Homo-PROTACs for the Chemical Knockdown of Cereblon
Stefanie Lindner
60th ASH Annual Meeting and Exposition December 1, 2018 San Diego , CA IMiDs modulate substrate specificity of the CRBN E3 ligase
Thalidomide Lenalidomide omalidomide IKZF1 Multiple IKZF3 Myeloma CRBN Proteasomal CK1α del(5q) MDS Neo- E3 ubiquitin ligase degradation substrate
SALL4 Teratogenicity
Ito et al., 2010, Science Krönke et al., 2014 Science Lu et al., 2014, Science Krönke et al., 2015, Nature Donovan et al., 2018, Elife Matyskiela et al., 2018, Nat Chem Biol. Proteolysis Targeting Chimeras (PROTACs)
Bifunctional molecules for degradation of proteins of interest (POI)
Thalidomide POI Lenalidomide ligand Pomalidomide linker
POI IMiD CRBN
PROTAC
Sakamoto et. al, 2001, PNAS Schneekloth et. al, 2004, J Am Chem Soc. Lu et al., 2015, Chem Biol Winter et al., 2017, Mol Cell. Proteolysis Targeting Chimeras (PROTACs)
Bifunctional molecules for degradation of proteins of interest (POI)
POI IMiD CRBN
Proteasomal PROTAC degradation
Sakamoto et. al, 2001, PNAS Schneekloth et. al, 2004, J Am Chem Soc. Lu et al., 2015, Chem Biol Winter et al., 2017, Mol Cell. Homodimeric pomalidomide-based PROTACs
Linker (size X)
O O O HN O O NH O O HN NH O N N O Homo-bifunctional molecules
O O
Chemical Formula: C36H40N6O11 Molecular Weight: 732,75 Pomalidomide Pomalidomide
Chemically CRBN Pom Pom CRBN induced CRBN knockdown Proteasomal POI = CRBN degradation Homodimeric pomalidomide-based PROTACs corresponding linker substructures
No. of linear CRBN IKZF1 Linker linker atoms degradation degradation d multiple myeloma cell line MM1S
O O 8 ++ +
O O 10 + ++ Compound - 15a15b 15c 15d 15e POM LEN
O O 12 + ++ CRBN O O O 13 + ++ IKZF1 O 5 ++ + IKZF3 O O 8 - ++ CUL4A O 8 - - O Tubulin O O 8 + +
O 8 ± + O
LEN: Lenalidomide POM: Pomalidomide Pomalidomide-based Homo-PROTACs induce degradation of CRBN
15a (24h) 15a (100 nM) MSO POM 10 0.001 0.01 0.1 1 10 (µM) DMSO POM 0.25 0.5 1 3 6 24 (h) CRBN CRBN
IKZF1 IKZF1 IKZF3 IKZF3
Tubulin kinetics Tubulin Impact of Homo-PROTAC 15a on the cellular proteome
T- based quantitative proteomics in MM1S cell line centration: 100nM bation: 3h
Homo-PROTAC 15 vs. DMSO (3h) Pomalidomide vs. DMSO (3h) TMT Ratio Pomalidomide (3h/DMSO) 2 Log
Rank Rank CRBN knockdown has no effect on cell proliferation
LP-1 RPMI 8226 OCI-AML5
100% 100% 100%
80% 80% 80% (%)
60% 60% 60% cells
40% 40% 40% Viable (%) Viable cells 20% 20% (%) Viable cells 20%
0% 0% 0% 15a (µM) 0 0.1 1.0 10 15a (µM) 0 0.1 1.0 10 15a (µM) 0 0.1 1.0 10
100% 100% 100% 80% 80% 80% (%)
60% 60% 60% cells
40% 40% 40% Viable cells (%) Viable cells (%) Viable cells Viable 20% 20% 20%
0% 0% 0% OM (µM) 0 0.1 1.0 10 POM (µM) 0 0.1 1.0 10 POM (µM) 0 0.1 1.0 10 mpound 15a antagonizes the effects of IMiDs on multiple myeloma cells
MM1S
15a (100nM) --++ 120% *** POM (1 µM) -+-+ 100% CRBN 80%
60% IKZF1
40%
Viable cells (%) cells Viable IKZF3 20% Tubulin 0% 15a (µM) - 0.1 - 0.1 POM (µM) - - 1.0 1.0 pre Conclusions
IMiD-based Homo-PROTACs induce specific CRBN ubiquitination and degradation Chemical CRBN degradation had no effect on cancer cell proliferation Homo-PROTAC abrogates IMiD effects in multiple myeloma
Homodimeric pomalidomide-based compounds may help to: • Identify CRBN‘s endogenous substrates and physiologic function • Investigate the molecular mechanism of IMiDs • Potential clinical application in obesity (Lee et al., 2013, Diabetes) Acknowledgment
Gütschow lab Krönke Lab Michael Gütschow Jan Krönke Christian Steinebach Hannes Kehm Simon Köpff Tatjana Meyer Linda Röhner Yuen Lam Dora Ng Broad Institute of MIT and Imke Bauhuf Harvard Stephan Bohl Steven A. Carr Namrata D. Udeshi Deepak Mani