In the 1950s, thalidomide [α-(N-phthalimido)glutarimide] was introduced and taken by many pregnant women as a sedative/anti-nausea drug (Bartlett et al. 2004; Ito et al. 2011; Shortt et al. 2013). In the early 1960s, however, the drug was banned from the market because of its teratogenic potential (Mcbride 1961; Lenz et al. 1962). Despite this notorious effect, intensive research has been carried out with thalidomide due to its efficacy of inhibiting tumor necrosis factor (TNF)-α secretion and treating multiple myeloma and other hematologic malignancies (Sheskin 1965; Singhal et al. 1999). In this context, attempts to augment the effect of the drug resulted in the development of its analogs, lenalidomide and pomalidomide. This class of compounds is referred to as immunomodulatory imide drugs or IMiDs, and these compounds share two structural elements, the glutarimide moiety and the phthaloyl moiety (Fig. 1a). Although apremilast was approved as an analog of IMiDs in 2014 by FDA, apremilast was not included in the current study. Because while both apremilast and thalidomide share a phthaloyl moiety structure, apremilast lacks the glutarimide moiety and thus fails to bind to cereblon, the target of thalidomide action.
Due to chirality, the drugs have two isomeric forms and it is difficult to isolate one enantiomer from the other, because both enantiomers rapidly interchange in vivo (Eriksson et al. 1995; Lepper et al. 2006). Since the discovery of the teratogenic potential of IMiDs, a number of studies have proposed many hypotheses about their mechanisms, including oxidative stress (Parman et al. 1999) and anti-angiogenesis (Therapontos et al. 2009). These efforts, however, have not completely elucidated the mechanism of IMiDs-induced teratogenicity and other therapeutic mechanisms (Bartlett et al. 2004; Ito et al. 2011). In 2010, the discovery that the protein cereblon was the primary target of IMiDs opened a new avenue in IMiD research (Ito et al.). Cereblon protein has three domains; the amino terminal domain (NTD), the α-helical bundle domain (HBD), and the carboxy-terminal domain (CBD), and its sequence is highly conserved between different species (Ito et al. 2011; Shortt et al. 2013). This protein is a part of damage-specific DNA binding protein 1 (DDB1)/cullin4 E3 ubiquitin ligase complex and acts as the recruitment site for the ubiquitylation of substrate proteins, thus promoting their degradation (Ito et al. 2011; Shortt et al. 2013). Recently, the crystal structures of cereblon bound to IMiDs were solved and these structures revealed that the (S)-enantiomers of IMiDs bind to the protein, and the myeloid ecotropoic viral insertion site homeobox 2 (MEIS2) protein was identified as a substrate of cereblon (Fischer et al. 2014; Chamberlain et al. 2014) (Fig. 1b). A detailed structural analysis revealed that the glutarimide moiety of these compounds recognizes three tryptophans (W380, W386, and W400) of the aromatic cage (tri-Trp pocket), which are a part of the Tbk1/Ikki binding domain (TBD), and the phthalimide moiety is exposed to the solvent (Fischer et al. 2014; Chamberlain et al. 2014) (Fig. 1c).
Similar to cereblon protein, a number of proteins contain such aromatic cages, suggesting that this protein is bound by other ligands (particularly endogenous ligands) (Chamberlain et al. 2014; Hartmann et al. 2014; Lupas et al. 2015). More recently, uridine, one of the pyrimidine nucleosides, was identified as the cellular ligand of cereblon, and was shown to cause teratogenicity similar to IMiDs (Hartmann et al. 2014). However, other pyrimidine nucleosides such as cytidine or thymidine are shown to have no effect on teratogenicity in vivo (Hartmann et al. 2014). Uridine is structurally similar to the glutarimide moiety of IMiDs, implying that glutarimide- or uridine- like moieties induce teratogenicity. Considering the similarity of the ligand binding sites between cereblon and other proteins containing aromatic cages, other cationic ligands such as methylated lysine and/or arginine residues and the ligands containing quaternary ammonium groups could competitively bind to the IMiDs binding pocket of cereblon.
In this study, using the crystal structures of cereblon, we carried out in silico docking simulations in order to replicate these experimental results previously reported; the enantiomeric selectivity of IMiDs, and the identification of the region of cereblon contributing to this selectivity. In addition, we aim to replicate the experimental results obtained with pyrimidine nucleosides suggesting the preference of uridine over cytidine or thymidine when binding to the cereblon.