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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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Structures of CC0651 bound to an allosteric pocket on the E2-conjugating enzyme Cdc34Top: crystal structure of the Cdc34–CC0651 binary complex (PDB code 3RZ3). The protein and its ligand are shown as molecular surface representations. Inset: Cdc34 residues (green carbons) forming the binding pocket and the CC0651 ligand (yellow carbons). Bottom: crystal structure of the ternary complex Cdc34A~Ub–CC0651 (PDB code 4MDK). CC0651 (yellow carbons) is bound embedded within the Cdc34A~Ub covalent conjugate protein. CC0651 suppresses the hydrolysis of the thioester bond between the catalytic cysteine residue of Cdc34 (green carbons, cysteine residue not shown) and ubiquitin (brown carbons, Lys48 side chain shown). Oxygen atoms are in red, nitrogen atoms are in blue, and chlorine atoms are in light green.
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Figure 11: Structures of CC0651 bound to an allosteric pocket on the E2-conjugating enzyme Cdc34Top: crystal structure of the Cdc34–CC0651 binary complex (PDB code 3RZ3). The protein and its ligand are shown as molecular surface representations. Inset: Cdc34 residues (green carbons) forming the binding pocket and the CC0651 ligand (yellow carbons). Bottom: crystal structure of the ternary complex Cdc34A~Ub–CC0651 (PDB code 4MDK). CC0651 (yellow carbons) is bound embedded within the Cdc34A~Ub covalent conjugate protein. CC0651 suppresses the hydrolysis of the thioester bond between the catalytic cysteine residue of Cdc34 (green carbons, cysteine residue not shown) and ubiquitin (brown carbons, Lys48 side chain shown). Oxygen atoms are in red, nitrogen atoms are in blue, and chlorine atoms are in light green.

Mentions: An interesting example shows how a high-throughput screen for CRL1Skp2 inhibitors aided the discovery of an E2 enzyme modulator instead [157]. Here, a screening campaign based on a previously developed ubiquitination assay [142] identified the small molecule CC0651 that inhibited ubiquitination of substrate p27Kip1. The compound was not selective towards p27Kip1 and demonstrated inhibitory properties against other CRL1-based substrate/receptor pairs; however, biochemical studies established that CC0651 targets the E2-conjugating enzyme Cdc34 operating together with CRL1 [13]. Importantly, the compound showed specificity for Cdc34 compared with other E2 enzymes despite their significant structural similarity. It is well known that the presence of a hydrolysable thioester bond between E2 and ubiquitin is crucial for the catalytic activity of the whole E3 ligase. The crystal structures of Cdc34 and the Cdc34–CC0651 complex revealed that CC0651 targets a previously unknown allosteric pocket at a distance from the catalytic cysteine residue. Ligand binding is observed to induce a significant conformational change in the E2 protein to open up such pocket. In so doing, CC0651 appears to stabilize the E2–ubiquitin interaction and lock the complex in an inactive conformation, therefore inhibiting the discharge of ubiquitin from the E2~Ub conjugate on to the substrate lysine residues (Figure 11). More recently, the same group solved the crystal structure of a Cdc34~Ub–CC0651 complex, and determined that CC0651 bridges the Cdc34~Ub link and suppresses hydrolysis of the weak thioester bond between the two proteins [158]. CC0651 was also found to negatively affect proliferation of cancer cells.


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

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

Structures of CC0651 bound to an allosteric pocket on the E2-conjugating enzyme Cdc34Top: crystal structure of the Cdc34–CC0651 binary complex (PDB code 3RZ3). The protein and its ligand are shown as molecular surface representations. Inset: Cdc34 residues (green carbons) forming the binding pocket and the CC0651 ligand (yellow carbons). Bottom: crystal structure of the ternary complex Cdc34A~Ub–CC0651 (PDB code 4MDK). CC0651 (yellow carbons) is bound embedded within the Cdc34A~Ub covalent conjugate protein. CC0651 suppresses the hydrolysis of the thioester bond between the catalytic cysteine residue of Cdc34 (green carbons, cysteine residue not shown) and ubiquitin (brown carbons, Lys48 side chain shown). Oxygen atoms are in red, nitrogen atoms are in blue, and chlorine atoms are in light green.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Structures of CC0651 bound to an allosteric pocket on the E2-conjugating enzyme Cdc34Top: crystal structure of the Cdc34–CC0651 binary complex (PDB code 3RZ3). The protein and its ligand are shown as molecular surface representations. Inset: Cdc34 residues (green carbons) forming the binding pocket and the CC0651 ligand (yellow carbons). Bottom: crystal structure of the ternary complex Cdc34A~Ub–CC0651 (PDB code 4MDK). CC0651 (yellow carbons) is bound embedded within the Cdc34A~Ub covalent conjugate protein. CC0651 suppresses the hydrolysis of the thioester bond between the catalytic cysteine residue of Cdc34 (green carbons, cysteine residue not shown) and ubiquitin (brown carbons, Lys48 side chain shown). Oxygen atoms are in red, nitrogen atoms are in blue, and chlorine atoms are in light green.
Mentions: An interesting example shows how a high-throughput screen for CRL1Skp2 inhibitors aided the discovery of an E2 enzyme modulator instead [157]. Here, a screening campaign based on a previously developed ubiquitination assay [142] identified the small molecule CC0651 that inhibited ubiquitination of substrate p27Kip1. The compound was not selective towards p27Kip1 and demonstrated inhibitory properties against other CRL1-based substrate/receptor pairs; however, biochemical studies established that CC0651 targets the E2-conjugating enzyme Cdc34 operating together with CRL1 [13]. Importantly, the compound showed specificity for Cdc34 compared with other E2 enzymes despite their significant structural similarity. It is well known that the presence of a hydrolysable thioester bond between E2 and ubiquitin is crucial for the catalytic activity of the whole E3 ligase. The crystal structures of Cdc34 and the Cdc34–CC0651 complex revealed that CC0651 targets a previously unknown allosteric pocket at a distance from the catalytic cysteine residue. Ligand binding is observed to induce a significant conformational change in the E2 protein to open up such pocket. In so doing, CC0651 appears to stabilize the E2–ubiquitin interaction and lock the complex in an inactive conformation, therefore inhibiting the discharge of ubiquitin from the E2~Ub conjugate on to the substrate lysine residues (Figure 11). More recently, the same group solved the crystal structure of a Cdc34~Ub–CC0651 complex, and determined that CC0651 bridges the Cdc34~Ub link and suppresses hydrolysis of the weak thioester bond between the two proteins [158]. CC0651 was also found to negatively affect proliferation of cancer cells.

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