The thalidomide saga

The Thalidomide Saga: From Embryotoxic to Anti-Cancer

Activity

Katie Strong

September 15th, 2014

Liotta Group Meeting

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“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”

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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

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History behind FDA Approval


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, 1489http://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

The Myth Behind the Chirality of Thalidomide

4Agranat, 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

Identification of the Primary Target: Cereblon (CRBN)

5Ito, 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.

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.

Downregulation of CRBN causes similar in vivo effects as thalidomide

7Ito, 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

Thalidomide Causes Degradation of IKZF1 and IKZF3 Transcription Factors

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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)

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.

Thalidomide Binding Pocket in DDB1-CRBN E3 Ubiquitin Ligase


• 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

Thalidomide Binding Pocket in DDB1-CRBN E3 Ubiquitin Ligase


• 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

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

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

IKZF1transcription

factor

Gene of interest: IKZF1 gene

C4 position of Thalidomide Analogs is Important for IKZF1 Degradation


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

MEIS2 is an endogenous ligand of CRCL4CRBN


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.

Thalidomide Interacts with the CRBN protein in a complex mechanism


Data suggests antagonist pathway is partly

responsible for pleiotropic effects

Lenalidomide interacts with IKAROS transcription factors

and is linked to myleoma


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

Conclusions


• 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.

The thalidomide saga

Science

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