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Protein neddylation is one type of posttranslational modifications that regulates the activity of the substrate proteins. Neddylation modification is catalyzed by NEDD8-activating enzyme (NAE, E1), NEDD8-conjugating enzyme (E2), and NEDD8 ligase (E3) to attach NEDD8, an ubiquitin-like molecule, to a lysine residue of a substrate protein. The best known neddylation substrates are cullin family members, which are scaffold components of cullin-RING ligases (CRLs), and cullin neddylation is required for activation of CRLs. In mammalian cells, there are one E1, two E2s (UBC12/UBE2M and UBE2F), and over a dozen E3s. MLN4924, the first-in-class small-molecule inhibitor of NAE, blocks the entire neddylation modification to inactivate activity of all CRLs. MLN4924 is currently in the Phase I/II clinical trials for anticancer application.In the last few years, targeting protein-protein interactions of the neddylation complexes has been pursued as a potential strategy to selectively inhibit the activity of individual CRL. Analysis of the co-crystal structures of DCN1, a co-E3 for neddylation, and its binding partners UBC12 (a neddylation E2) suggested that it may be amenable for the design of potent, small-molecule inhibitors. In this chapter, we will review the discovery of small-molecule inhibitors that block the interactions of DCN1 with UBC12 (hereafter called DCN1 inhibitors) from a number of laboratories, including ours, leading to selective inactivation of CRL-1 and/or CRL-3. We will also discuss potential therapeutic applications of these small-molecule inhibitors.

MLN4924, also known as pevonedistat, is a highly selective small-molecule inhibitor of NEDD8 (neuronal precursor cell-expressed developmentally downregulated protein 8)-activating enzyme (NAE) to block the entire neddylation modification cascade, leading to inactivation of cullin-RING ligases (CRLs), since activation of CRLs requires cullin neddylation. MLN4924 showed impressive anticancer activity in many preclinical studies and is currently in several Phase I/II clinical trials for anticancer therapy as a single agent or in combination with chemotherapeutic drugs.In addition to well-characterized anti-neddylation activity, recent studies showed that MLN4924 has several neddylation-independent activities. First, MLN4924 triggers EGFR dimerization to activate EGFR and its downstream RAS/MAPK and PI3K/AKT1 signals, leading to enhanced tumor sphere formation, accelerated EGF-mediated wound healing, and inhibited ciliogenesis. Second, MLN4924 induces PKM2 tetramerization to promote glycolysis, thus affecting energy metabolism. Third, MLN4924 inhibits the interaction between ACT1 (NF-κB activator 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) and attenuates IL-17A-mediated activation of NF-κB to reduce pulmonary inflammation. Fourth, MLN4924 inhibits IRF3 binding to the IFN-β promoter to inhibit IFN-β production. And finally, MLN4924 activates the JNK signaling pathway to reduce c-FLIP levels, thus enhancing TRAIL-induced apoptosis. This chapter will summarize these neddylation-independent activities of MLN4924 and discuss the underlying mechanisms and potential therapeutic applications.

Neddylation is a posttranslational modification that conjugates a ubiquitin-like protein NEDD8 to substrate proteins. The best-characterized substrates of neddylation are the cullin subunits of cullin-RING E3 ubiquitin ligase complexes (CRLs). CRLs as the largest family of E3 ubiquitin ligases control many important biological processes, including tumorigenesis, through promoting ubiquitylation and subsequent degradation of a variety of key regulatory proteins. The process of protein neddylation is overactivated in multiple types of human cancers, providing a sound rationale as an attractive anticancer therapeutic strategy, evidenced by the development of the NEDD8-activating enzyme (NAE) inhibitor MLN4924 (also known as pevonedistat). Recently, increasing evidence strongly indicates that neddylation inhibition by MLN4924 exerts anticancer effects mainly by triggering cell apoptosis, senescence, and autophagy and causing angiogenesis suppression, inflammatory responses, and chemo-/radiosensitization in a context-dependent manner. Here, we briefly summarize the latest progresses in this field, focusing on the preclinical studies to validate neddylation modification as a promising anticancer target.

Cullin-RING ubiquitin ligases (CRLs) represent the largest superfamily of multi-subunit E3s conserved in all eukaryotes. Soon after the discovery of these important ubiquitin ligase machineries, structural studies have made tremendous contributions to our understanding of their functions. Identification of the key components of CRLs by early studies raised immediate questions as to how these multi-subunit complexes assemble to promote the polyubiquitination of substrates. Specifically, how do the CRL subunits interact with each other to form a versatile E3 platform? How do they recognize specific substrates? How are the CRL-substrate interactions regulated in response to upstream signals? How are the CRL E3s themselves activated and deactivated, and how are substrate receptor subunits of CRLs exchanged in the cell? Even though we might not yet have complete answers to these questions, extensive structural analyses of CRL complexes in the past two decades have begun to unveil the themes and variations of CRL biology. In this chapter we will discuss both classic and emerging structures that help elucidate the overall architecture of CRLs, their substrate recognition modes, and regulatory mechanism of CRLs by NEDD8 modification.