DEG_CRBN_cyclicCter_1

The drug thalidomide was used in the 1950s-1960s as a sedative for pregnant women suffering acute morning sickness. Unfortunately, it proved strongly teratogenic, causing developmental abnormalities. For many years the molecular mechanism of the drug was unknown, although it had become clear that the drug might be a useful cancer treatment. In 2010, Ito et al. reported that thalidomide bound to the protein cereblon (CRBN), also demonstrating that the protein is an E3 ligase (Ito,2010), thus paving the way to understanding the molecular and cellular processes modulated by thalidomide.

Cereblon (CRBN) is a ubiquitously expressed protein that acts as a substrate receptor of the Cullin-4 E3 ubiquitin ligase complex, CUL4-RBX1-DDB1 (CRL4). The CRBN gene was initially identified in connection with memory and learning. Mutations in this gene cause a mild type of recessive nonsyndromic mental retardation (Higgins,2004). Later, it was reported to be involved in various biological activities such as the regulation of ion transport, modulation of the AMPK signalling pathway, and cell metabolism. Thalidomide and its analogues are termed Immunomodulatory imide drugs (IMiDs) as they adjust immune responses. It was discovered that IMiDs enable certain proteins to bind CRBN and be destroyed by CUL4-CRBN. These proteins are termed neo-substrates and include IRF4, IKZF1 and IKZF3, MYC and SALL4 (Costacurta,2021).

Repurposing its interactome by interacting with IMiDs like thalidomide and its analogues or proteolysis targeting chimeras (PROTACs) helps in the control of tumour growth and angiogenesis in adults (Ito,2021). By contrast, thalidomide is teratogenic for the developing foetus (Asatsuma-Okumura,2020).
The more recently identified cellular ligands also share the same binding groove as Thalidomide and its derivatives (Heim,2022). The C-terminal cyclic amides aspartimide and aminoglutarimide residues are the first natural degron motifs that are recognized by CRBN with high specificity. This post-translational modification is formed as a result of the intramolecular cyclization of glutamine or asparagine residues and concomitant cleavage of the peptide backbone. This reaction results in a fragmented protein which may have lost functionality, especially for any multidomain proteins where the modules interact with each other. Cyclic amides can arise spontaneously and are likely to accumulate in ageing proteins, and their detection acts as a mechanism for the efficient removal of malfunctional and damaged proteins. Moreover, these cyclic imides readily interact with free amino groups and can form additional unwanted cross-links that are frequently seen in many age-related diseases (Friedrich,2019). The best characterized cyclic imides are from Beta-crystallin and Amyloid-beta precursor proteins (Heim,2022; Ichikawa,2022). The CULT domain (IPR034750) of CRBN is present in bacteria as well as eukaryotes (though absent from fungi), suggesting that the removal of cyclic C-termini is also important in prokaryotes.

The C-terminal thalidomide-binding domain (TBD), also known as the CULT domain (Cereblon domain of Unknown activity, binding cellular Ligands and Thalidomide), acts as a binding site for the cellular degrons and IMiDs. It is highly conserved across species with very few residue substitutions and contains three strictly conserved tryptophan residues (tri-tryptophan pocket) that are important for ligand binding. In human cereblon (Q96SW2), Valine 388 is an important residue as the substitution of this residue to a smaller (V388A) or larger (V388I) side chain impaired neo-substrate binding and causes the teratogenic effect of thalidomide differently. Thalidomide is teratogenic in humans, zebrafish, and chicks but not in rodents (Heim,2023). Degradation of the SALL4 neosubstrate is likely to be particularly important for the teratogenicity of IMiDs (Donovan,2018).

Immunomodulatory drugs (IMiDs) or cereblon (CRBN) E3 ligase modulatory drugs (CELMoDs) are molecular glues. They recruit neo-substrate proteins to CRBN for ubiquitination and subsequent proteasomal degradation. The glutarimide ring of the cereblon binding moiety is slotted into the cage while the remaining molecules protrude out and act as a binding site for various neo-substrates with its specificity fine-tuned by the protruding moiety (Boichenko,2018). Several structures of neo-substrates in complex with CRBN and IMiDs are available (5HXB; 5FQD; 7U8F; 6H0F; 7BQV). These all show substantial interactions of globular domains; for example, C2H2 Zinc fingers insert into the CRBN interaction groove in the case of SALL4 and Ikaros transcription factors.