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Targeting Cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation.

Bulatov E, Ciulli A - Biochem. J. (2015)

Bottom Line: In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity.A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included.This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.

View Article: PubMed Central - PubMed

Affiliation: *College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.

ABSTRACT
In the last decade, the ubiquitin-proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin-RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.

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Crystal structure of the NAE1–UBA3–NEDD8~MLN4924 complex (PDB code 3GZN)The NAE1 regulatory subunit and the UBA3 catalytic subunit form the heterodimeric NAE. Left: NAE complexed with NEDD8~MLN4924 covalent adduct, where MLN4924 inhibits the active site of NAE. Right: the residues forming interface between NAE1 (grey carbons), UBA3 (green carbons), NEDD8 (cyan carbons) and MLN4924 (yellow carbons). Oxygen atoms are in red, nitrogen atoms are in blue, and sulfur atoms are in orange.
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Figure 12: Crystal structure of the NAE1–UBA3–NEDD8~MLN4924 complex (PDB code 3GZN)The NAE1 regulatory subunit and the UBA3 catalytic subunit form the heterodimeric NAE. Left: NAE complexed with NEDD8~MLN4924 covalent adduct, where MLN4924 inhibits the active site of NAE. Right: the residues forming interface between NAE1 (grey carbons), UBA3 (green carbons), NEDD8 (cyan carbons) and MLN4924 (yellow carbons). Oxygen atoms are in red, nitrogen atoms are in blue, and sulfur atoms are in orange.

Mentions: The ubiquitin-like protein NEDD8 enhances CRL activity by inducing dynamics of the complex and activating conformational shift of Rbx–E2~Ub to bring ubiquitin in close proximity to the substrate bound at the opposite terminus of the E3 ligase [100,194]. CRLs are the main targets for NEDDylation [195], a process of covalent NEDD8 modification of a conserved lysine residue at the Cullin CTD. The conjugation occurs through a pathway involving its own E1 and E2 enzymes. A group of scientists at Millennium Pharmaceuticals developed a highly selective inhibitor of NAE (NEDD8-activating enzyme), which functions as the heterodimeric complex NAE1–UBA3, adenosine sulfamate MLN4924, which is structurally similar to the adenylate intermediate of the NAE catalytic reaction [19]. Early crystal structures of NAE with bound MLN4924 were not particularly informative in terms of explaining the high potency and selectivity of the drug. However, a later crystal structure of the NAE–NEDD8~MLN4924 ternary complex obtained in the presence of NEDD8 and ATP revealed the formation of covalent NEDD8~MLN4924 adduct that blocks the active site of NAE [196] (Figure 12). The selectivity for NAE over other E1s was shown to be achieved via substrate-assisted inhibition [197]. The MLN4924-induced inhibition of NEDD8 transfer leads to an increase in non-NEDDylated CRLs with suppressed ubiquitination activity against their substrates. The compound is now being tested in Phase I clinical trials against a number of cancers [198].


Targeting Cullin-RING E3 ubiquitin ligases for drug discovery: structure, assembly and small-molecule modulation.

Bulatov E, Ciulli A - Biochem. J. (2015)

Crystal structure of the NAE1–UBA3–NEDD8~MLN4924 complex (PDB code 3GZN)The NAE1 regulatory subunit and the UBA3 catalytic subunit form the heterodimeric NAE. Left: NAE complexed with NEDD8~MLN4924 covalent adduct, where MLN4924 inhibits the active site of NAE. Right: the residues forming interface between NAE1 (grey carbons), UBA3 (green carbons), NEDD8 (cyan carbons) and MLN4924 (yellow carbons). Oxygen atoms are in red, nitrogen atoms are in blue, and sulfur atoms are in orange.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403949&req=5

Figure 12: Crystal structure of the NAE1–UBA3–NEDD8~MLN4924 complex (PDB code 3GZN)The NAE1 regulatory subunit and the UBA3 catalytic subunit form the heterodimeric NAE. Left: NAE complexed with NEDD8~MLN4924 covalent adduct, where MLN4924 inhibits the active site of NAE. Right: the residues forming interface between NAE1 (grey carbons), UBA3 (green carbons), NEDD8 (cyan carbons) and MLN4924 (yellow carbons). Oxygen atoms are in red, nitrogen atoms are in blue, and sulfur atoms are in orange.
Mentions: The ubiquitin-like protein NEDD8 enhances CRL activity by inducing dynamics of the complex and activating conformational shift of Rbx–E2~Ub to bring ubiquitin in close proximity to the substrate bound at the opposite terminus of the E3 ligase [100,194]. CRLs are the main targets for NEDDylation [195], a process of covalent NEDD8 modification of a conserved lysine residue at the Cullin CTD. The conjugation occurs through a pathway involving its own E1 and E2 enzymes. A group of scientists at Millennium Pharmaceuticals developed a highly selective inhibitor of NAE (NEDD8-activating enzyme), which functions as the heterodimeric complex NAE1–UBA3, adenosine sulfamate MLN4924, which is structurally similar to the adenylate intermediate of the NAE catalytic reaction [19]. Early crystal structures of NAE with bound MLN4924 were not particularly informative in terms of explaining the high potency and selectivity of the drug. However, a later crystal structure of the NAE–NEDD8~MLN4924 ternary complex obtained in the presence of NEDD8 and ATP revealed the formation of covalent NEDD8~MLN4924 adduct that blocks the active site of NAE [196] (Figure 12). The selectivity for NAE over other E1s was shown to be achieved via substrate-assisted inhibition [197]. The MLN4924-induced inhibition of NEDD8 transfer leads to an increase in non-NEDDylated CRLs with suppressed ubiquitination activity against their substrates. The compound is now being tested in Phase I clinical trials against a number of cancers [198].

Bottom Line: In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity.A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included.This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.

View Article: PubMed Central - PubMed

Affiliation: *College of Life Sciences, University of Dundee, Dundee DD1 5EH, U.K.

ABSTRACT
In the last decade, the ubiquitin-proteasome system has emerged as a valid target for the development of novel therapeutics. E3 ubiquitin ligases are particularly attractive targets because they confer substrate specificity on the ubiquitin system. CRLs [Cullin-RING (really interesting new gene) E3 ubiquitin ligases] draw particular attention, being the largest family of E3s. The CRLs assemble into functional multisubunit complexes using a repertoire of substrate receptors, adaptors, Cullin scaffolds and RING-box proteins. Drug discovery targeting CRLs is growing in importance due to mounting evidence pointing to significant roles of these enzymes in diverse biological processes and human diseases, including cancer, where CRLs and their substrates often function as tumour suppressors or oncogenes. In the present review, we provide an account of the assembly and structure of CRL complexes, and outline the current state of the field in terms of available knowledge of small-molecule inhibitors and modulators of CRL activity. A comprehensive overview of the reported crystal structures of CRL subunits, components and full-size complexes, alone or with bound small molecules and substrate peptides, is included. This information is providing increasing opportunities to aid the rational structure-based design of chemical probes and potential small-molecule therapeutics targeting CRLs.

Show MeSH
Related in: MedlinePlus