The R -enantiomer, shown in the figure, has sedative effects, whereas the S -isomer is teratogenic. Under biological conditions, the isomers interconvert, so separating the isomers before use is ineffective.
More recently, thalidomide has proven useful for treating cancer and leprosy and is approved for these uses. But although more than papers have been written about its mechanism of teratogenic action, it was not until the past few years that this mechanism was established. In , H. Handa and colleagues at the Tokyo Institute of Technology showed that its biological target is cereblon, a component of an E3 ubiquitin ligase complex. Earlier this year, N. Learn more about this molecule from CAS , the most authoritative and comprehensive source for chemical information.
If your favorite molecule is not in our archive , please send an email to motw acs. The molecule can be notable for its current or historical importance or for any quirky reason. Thank you! Comparison of R -thalidomides in the free form and in the CRBN-bound form found in the complex crystal.
Superposition of the free form of R -thalidomide grey on the CRBN-bound form of R -thalidomide green found in the crystal. The free form of R -thalidomide has its glutarimide ring in a C4- exo conformation, while R -thalidomide green bound to the tri-Trp pocket of CRBN displays a twisted conformation see text. The C4- exo conformation of the free form causes serious steric clash between the phthalimidocarbonyl group and the ring carbon atom of Trp 2.
The twisted conformation of the bound R -thalidomide avoids this clash by shifting the phthalimide ring toward the endo direction so that the clashes are relaxed 4. It was apparent that with thalidomide-treated embryos, the development of pectoral fins and otic vesicles was disturbed Fig.
Thus, S -thalidomide exerts greater teratogenic effects on fin development of zebrafish, which is consistent with the results of our binding and ubiquitylation-inhibition assays using deuterium-substituted thalidomide enantiomers Fig. S -thalidomide treatment causes more sever developmental defects in zebrafish than R -thalidomide. Fins are indicated by arrowheads. The teratogenic effects resulting from treatment with thalidomide are classified into three categories.
Based on our complex structures, we are now able to assess the CRBN-binding affinity of thalidomide derivatives. The glutarimide ring in a relaxed six-membered ring conformation is important for CRBN binding with the imide group, serving as both a hydrogen donor and acceptor.
Thus, ring puckering usually occurs at the C4 carbon atom. In the isolated state, the C4- endo conformation of the S -thalidomide glutarimide ring minimizes the conformational energy by allowing the N-C bond at the chiral C3 atom to be oriented in a stable equatorial conformation, whereas R -thalidomide favors the C4- exo conformer of the glutarimide ring, allowing the N-C bond to adopt an equatorial conformation.
In the C4- endo conformer of the S -thalidomide glutarimide ring, the chiral centre tilts the phthalimido group slightly toward the endo direction bottom in Fig. Therefore, small adjustment of the orientation of the phthalimido group is sufficient for CRBN binding middle in Fig.
In the C4- exo conformer of the R -thalidomide glutarimide ring, however, the chiral centre tilts the phthalimido group slightly toward the exo direction bottom in Fig. In this case, small adjustment of the orientation of the phthalimido group is insufficient for CRBN binding, and therefore the glutarimide ring pucker of R -thalidomide needs to be changed to the twist conformation, C4- exo -C5- endo , to shift the phthalimido group up toward the endo direction middle in Fig.
It should be noted that this enantiomer-specific discrimination by the tri-Trp pocket is mediated by hydrogen bonding interactions between the amide group of the glutarimide ring and the inside of the pocket, in addition to contacts between carbonyl groups of the phthalimido group and the entrance of the pocket. Lenalidomide and pomalidomide have attracted much attention from pharmaceutical scientists and physicians as hopeful IMiDs in the treatment of multiple myeloma and other cancers 11 , Interestingly, this shift accompanies a movement of the phthalimido group toward the endo direction as seen in S -thalidomide Fig.
As with previously reported structures 28 , 29 , binding to CRBN is primarily mediated by the glutarimide ring. Correspondingly, glutarimide alone is able to bind CRBN and inhibit ubiquitylation to a similar extent as R -thalidomide Fig.
Bulky modification of the glutarimide C4-carbon atom also results in loss of CRBN binding due to steric clashes cycloheximide in Fig.
The primary metabolic derivatives generated by cytochrome P isozymes are derived from 4- or 5-hydroxylation of the phthalimido moiety or 5-hydroxylation of the glutarimide moiety, which subsequently undergo spontaneous hydrolysis 12 , 41 Supplementary Fig. Based on our structural data, metabolites with phthalimido modifications may still bind CRBN, whereas 4- or 5-hydroxylation of the glutarimide moiety is likely to result in loss of binding due to steric clashes.
