Name: chemical inactivation of the e3 ubiquitin ligase cereblon by pomalidomide-based homo-protacs
Uploaded: 2019-05-15T14:00:00
Description: Watch this Scientific Journal Video about Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs at JoVE.com

This work was supported by the Deutsche Forschungsgemeinschaft (Emmy-Noether Program Kr-3886/2-1 and SFB-1074 to J.K.; FOR2372 to M.G.)

1. Preparation of PROTAC molecules
CAUTION: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are toxic and carcinogenic. Please use all appropriate safety practices and personal protective equipment.
<ol>
	<li>Preparation of tert-butyl N-(2,6-dioxo-3-piperidyl)carbamate (compound 1)
	<ol>
		<li>Add 1,1&#39;-carbonyldiimidazole (1.95 g, 12 mmol) and a catalytic amount of 4-(dimethylam...

<ol>
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K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+agents+lenalidomide+and+pomalidomide+co-stimulate+T+cells+by+inducing+degradation+of+T+cell+repressors+Ikaros+and+Aiolos+via+modulation+of+the+E3+ubiquitin+ligase+complex+CRL4(CRBN).">Immunomodulatory agents lenalidomide and pomalidomide co-stimulate T cells by inducing degradation of T cell repressors Ikaros and Aiolos via modulation of the E3 ubiquitin ligase complex CRL4(CRBN).</a> British Journal of Haematology. 164 (6), 811-821 (2014).</li><li>Kronke, J., Hurst, S. N., Ebert, B. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thalidomide+promotes+degradation+of+SALL4,+a+transcription+factor+implicated+in+Duane+Radial+Ray+syndrome.">Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome.</a> eLife. 7, (2018).</li><li>Matyskiela, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SALL4+mediates+teratogenicity+as+a+thalidomide-dependent+cereblon+substrate.">SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate.</a> Nature Chemical Biology. 14 (10), 981-987 (2018).</li><li>Kronke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lenalidomide+induces+ubiquitination+and+degradation+of+CK1alpha+in+del(5q)+MDS.">Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS.</a> Nature. 523 (7559), 183-188 (2015).</li><li>Sakamoto, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protacs:+chimeric+molecules+that+target+proteins+to+the+Skp1-Cullin-F+box+complex+for+ubiquitination+and+degradation.">Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.</a> Proceedings of the National Academy of Sciences of the United States of America. 98 (15), 8554-8559 (2001).</li><li>Sakamoto, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+Protacs+to+target+cancer-promoting+proteins+for+ubiquitination+and+degradation.">Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation.</a> Molecular &amp; Cellular Proteomics. 2 (12), 1350-1358 (2003).</li><li>Schneekloth, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+control+of+protein+levels:+selective+in+vivo+targeted+degradation.">Chemical genetic control of protein levels: selective in vivo targeted degradation.</a> Journal of the American Chemical Society. 126 (12), 3748-3754 (2004).</li><li>Gu, S., Cui, D., Chen, X., Xiong, X., Zhao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PROTACs:+An+Emerging+Targeting+Technique+for+Protein+Degradation+in+Drug+Discovery.">PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery.</a> Bioessays. 40 (4), e1700247 (2018).</li><li>Collins, I., Wang, H., Caldwell, J. J., Chopra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+approaches+to+targeted+protein+degradation+through+modulation+of+the+ubiquitin-proteasome+pathway.">Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway.</a> Biochemical Journal. 474 (7), 1127-1147 (2017).</li><li>Neklesa, T. K., Winkler, J. D., Crews, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+protein+degradation+by+PROTACs.">Targeted protein degradation by PROTACs.</a> Pharmacology &amp; Therapeutics. 174, 138-144 (2017).</li><li>Winter, G. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phthalimide+conjugation+as+a+strategy+for+in+vivo+target+protein+degradation.">Phthalimide conjugation as a strategy for in vivo target protein degradation.</a> Science. 348 (6241), 1376-1381 (2015).</li><li>Maniaci, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homo-PROTACs:+bivalent+small-molecule+dimerizers+of+the+VHL+E3+ubiquitin+ligase+to+induce+self-degradation.">Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation.</a> Nature Communications. 8 (1), 830 (2017).