Abstract
Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins. Neddylation of individual cullin proteins is essential for the activation of each CRL. We report herein the discovery of DI-1548 and DI-1859 as two potent, selective and covalent DCN1 inhibitors. These inhibitors selectively inhibit neddylation of cullin 3 in cells at low nanomolar concentrations and are 2–3 orders of magnitude more potent than our previously reported reversible DCN1 inhibitor. Mass spectrometric analysis and co-crystal structures reveal that these compounds employ a unique mechanism of covalent bond formation with DCN1. DI-1859 induces a robust increase of NRF2 protein, a CRL3 substrate, in mouse liver and effectively protects mice from acetaminophen-induced liver damage. Taken together, this study demonstrates the therapeutic potential of selective inhibition of cullin neddylation.
Highlights
Cullin-RING E3 ligases (CRLs) regulate the turnover of approximately 20% of mammalian cellular proteins
We explored the possibility of designing a class of covalent DCN1 inhibitors based upon DI-591
Analysis of the cocrystal structure of DI-591 (1) in a complex with human DCN1 protein showed that the sulfur atom of Cys[115] in DCN1 is located 4.1 Å from the α carbon of the 3morpholinopropanamide unit in DI-591 (Fig. 1a), providing an opportunity for the design of covalent DCN1 inhibitors
Summary
Structure-based design of highly potent covalent DCN1 inhibitors. In addition to noncovalent DCN1 inhibitors, covalent DCN1 inhibitors have been reported[25]. Consistent with its slower reaction kinetics, compound 10 is ten times less potent than DI-1548 in cell in inhibition of cullin 3 neddylation (Supplementary Fig. 12) Among those three cysteine residues (Cys[90], Cys[115], and Cys131) in DCN1 protein, Cys[115] was predicted to form a covalent bond with these covalent inhibitors. In contrast to DI-1548 and DI-1859, a covalent complex was formed between compound 10 and DCN1 protein without loss of the imidazole group in 10 based on mass spectrometry data (Fig. 2b and Supplementary Fig. 11). The cocrystal structure for compound 4 shows that there is no covalent bond formed between 4 and DCN1 protein (Fig. 3c), which is consistent with its very slow reaction kinetics in the mass spectral analysis (Fig. 2a). We evaluated the pharmacodynamics (PD) of DI-1548 and DI-1859, our two most potent DCN1 inhibitors, in mice
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