We suggest classifying the hydrolysis processes into three pathways A, B and C. CRBN binding and E3 inhibition by thalidomide derivatives. Phthalimide exhibits no binding. The method used was that described in Fig. The intact glutarimide ring in a relaxed six-membered ring conformation is important for CRBN binding with the imide group serving as both a hydrogen donor and acceptor. Correspondingly, glutarimide still binds CRBN, although phthalimide and glutaric anhydride, which lack the ring amide of glutarimide, exhibited no binding.
Bulky modification of the glutarimide C4-carbon atom also results in loss of CRBN binding due to steric clash cycloheximide. Full-length blots in a , b and c are presented in Supplementary Fig. In conclusion, our study provides a structural framework for further investigations on the mechanisms of the pharmaceutical and teratogenic actions of this drug and for the development of more effective IMiDs.
The E. Purified CRBN protein sample was dialyzed against a buffer solution containing 0. Crystallization conditions were searched for using the sitting-drop vapour diffusion method and Hydra II-Plus-One crystallization robot Matrix Technology with a commercial crystallization solution kit. Diffraction data were processed using the HKL program Crystals of the three thalidomide-bound forms belong to space group R 3 H , with V M values in the range 2.
The secondary structures of models were calculated using DSSP The refinement statistics are summarized in Table 2. These crystals contain 16 molecules in the asymmetric unit. Of these, 15 molecules form 5 trimers similar to those of the free form but with non-crystallographic three-fold axes, while one molecule forms a trimer with symmetry-related molecules related by a crystallographic three-fold axis. No significant structural deviations were found among these molecules with small averaged rms deviations 0.
No outliers were flagged in the Ramachandran plots using MolProbity Anti-mouse monoclonal CRBN antibody was generated in house epitope 1— Racemic thalidomide was purchased from Tocris Biosciences.
S - and R -thalidomides and their deuterated D-thalidomide enantiomers were synthesized as previously described 30 , Binding assays were performed essentially as previously described Cell extracts were incubated with thalidomide-immobilized beads, and bound material was eluted with buffer containing thalidomide, thalidomide-related compounds, or SDS.
Lysates input and bead-affinity-purified AP materials were immunoblotted IB. Subsequent binding assays were performed as described above. Fish were kept at Following extensive washing three times with 0. Details of each titration are given in the figure legends of Supplementary Figs 2 and 5. We thank R. Kurata and Y. We also thank Dr I. Ooki for help with the data collection at SPring Tomoyuki Mori and Takumi Ito contributed equally to this work.
Electronic supplementary material. Supplementary information accompanies this paper at Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Hiroshi Handa, Email: pj. Toshio Hakoshima, Email: pj. National Center for Biotechnology Information , U. Sci Rep. Published online Jan Author information Article notes Copyright and License information Disclaimer.
Corresponding author. Received Mar 28; Accepted Dec This article has been cited by other articles in PMC. Associated Data Supplementary Materials Supplementary figures. Abstract Thalidomide possesses two optical isomers which have been reported to exhibit different pharmacological and toxicological activities. Introduction More than 50 years have passed since thalidomide was first prescribed as a sedative and antiemetic to provide effective relief from morning sickness during early pregnancy.
Results Differences in CRBN binding affinity between S - and R - thalidomides assayed using deuterium-substituted enantiomers Since interconversion of thalidomide enantiomers could occur under physiological conditions, special precautions are required to delineate any enantiomer-specific differences in the biological activity of thalidomide.
Open in a separate window. Figure 1. Figure 2. Figure 3. Figure 4. Conformational differences between bound S - and R -thalidomides The electron density maps of our complex structures are sufficiently clear to define the conformation of the bound thalidomide molecules Fig. Figure 5. Figure 6. Figure 7. Discussion Based on our complex structures, we are now able to assess the CRBN-binding affinity of thalidomide derivatives.
Figure 8. Crystallization Crystallization conditions were searched for using the sitting-drop vapour diffusion method and Hydra II-Plus-One crystallization robot Matrix Technology with a commercial crystallization solution kit. Thalidomide and related compounds Racemic thalidomide was purchased from Tocris Biosciences.
In vivo binding assay using thalidomide-immobilized beads Binding assays were performed essentially as previously described Thalidomide treatment of zebrafish Fish were kept at Electronic supplementary material Supplementary figures 1. Acknowledgements We thank R. Author Contributions T. Notes Competing Interests The authors declare that they have no competing interests.
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Download references. The authors would like to thank Ms. Yuka Suzuki for her contributions during the early stages of this work, and Mr.
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