A presentation of the history of thalidomide with an emphasis on the most recent crystal structure of thalidomide bound to CRBN.

1. The Thalidomide Saga: From
Embryotoxic to Anti-Cancer
Activity
Katie Strong
September 15th, 2014
Liotta Group Meeting
1
“The 55-year history of the drug thalidomide is Shakespearean in scope, awash in
unintended consequences, tragedy, resilience, driven characters, and redemption.”
- A. Keith Stewart in “How Thalidomide Works Against Cancer”

2. 2
History behind FDA Approval
1954: Thalidomide was introduced to the market by the company Chemie Grunenthal
1956: The first known victim, the daughter of a Grunenthal employee, was born
1961: The drug was banned worldwide after causing an estimated 10,000 birth defects
1965: Thalidomide was serendipitously discovered by Dr. Jacob Sheskin to treat
complications associated with erythema nodosum leprosum (ENL)
1998: Thalidomide was approved by the FDA for treatment of ENL
Melchert, M. List, A. The thalidomide saga. Int J Biochem Cell B 2007, 39, 1489

3. 3
History behind FDA Approval
1998: Thalidomide was approved by the FDA for treatment of ENL
May 2006: A combination of thalidomide and dexamethasone was FDA approved for the
treatment of multiple myeloma
June 2006: Lenalidomide (Revlimid) and pomalidomide (Actimid), derivatives of
thalidomide, were FDA approved for the treatment of multiple myeloma
The use of thalidomide is incredibly restricted and requires participation in
the System for Thalidomide Education and Prescription Safety (STEPS)
program
Melchert, M. List, A. The thalidomide saga. Int J Biochem Cell B 2007, 39, 1489
http://www.fda.gov/ohrms/dockets/ac/04/briefing/4017b1-06b%20overview%20steps%20section%20c%20tab%207.htm
Thalidomide and its related analogs are referred to as immunomodulatory drugs (IMiDs)
Thalidomide Lenalidomide Pomalidomide

4. The Myth Behind the Chirality of Thalidomide
4
Agranat, I.; Caner, H.; Caldwell, J. Putting Chirality to Work: The Strategy of Chiral Switches. Nature Rev Drug Discov 2002, 1, 753.
Reist, M.; Carrupt, P-A.; Francotte, E.; Testa, B. Chem Res Toxicol 1998, 11, 1521
“The thalidomide tragedy would probably never have occurred if, instead of using the
racemate, the (R)-enantiomer had been brought on to the market. In studies … it was
shown that after i.p. administration only the (S)-(–)-enantiomer exerts an embryotoxic and
teratogenic effect. The (R)-(+)-enantiomer is devoid of any of those effects under the same
experimental conditions”.
- C.J. De Ranter in X-Ray Crystallography and Drug Action
- Alluded to in the citation for the 2001 Nobel Prize in Chemistry
1) (S)-(-) was only more teratogenic in mice, but in rabbits, the most sensitive
species, both enantiomers were active.
2) Chiral center is unstable in protonated media and undergoes a rapid
configurational inversion, along with degradation of the glutarimide ring

5. Identification of the Primary Target: Cereblon (CRBN)
5
Ito, T. et al. Identification of a Primary Target of Thalidomide Tetragenicity. Science 2010, 327, 1345.
Through a series of experiments using affinity purification, cereblon (CRBN) was identified
as a thalidomide binding protein. CRBN is a part of the CRCL4 E3 ubiquitin ligase.

6. Identification of the Primary Target: Cereblon (CRBN)
6Ito, T. et al. Identification of a Primary Target of Thalidomide Tetragenicity. Science 2010, 327, 1345.
http://yamaguchi.bio.titech.ac.jp/english/research/detail_25.html
Thalidomide binds to CRBN, which prohibits an endogenous substrate from binding. This
substrate, or target protein, does not undergo ubiquitination, and it is not tagged for
degradation, which leads to abnormal development. At this time, the identity of the
substrate was not known.

7. Downregulation of CRBN causes similar in vivo effects as thalidomide
7
Ito, T. et al. Identification of a Primary Target of Thalidomide Tetragenicity. Science 2010, 327, 1345.
Researchers used zebrafish as a model system for thalidomide embrotoxicity
Pectoral fins at 75 hours post
fertilization
Otic vesicle size 30 hours post
fertilization
zcrbn AMO: Embryos
injected with an antisense
morpholino oligonucleotide
for zcrbn, which is a way to
knockdown the gene

8. Thalidomide Causes Degradation of IKZF1 and IKZF3 Transcription Factors
8
Kronke, J. et al. Lenalidomide Causes Selective Degradation of IKZF1 and IKZF3 in Multiple Myeloma Cells. Science 2014, 343, 301.
Lu, G. et al. The Myeloma Drug Lenalidomide Promotes the Cereblon-Dependent Destruction of Ikaros Proteins. Science 2014, 343, 305.
Stewart, K. How Thalidomide Works Against Cancer. Science 2014, 343, 256.
2006: Lenalidomide (Revlimid) and pomalidomide (Actimid), derivatives of thalidomide,
were FDA approved for the treatment of mantle cell lymphoma (MCL)

9. Crystal Structure of Thalidomide and DDB1-CRBN E3 Ubiquitin Ligase
9Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.

