COH inhibited global SUMOylation in cells and blocked
COH000 inhibited global SUMOylation in Fmoc-Cys(Trt)-ol 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).
Introduction The ubiquitin-activating enzyme Uba1 (E1) constitutes the first step in the covalent cascade modification of target proteins with ubiquitin (Ub). Ubiquitin itself, discovered less than 50 years ago, tags thousands of diseased proteins for destruction , . It is small (only 76 amino acids), and is found unchanged in mammals, birds, fish and even worms. Because of its universality, Ub is a valuable proving ground for universal biophysical theories discussing protein amino acid sequences, structure and function . Indeed key features of Ub functionality (hydropathic waves) were identified using critical point thermodynamic scaling theory . The general biochemical logistics of Ub activation, conjugation and ligation are orchestrated sequentially by the Ub conjugation cascade of E1, E2 and E3 enzymes. Humans are known to harbor two E1, ∼30 E2 and ∼600 E3 enzymes in the Ub conjugation cascade . While Ub is “perfect”, Uba1 (E1) has evolved only modestly from slime mold to humans. The details of this evolution express several leading features of enzyme functionality. Uba1 (E1) is a large protein (>1000 amino acids), but it is readily treated by critical point thermodynamic scaling theory, with its firm foundations in statistical mechanics and its bioinformatically determined universal parameters . It turns out that hydropathic waves are also useful for Uba1 (E1), which is >14 times larger than Ub.