Structural studies reveal thalidomide's mechanism of action and clinical effects: crystal clear or clearly complexed?

Abstract

Another piece of the thalidomide puzzle is unraveled through structural studies of thalidomide and its derivatives bound to its protein target cereblon. Two recent studies published in Nature (Fischer et al.) and Nature Structural & Molecular Biology (Chamberlain et al.) have shed light on the drug's mechanisms of action and its complex biological effects.

Keywords:

• angiogenesis
• cereblon
• IMiDs
• immunomodulation
• lenalidomide
• pomalido- mide
• teratogenesis
• Thalidomide
• ubiquitin ligase

Thalidomide was initially introduced to the market in the 1950s as an anti-emetic for pregnant women suffering from morning sickness; however, in the early 1960s it was subsequently banned for causing severe congenital limb deformities. Despite its teratogenic properties, interest in thalidomide and its analogs, lenalidomide and pomalidomide (collectively known as immunomodulatory drugs or IMiDs), resurfaced when thalidomide was shown to effectively treat erythema nodosum leprosum and was discovered to have anti-inflammatory and anti-angiogenic properties. Thalidomide and its IMiD derivatives are currently approved for the treatment of multiple myeloma (MM) and 5q-deletion-associated myelodysplastic syndrome; however, the true mechanism of action remains controversial. Several recent publications highlight the anti-proliferative and immunomodulatory role of IMiDs in treating these hematological cancers in an effort to elucidate the mechanisms by which these compounds operate.

In 2010, Ito et al. identified cereblon (CRBN) as a thalidomide binding protein and determined that CRBN, a ubiquitously expressed protein, forms part of the cullin-4-containing E3 ubiquitin ligase complex with RING box-domain protein (RBX1) and adaptor protein damaged DNA binding protein (DDB1) (CUL4–RBX1–DDB1–CRBN known as CRL4^CRBN).¹ The E3 ubiquitin ligase complex is in part responsible for limb outgrowth and expression of Fgf8, a fibroblast growth factor, in zebrafish and chicks. Thalidomide initiates its teratogenic effects by binding to CRBN and inhibiting the associated ubiquitin ligase activity. Building off the research of Ito et al., Kronke et al. ² and Lu et al. ³ independently demonstrated that the binding of IMiDs to CRBN alters the selectivity and potentiates the ubiquitination and proteosomal-dependent degradation of 2 transcription factors: IKZF1 and IKZF3. These 2 specific B cell transcription factors are necessary and sufficient for lenalidomide's activity in myeloma cells. The findings of Kronke et al. and Lu et al. reveal the first hint that antitumor and teratogenic effects of thalidomide and other IMiDs are dissociable.

Two recent studies published in Nature (Fischer et al.)⁴ and Nature Structural & Molecular Biology (Chamberlain et al.)⁵ determined the crystal structures of the DDB1-CRBN complex bound to either thalidomide or one of its derivatives, and further shed light on the drug's mechanism. Fischer et al. presented crystal structures of the human DDB1 in complex with G. Gallus (chicken) CRBN bound to thalidomide, lenalidomide, and pomalidomide.⁴ The high sequence identity conservation between human and chicken CRBN allows structural insight into human CRBN to be inferred directly from G. gallus CRBN and reveals that important structural features are conserved. This crystal structure established that CRBN is a substrate receptor within CRL4^CRBN and that binding to IMiDs is enantiomer selective (in favor of the binding of the (S)-enantiomer over the (R)-enantiomer). Using a protein array-based ubiquitination screen, the study identified MEIS2 as an endogenous substrate of CRL4 ^CRBN, and the binding of MEIS2 and IMiDs to CRBN is mutually exclusive. Structure-function analysis indicated that IMiDs block endogenous substrates, such as MEIS2, from binding to CRL4 ^CRBN and prevent substrate degradation while simultaneously activating the DDB1-CRBN complex to recruit transcription factors IKZF1 and IKZF3 to promote their degradation. In combination with the existing understanding of thalidomide-cereblon interactions, this new research may help explain the dual nature of IMiDs as agonists and antagonists. An independent study by Chamberlain et al. also presented the crystal structure of human CRBN bound to DDB1 and lenalidomide, further confirming the binding domains and identifying specific residues responsible for binding.⁵ Site-specific mutagenesis studies in MM cell lines indicate that the IMiD compound-binding site is critical for the drug-induced anti-proliferative effects.

The structural characterization of the cereblon-IMiD binding complex not only enables us to better understand the mechanism of action of IMiDs, but also helps explain the pleiotropic clinical effects of the drugs. Furthermore, the observed enantioselective binding suggests the teratogenic effects involve the (S)-enantiomer, even though under biological conditions the isomers interconvert. Thus synthesising a non-teratogenic IMiDs would be highly improbable because the effects of the compound all utilize the cereblon pathway to simultaneously upregulate the endogenous substrates and decrease the amount of neo-substrates. Future research should focus on how and why the complex initiates the observed agonist activity, as evidence of the gain of function, and antagonist response, seen by the loss of ubiquitination. Moving beyond the discussion of thalidomide, insights into the structural studies of IMiDs bound to cereblon demonstrate that E3 ubiquitin ligases are druggable targets, implicate a potential for the rational design novel small molecule modulators of E3 ubiquitin ligases, and provide a direction for future drug discovery efforts.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

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