10. Crystal Structure of Thalidomide and DDB1-CRBN E3 Ubiquitin Ligase
10Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.

11. Thalidomide Binding Pocket in DDB1-CRBN E3 Ubiquitin Ligase
11Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.
• Glutarimide C2 and C6 carbonyls, along with amide, form hydrogen bonds
• Glutarimide C3, C4, and C5 are in tight Van der Waals contact with hydrophobic pocket
• Mutations of Trp 388 ablates the binding of thalidomide
• Phthaloyl C1 contributes to hydrogen bonding and the ring stacks with Pro 354

12. Thalidomide Binding Pocket in DDB1-CRBN E3 Ubiquitin Ligase
12Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.
• While all three IMiDs target the IKAROS transcription factors, thalidomide is
much less effective
• All three IMiDs have comparable affinities for CRBN and similar membrane
permeabilities
• The largest structural difference is in the C4 aniline functionality

13. 13Allard, S.T.M.; Kopish, K. Luciferase Reporter Assays: Powerful, Adaptable Tools for Cell Biology Research. Cell Notes 2008, 21, 23.
Researchers wanted to synthesize thalidomide analogs where the C4, C5, and C6
positions were modified and then test these for IKAROS degradation.
Dual Reporter Luciferase Assay to Determine IKZF1 Transcription Factor Degradation

14. 14Allard, S.T.M.; Kopish, K. Luciferase Reporter Assays: Powerful, Adaptable Tools for Cell Biology Research. Cell Notes 2008, 21, 23.
Dual Reporter Luciferase Assay to Determine IKZF1 Transcription Factor Degradation
IKZF1
transcription
factor
Gene of interest: IKZF1 gene

15. C4 position of Thalidomide Analogs is Important for IKZF1 Degradation
15Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.
A series of thalidomide analogs were tested in the dual reporter luciferase assay
Bulky groups at C4 interfere
with IKZF1 binding, while
small groups at C4 promote
degradation
The binding affinities of thalidomide analogs were also determined
These modifications, while
impacting degradation, are
not affecting CRBN binding

16. MEIS2 is an endogenous ligand of CRCL4CRBN
16Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.
Approximately 9,000 proteins were subjected to a on-chip ubiquitination, and 5 genes
were candidates for further screening. Research continued with MEIS2, a transcription
factor involved in various aspects of human development.
MEIS2 is ubiquitinated in vitro,
and this ubiquitination is
inhibited by thalidomide
derivatives.
The MEIS2 protein levels in
M059J cells were elevated in the
presence of thalidomide
derivatives.
It is likely that multiple substrates for CRCL4CRBN exist, although mutations is MEIS2
have been related to mental retardation, cleft palate, lens and retina development
and congenital cardiac defects. More research is required in this area though.

17. Thalidomide Interacts with the CRBN protein in a complex mechanism
17
Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.
Data suggests antagonist
pathway is partly
responsible for pleiotropic
effects
Lenalidomide interacts with
IKAROS transcription factors
and is linked to myleoma

18. 1998: Thalidomide was approved by the FDA for treatment of ENL
2006: A combination of thalidomide and dexamethasone was FDA approved for the
treatment of multiple myeloma
2006: Lenalidomide (Revlimid) and pomalidomide (Actimid), derivatives of thalidomide,
were FDA approved for the treatment of mantle cell lymphoma (MCL)
Thalidomide’s Story is Continually Evolving
2010: Cereblon (CRBN) was identified as the primary target responsible for thalidomide’s
embryotoxic effects
Jan 2014: Degradation of transcription factors IKZF1 and IKZF3 was identified as the main
event resulting in a toxic outcome for multiple myeloma cells
1954: Thalidomide was introduced to the market by the company Chemie Grunenthal
July 2014: Crystal structure of thalidomide bound to CRCL4CRBN complex sheds light on the
complex mechanism behind thalidomide’s teratogenic and anti-cancer effects

19. Conclusions
19
Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.
• The mechanism behind thalidomide’s teratogenic and immunomodulatory effects
are discernable, however both go through the CRBN pathway. Developing a drug
with only anti-cancer properties may prove difficult.
• Transcription factors are typically thought of as “undruggable,” so developing
compounds that modulate the activity of transcription factors through selective
ubiquition may be possible.

20. Crystal Structure of Thalidomide and DDB1-CRBN E3 Ubiquitin Ligase
20Fischer, E.S. et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, Advance Online Publication.