of SUMOylation from different angles. The inhibitor ML-792 acts by forming a covalent adduct with SUMO in a SUMO E1-dependent manner, which then acts as a competitive inhibitor for SUMO E1 (He et al., 2017). This mechanism of action also depletes the amount of free SUMO available for conjugation. In contrast, COH000 allosterically inhibits SUMO E1, but does not affect SUMO itself, which may allow for other E1s to act as alternative activators as the cytoplasmic pool of SUMO increases. Indeed, even with cell treatment of 20 μM COH000, there is still residual SUMOylation (Li et al., 2019). The different mechanisms of the two inhibitors will allow for a detailed examination of SUMOylation dynamics of substrates and differential effects of targeting the pathway in their distinct manner. The crystal structure of the compound will aid further improvements in potency for COH000 as well as novel strategies of inhibition. Intriguingly, when bound to COH000, the catalytic cysteine of SUMO E1 is only 12 Å away from the COH000 binding pocket (Lv et al., 2018). This raises the possibility of elaborating the compound to target the catalytic cysteine with the SCCH domain in an inactive conformation. The inhibitor also opens new avenues of research into a novel class of allosteric E1 inhibitors. All other E1s in the human proteome (aside from ATG7) have a conserved cysteine with SUMO E1 Cys30 and have around 70% conservation in the allosteric pocket where COH000 binds. This lends itself to development of other E1 inhibitors based on the scaffold of COH000. It could also be instructive to probe whether other E1s without well-characterized inhibitors can have their pockets mutated to be inhibited by COH000 or close analogs, which would allow for bump-and-hole strategies in cells to explore these pathways through chemical inhibition (Wertz and Wang, 2019).