</li><li>Crew, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+Characterization+of+Von+Hippel-Lindau-Recruiting+Proteolysis+Targeting+Chimeras+(PROTACs)+of+TANK-Binding+Kinase+1.">Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1.</a> Journal of Medicinal Chemistry. , (2017).</li><li>Lu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hijacking+the+E3+Ubiquitin+Ligase+Cereblon+to+Efficiently+Target+BRD4.">Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4.</a> Chemistry &amp; Biology. 22 (6), 755-763 (2015).</li><li>Steinebach, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PROTAC-mediated+crosstalk+between+E3+ligases.">PROTAC-mediated crosstalk between E3 ligases.</a> Chemical Communications. 55 (12), 1821-1824 (2019).</li><li>Tinworth, C. P., Lithgow, H., Churcher, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule-mediated+protein+knockdown+as+a+new+approach+to+drug+discovery.">Small molecule-mediated protein knockdown as a new approach to drug discovery.</a> Medchemcomm. 7 (12), 2206-2216 (2016).</li><li>Steinebach, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homo-PROTACs+for+the+Chemical+Knockdown+of+Cereblon.">Homo-PROTACs for the Chemical Knockdown of Cereblon.</a> ACS Chemical Biology. 13 (9), 2771-2782 (2018).</li><li>Ambrozak, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Antiangiogenic+Properties+of+Tetrafluorophthalimido+and+Tetrafluorobenzamido+Barbituric+Acids.">Synthesis and Antiangiogenic Properties of Tetrafluorophthalimido and Tetrafluorobenzamido Barbituric Acids.</a> ChemMedChem. 11 (23), 2621-2629 (2016).</li><li>Zhou, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+a+Small-Molecule+Degrader+of+Bromodomain+and+Extra-Terminal+(BET)+Proteins+with+Picomolar+Cellular+Potencies+and+Capable+of+Achieving+Tumor+Regression.">Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression.</a> Journal of Medicinal Chemistry. , (2017).</li><li>Zhang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+Targeting+Chimeras+(PROTACs)+of+Anaplastic+Lymphoma+Kinase+(ALK).">Proteolysis Targeting Chimeras (PROTACs) of Anaplastic Lymphoma Kinase (ALK).</a> European Journal of Medicinal Chemistry. 151, 304-314 (2018).</li><li>Runcie, A. C., Chan, K. H., Zengerle, M., Ciulli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetics+approaches+for+selective+intervention+in+epigenetics.">Chemical genetics approaches for selective intervention in epigenetics.</a> Current Opinion in Chemical Biology. 33, 186-194 (2016).</li><li>Kronke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lenalidomide+causes+selective+degradation+of+IKZF1+and+IKZF3+in+multiple+myeloma+cells.">Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells.</a> Science. 343 (6168), 301-305 (2014).</li><li>Eichner, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+drugs+disrupt+the+cereblon-CD147-MCT1+axis+to+exert+antitumor+activity+and+teratogenicity.">Immunomodulatory drugs disrupt the cereblon-CD147-MCT1 axis to exert antitumor activity and teratogenicity.</a> Nature Medicine. 22 (7), 735-743 (2016).</li><li>Zhu, Y. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cereblon+expression+is+required+for+the+antimyeloma+activity+of+lenalidomide+and+pomalidomide.">Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide.</a> Blood. 118 (18), 4771-4779 (2011).</li><li>Kortum, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+sequencing+of+refractory+myeloma+reveals+a+high+incidence+of+mutations+in+CRBN+and+Ras+pathway+genes.">Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes.</a> Blood. 128 (9), 1226-1233 (2016).</li><li>Gil, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cereblon+deficiency+confers+resistance+against+polymicrobial+sepsis+by+the+activation+of+AMP+activated+protein+kinase+and+heme-oxygenase-1.">Cereblon deficiency confers resistance against polymicrobial sepsis by the activation of AMP activated protein kinase and heme-oxygenase-1.</a> Biochemical and Biophysical Research Communications. 495 (1), 976-981 (2018).</li><li>Kim, H. 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The design of such homo-PROTACs as described here for CRBN relies on the specific affinity of pomalidomide to CRBN, which has been successfully utilized in numerous heterobifunctional PROTACs and resulted in the development of PROTAC 8 as a highly selective CRBN degrader. The specificity of our molecule has already been confirmed by proteomic analyses24. For genetically mediated knockout, exclusion and validation of side effects is challenging and time consuming. In addition, a ch...

