Structural basis of thalidomide enantiomer binding to cereblon

Mori, Tomoyuki; Ito, Takumi; Liu, Shujie; Ando, Hideki; Sakamoto, Satoshi; Yamaguchi, Yuki; Tokunaga, Etsuko; Shibata, Norio; Handa, Hiroshi; Hakoshima, Toshio

doi:10.1038/s41598-018-19202-7

Cited by 90 publications

(74 citation statements)

References 52 publications

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“…Only a handful out of ~7,000 proteins quantified in the experiments were found to be significantly affected by the IMiDs treatment. Among them we could observe a clear temperature-and concentration-dependent stabilization of CRBN for all three compounds, confirming binding to the endogenous target12,28,29 . Notably, for proteins ZFP91, IKZF1 and RNF166 the same concentration dependent reduction of soluble protein amount was observed for all sample temperatures, suggesting compound-induced protein degradation rather than destabilization, which is well in line with previous findings 14,16,30 .…”

supporting

confidence: 54%

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

Chernobrovkin

Cázares-Körner

Friman

et al. 2020

Preprint

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Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance and reaching "undruggable" proteins compared to traditional small molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation, instead one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs) understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The Cellular Thermal Shift Assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS) CETSA can simultaneously evaluate compound induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g. IMiDs) and PROTACs to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e. binding to the PoI and ligases) and functional readout (i.e. degrader induced protein degradation).

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supporting

confidence: 54%

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

Chernobrovkin

Cázares-Körner

Friman

et al. 2020

Preprint

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“…This report opened a new era for thalidomide in science and completely redirected the research on this drug 24 – 27 . In 2018, Hakoshima, Handa, and co-workers, including two of the authors of the present study, finally put an end to the debate on the teratogenicity of ( S )-thalidomide via structural and biochemical studies of the ( S )- and ( R )-enantiomers of thalidomide coordinated to CRBN 28 . The biochemical studies were carried out on deuterium-substituted enantiomers of thalidomide 29 to suppress any enantiomeric interconversion.…”

Section: Introductionmentioning

confidence: 85%

Understanding the Thalidomide Chirality in Biological Processes by the Self-disproportionation of Enantiomers

Tokunaga

Yamamoto

Ito

et al. 2018

Sci Rep

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111

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Twenty years after the thalidomide disaster in the late 1950s, Blaschke et al. reported that only the (S)-enantiomer of thalidomide is teratogenic. However, other work has shown that the enantiomers of thalidomide interconvert in vivo, which begs the question: why is teratogen activity not observed in animal experiments that use (R)-thalidomide given the ready in vivo racemization (“thalidomide paradox”)? Herein, we disclose a hypothesis to explain this “thalidomide paradox” through the in-vivo self-disproportionation of enantiomers. Upon stirring a 20% ee solution of thalidomide in a given solvent, significant enantiomeric enrichment of up to 98% ee was observed reproducibly in solution. We hypothesize that a fraction of thalidomide enantiomers epimerizes in vivo, followed by precipitation of racemic thalidomide in (R/S)-heterodimeric form. Thus, racemic thalidomide is most likely removed from biological processes upon racemic precipitation in (R/S)-heterodimeric form. On the other hand, enantiomerically pure thalidomide remains in solution, affording the observed biological experimental results: the (S)-enantiomer is teratogenic, while the (R)-enantiomer is not.

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“…Another example is thalidomide-induced teratogenicity; zebrafish embryos appear to be sensitive to this chemical, which causes pectoral fin malformations within 48 to 72 hpf, being similar to the limb malformations seen in thalidomide embryopathy in humans (Ito et al, 2010). An enantiomer selectivity of thalidomide teratogenicity was also indicated in zebrafish (Mori et al, 2018).…”

Section: Introductionmentioning

confidence: 93%

Transcriptional profiling of cytochrome P450 genes in the liver of adult zebrafish, <i>Danio rerio</i>

et al. 2019

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Increasing use of zebrafish in biomedical, toxicological and developmental studies requires explicit knowledge of cytochrome P450 (CYP), given the central role of CYP in oxidative biotransformation of xenobiotics and many regulatory molecules. A full complement of CYP genes in zebrafish and their transcript expression during early development have already been examined. Here we established a comprehensive picture of CYP gene expression in the adult zebrafish liver using a RNAseq technique. Transcriptional profiling of a full complement of CYP genes revealed that CYP2AD2, CYP3A65, CYP1A, CYP2P9 and CYP2Y3 are major CYP genes expressed in the adult zebrafish liver in both sexes. Quantitative real-time RT-PCR analysis for selected CYP genes further supported our RNAseq data. There were significant sex differences in the transcript levels for CYP1A, CYP1B1, CYP1D1 and CYP2N13, with males having higher expression levels than those in females in all cases. A similar feature of gender-specific expression was observed for CYP2AD2 and CYP2P9, suggesting sex-specific regulation of constitutive expression of some CYP genes in the adult zebrafish liver. The present study revealed several "orphan" CYP genes as dominant isozymes at transcript levels in the adult zebrafish liver, implying crucial roles of these CYP genes in liver physiology and drug metabolism. The current results establish a foundation for studies with zebrafish in drug discovery and toxicology.

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Structural basis of thalidomide enantiomer binding to cereblon

Cited by 90 publications

References 52 publications

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

Understanding the Thalidomide Chirality in Biological Processes by the Self-disproportionation of Enantiomers

Transcriptional profiling of cytochrome P450 genes in the liver of adult zebrafish, <i>Danio rerio</i>

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