No abstract

The NEDD8-activating enzyme (NAE) initiates a protein homeostatic pathway essential for cancer cell growth and survival. MLN4924 is a selective inhibitor of NAE currently in clinical trials for the treatment of cancer. Here, we show that MLN4924 is a mechanism-based inhibitor of NAE and creates a covalent NEDD8-MLN4924 adduct catalyzed by the enzyme. The NEDD8-MLN4924 adduct resembles NEDD8 adenylate, the first intermediate in the NAE reaction cycle, but cannot be further utilized in subsequent intraenzyme reactions. The stability of the NEDD8-MLN4924 adduct within the NAE active site blocks enzyme activity, thereby accounting for the potent inhibition of the NEDD8 pathway by MLN4924. Importantly, we have determined that compounds resembling MLN4924 demonstrate the ability to form analogous adducts with other ubiquitin-like proteins (UBLs) catalyzed by their cognate-activating enzymes. These findings reveal insights into the mechanism of E1s and suggest a general strategy for selective inhibition of UBL conjugation pathways.

Ubiquitin-activating enzyme (UAE or E1) activates ubiquitin via an adenylate intermediate and catalyzes its transfer to a ubiquitin-conjugating enzyme (E2).Mechanistic studies on Compound I and its purified ubiquitin adduct demonstrate that the proposed substrate-assisted inhibition via covalent adduct formation is entirely consistent with the three-step ubiquitin activation process and that the adduct is formed via nucleophilic attack of UAE thioester by the sulfamate group of Compound I after completion of step 2. Kinetic and affinity analysis of Compound I, MLN4924, and their purified ubiquitin adducts suggest that both the rate of adduct formation and the affinity between the adduct and E1 contribute to the overall potency. Because all E1s are thought to use a similar mechanism to activate their cognate ubiquitin-like proteins, the substrate-assisted inhibition by adenosine sulfamate analogues represents a promising strategy to develop potent and selective E1 inhibitors that can modulate diverse biological pathways.Post-translational modification by ubiquitin plays an essential role in a wide range of cellular processes. One of the most intensively studied pathways is the ubiquitin-proteasome system that attaches a polyubiquitin chain to a lysine residue on a target protein and directs it to proteasome-mediated proteolysis. The ubiquitin-proteasome system is a key system responsible for maintaining cellular protein homeostasis, an emerging research area that could potentially transform our understanding of human diseases (1-3). Bortezomib (VELCADE), a proteasome inhibitor, is currently approved in the treatment of patients with multiple myeloma and relapsed mantle cell lymphoma (4, 5). The clinical success of bortezomib suggests that targeting other components in the ubiquitin-proteasome system pathway might represent an opportunity to develop novel anti-cancer therapeutics (6 -9).Conjugating ubiquitin to a protein substrate is mediated by an enzymatic cascade initiated by ubiquitin-activating enzyme (UAE) 2 (or Ube1 in humans, also known as E1) (10). Previous mechanistic studies show that, in vitro, UAE activates ubiquitin by a three-step process using ATP as a cofactor (Fig. 1A) (11,12). In step 1, UAE binds ATP and ubiquitin, catalyzes formation of ubiquitin adenylate intermediate, and releases inorganic pyrophosphate (PP i ). The ubiquitin adenylate activates the C-terminal carboxyl group of ubiquitin for nucleophilic substitution. In step 2, the catalytic cysteine residue in UAE attacks the adenylate to form a thioester intermediate (UAE-Sϳubiq-uitin, ϳ denotes the thioester bond between the C-terminal carboxyl group of ubiquitin and Cys 632 of human UAE) with AMP as the by-product. In step 3, UAE-Sϳubiquitin binds another equivalent of ATP and ubiquitin and in a second round of adenylation, forms a UAE-Sϳubiquitin⅐ubiquitin-adenylate ternary complex. Although UAE-Sϳubiquitin is capable of transferring ubiquitin to the conjugating enzyme (E2) via a transthiolation reaction, E1-E2 transthiolation is grea...

MLN4924 is an investigational small-molecule inhibitor of NEDD8-activating enzyme (NAE) in clinical trials for the treatment of cancer. MLN4924 is a mechanism-based inhibitor, with enzyme inhibition occurring through the formation of a tight-binding NEDD8-MLN4924 adduct. In cell and xenograft models of cancer, we identified treatment-emergent heterozygous mutations in the adenosine triphosphate binding pocket and NEDD8-binding cleft of NAEβ as the primary mechanism of resistance to MLN4924. Biochemical analyses of NAEβ mutants revealed slower rates of adduct formation and reduced adduct affinity for the mutant enzymes. A compound with tighter binding properties was able to potently inhibit mutant enzymes in cells. These data provide rationales for patient selection and the development of next-generation NAE inhibitors designed to overcome treatment-emergent NAEβ mutations.

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.