Targeting Cullin-RING E3 Ubiquitin Ligase 4 by Small Molecule Modulators

Abstract

Cullin-RING E3 ubiquitin ligase 4 (CRL4) plays an essential role in cell cycle progression. Recent efforts using high throughput screening and follow up hit-to-lead studies have led to identification of small molecules 33-11 and KH-4-43 that inhibit E3 CRL4's core ligase complex and exhibit anticancer potential. This review provides: 1) an updated perspective of E3 CRL4, including structural organization, major substrate targets and role in cancer; 2) a discussion of the challenges and strategies for finding the CRL inhibitor; and 3) a summary of the properties of the identified CRL4 inhibitors as well as a perspective on their potential utility to probe CRL4 biology and act as therapeutic agents.

Keywords: Cdt1; Cell cycle; E3 CRL4; Small molecule inhibitors; Tumor inhibition.

Figure 1:

The structural organization of E3 CRL4^SV5-V (PDB: 2HYE).

Figure 2:

The E3 CRL4^DCAF2-Cdt1 pathway. A list of bullet points highlights Cdt1’s function as well as physiological and pathological defects caused by aberrant accumulation of Cdt1.

Figure 3:

FRET strategies to target E3 CRLs. A) FRET K48 di-Ub assay scheme. It begins with formation of a thiolester complex by E1 and Ub-Q31C/K48R-iFluo 555. This is followed by transthiolation to produce E2 Cdc34~Ub-Q31C/K48R-iFluo 555. In the presence of E3 sub-complex ROC1-CUL1 CTD, the K48 residue of Ub-E64C/ΔGG-iFluo 647 attacks Cdc34~Ub-Q31C/K48R-iFluo 555, yielding a di-Ub covalent product. As a result, Ub-linked fluorophores iFluo 555 and iFluo 647 are brought into proximity that generates FRET upon donor fluorophore excitation. B) FRET di-ubiquitination scheme. The final outcome is a result of mixing two reaction assemblies. Assembly 1 (right hand) is a complex formed by an E3 and a substrate-Ub fusion molecule. Ub is labeled with red coded acceptor fluorophore. Assembly 2 (left hand) involves reactions of the donor Ub with E1 and E2 to form E2~donor Ub thiol ester complex. The donor Ub contains a lysine substitution that eliminates Ub chain assembly and is labeled with blue coded donor fluorophore. The combination of assemblies 1 and 2 produces a uniform substrate-di-Ub product, resulting in close proximity between the red and blue fluorophores. When the donor fluorophore is excited, its emission causes the excitation of the acceptor fluorophore, which emits a distinct fluorescent signal.

Figure 4:

33-11/KH-4-43: inhibitors of E3 CRL4 with anticancer potential. Newly identified CRL4 inhibitors 33-11 and KH-4-43 target E3’s core ligase module. Highlighted are the properties of the inhibitors.