The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

The invariant chain, which associates with the major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, serves two functions important in antigen processing. First, it prevents class II molecules from binding peptides in the early stages of intracellular transport. Second, it contains a cytoplasmic signal that targets the class II-invariant chain complex to an acidic endosomal compartment. Proteolytic cleavage and subsequent dissociation of the invariant chain then occurs, allowing peptides derived from endocytosed proteins to bind to released class II molecules before their expression at the cell surface. Certain human cell lines that are mutant in one or more MHC-linked genes are defective in class II-restricted antigen processing. Here we show that in transfectants of one of these cell lines, T2, this deficiency results in the association of a large proportion of class II molecules with a nested set of invariant-chain-derived peptides (class II-associated invariant chain peptides, or CLIP). HLA-DR3 molecules isolated from T2 transfectants can be efficiently loaded with antigenic peptides by exposure to a low pH in vitro, perhaps reflecting the in vivo conditions in which peptides associate with class II molecules. Addition of synthetic CLIP inhibits the loading process, indicating that CLIP may define the region of the invariant chain responsible for obstructing the class II binding site.

The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.

Small ubiquitin-like modifier (SUMO) family proteins regulate target-protein functions by post-translational modification. However, a potent and selective inhibitor targeting the SUMO pathway has been lacking. Here we describe ML-792, a mechanism-based SUMO-activating enzyme (SAE) inhibitor with nanomolar potency in cellular assays. ML-792 selectively blocks SAE enzyme activity and total SUMOylation, thus decreasing cancer cell proliferation. Moreover, we found that induction of the MYC oncogene increased the ML-792-mediated viability effect in cancer cells, thus indicating a potential application of SAE inhibitors in treating MYC-amplified tumors. Using ML-792, we further explored the critical roles of SUMOylation in mitotic progression and chromosome segregation. Furthermore, expression of an SAE catalytic-subunit (UBA2) S95N M97T mutant rescued SUMOylation loss and the mitotic defect induced by ML-792, thus confirming the selectivity of ML-792. As a potent and selective SAE inhibitor, ML-792 provides rapid loss of endogenously SUMOylated proteins, thereby facilitating novel insights into SUMO biology.