Neddylation of the Cullin-RING E3 ligases (CRLs) regulates the homeostasis of approximately 20% of cellular proteins. Defective in cullin neddylation 1(DCN1), as a co-E3 ligase, interacts with UBE2M to enhance the activation of CRLs and this interaction is emerging as a therapeutic target for human diseases. Here, we present a series of pyrimidin-based small molecular inhibitors targeting DCN1-UBE2M interaction. After finding a novel inhibitor DC-1 with IC50 = 1.2µM, we performed a series of chemical optimization, which finally led to the discovery of a potent thiazole contained 5-cyano-6-phenylpyrimidin-based inhibitor DC-2 (IC50 = 15nM). Next, using protein and cellular thermal shift assays, co-immunoprecipitation, molecular docking and site specific mutation experiments, we further proved that DC-2 specifically inhibited the interaction of UBE2M and DCN1 at molecule and cellular levels, resulting in the decrease of cullin3 neddylation and accumulation of its substrate: NRF2. Our findings indicate that DC-2 may serve as a novel lead compound for specific derivatives targeting DCN1-UBE2M interaction. / 64 INTRODUCTION The ubiquitin-proteasome system (UPS) is integral to maintaining cellular protein homeostasis by regulating degradation of intracellular proteins. 1-7 The ubiquitylation pathway is executed by the coordinated efforts of the E1 (ubiquitin-activating enzyme), E2 (ubiquitinconjugating enzyme) and E3 (ubiquitin ligase enzyme) proteins. 5 Like ubiquitination, 8 neddylation is a novel type of posttranslational modification, in which the ubiquitin-like molecule NEDD8 is added to its target proteins and thus regulates their functions. 9, 10 Neddylation also consists of a tripartite enzymatic cascade, including the NEDD8-activating enzyme E1 (NAE, APPBP1 (NAE1) and UBA3 heterodimer), two NEDD8-conjugating enzymes E2s (UBE2F and UBE2M, also known as UBC12) and NEDD8-E3 ligases, 9, 10 with cullins being the best-characterized substrates. 11-22 Up to now, neddylation has been recognized to be highly activated in various cancers, 20, 27-33 which draws much attraction for oncologic drug discovery, especially on the finding of NEDD8 E1 inhibitor Pevonedistat(MLN4924). 10, 23-26 MLN4924 covalently binds to NEDD8 E1, blocking all CRLs neddylation, 7 thus causing accumulation of CRL substrates. 7, 10 Since MLN4924 possesses immense anti-cancer effects both in vitro and in vivo, 10, 27, 28-43 it has been approved for Phase II clinical trials for treatment of human acute myeloid leukemia, non-small cell lung cancer and mesothelioma. 29 However, as a result of its broad ablation of neddylation, MLN4924 has a series of toxicities. Therefore, alternative targeting specific CRLs may be more potential and safer for cancer treatment. 30-32 DCN1 (defective in cullin neddylation 1), also called DCUN1D1, DCNL1 or SCCRO, is a 4 / 64 highly conserved gene and amplified along the 3q26.3 in most squamous cell carcinomas and some other human cancers. 33-39 As a co-E3 ligase, it binds to the activation complex of Cullin-RBX1-UBE2M...