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all approved for the treatment of multiple myeloma, bind to the E3 ubiquitin ligase cereblon (CRBN), a substrate adaptor for cullin4A-RING E3 ubiquitin ligase (CRL4CRBN)1,2,3. Binding of IMiDs enhances the affinity of CRL4CRBN to the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3), leading to their ubiquitination and degradation (Figure 1)4,5,6,7,8. Since IKZF1 and IKZF3 are essential for multiple myeloma cells, their inactivation results in growth inhibition. SALL4 was recently found as an additional IMiD-induced neo-substrate of CRBN that is likely responsible for the teratogenicity and the so-called Contergan catastrophe in the 1950s caused by thalidomide9,10. In contrast, casein kinase 1&#945; (CK1&#945;) is a lenalidomide-specific substrate of CRBN that is implicated in the therapeutic effect in myelodysplastic syndrome with chromosome 5q deletions11.
The ability of small-molecules to target a specific protein for degradation is an exciting implication for modern drug development. While the mechanism of thalidomide and its analogs was discovered after their first use in humans, so called Proteolysis Targeting Chimeras (PROTACs) have been designed to specifically target a protein of interest (POI) (Figure 2)12,13,14,15,16,17,18. PROTACs are heterobifunctional molecules that consist of a specific ligand for the POI connected via a linker to a ligand of an E3 ubiquitin ligase like CRBN or von-Hippel-Lindau (VHL)18,19,20,21,22. PROTACs induce the formation of a transient ternary complex, directing the POI to the E3 ubiquitin ligase, resulting in its ubiquitination and proteasomal degradation. The major advantages of PROTACs over conventional inhibitors is that binding to a POI is sufficient rather than its inhibition and therefore PROTACs can potentially target a far wider spectrum of proteins including those that were considered to be undruggable like transcription factors15. In addition, chimeric molecules act catalytically and therefore have a high potency. After ubiquitin transfer to the POI, the ternary complex dissociates and is available for the formation of new complexes. Thus, very low PROTAC concentrations are sufficient for the degradation of the target protein23.
Here we describe the synthesis of a pomalidomide-pomalidomide conjugated homo-PROTAC (compound 8) that recruits CRBN for the degradation of itself24. The E3 ubiquitin ligase CRBN serves as both the recruiter and the target at the same time (Figure 3). To validate our data, we also synthesized a negative binding control (compound 9). Our data confirm that the newly synthesized homo-PROTAC is specific for CRBN degradation and has only minimal effects on other proteins.

<table>
	<tbody>
		<tr>
			<td>1,1&#39;-Carbonyldiimidazole</td>
			<td>TCI chemicals</td>
			<td>C0119</td>
			<td></td>
		</tr>
		<tr>
			<td>2,2&#8242;-(Ethylenedioxy)-bis(ethylamine)</td>
			<td>Sigma-Aldrich</td>
			<td>385506</td>
			<td>Compound 6</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Fluorophthalic anhydride, 98 %</td>
			<td>Alfa Aesar</td>
			<td>A12275</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine, 99 %</td>
			<td>Acros</td>
			<td>148270250</td>
			<td>Toxic</td>
		</tr>
		<tr>
			<td>Acrylamidstamml&#246;sung/ Bisacrylamid (30%/0,8%)</td>
			<td>Carl Roth</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Aiolos (D1C1E) mAB</td>
			<td>Cell signaling</td>
			<td>15103S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-CRBN antibody produced in rabbit</td>
			<td>Sigma</td>
			<td>HPA045910</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG HRP-linked antibody</td>
			<td>Sigma</td>
			<td>7074S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate</td>
			<td>Roth</td>
			<td>9592.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Boc-Gln-OH</td>
			<td>TCI chemicals</td>
			<td>B1649</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTiter-Glo Luminescent Cell Viability Assay</td>
			<td>Promega</td>
			<td>G7571</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc XRS+</td>
			<td>Bio-Rad</td>
			<td>1708265</td>
			<td></td>
		</tr>
		<tr>
			<td>DMF, anhydrous, 99.8 %</td>
			<td>Acros</td>
			<td>348435000</td>
			<td>Extra Dry over Molecular Sieve</td>
		</tr>
		<tr>
			<td>DMSO, anhydrous, 99.7 %</td>
			<td>Acros</td>
			<td>348445000</td>
			<td>Extra Dry over Molecular Sieve</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>15523-1L-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse (HRP conjugated)</td>
			<td>Santa Cruz biotechnology</td>
			<td>sc-2005</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease &#38; Phosphatase Inhibitor Single-use Cocktail (100X)</td>
			<td>Thermo Scientific</td>
			<td>1861280</td>
			<td></td>
		</tr>
		<tr>
			<td>Ikaros (D6N9Y) Mab</td>
			<td>Cell signaling</td>
			<td>14859S</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmobilonP Transfer Membrane (0,45&#181;m)</td>
			<td>Merck</td>
			<td>IPVH000010</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodomethane, 99 %</td>
			<td>Sigma-Aldrich</td>
			<td>I8507</td>
			<td>Highly toxic</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>32213-2.5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg132</td>
			<td>Selleckchem</td>
			<td>S2619</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot electrophoretic transfer cell</td>
			<td>Bio-Rad</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>1658004</td>
			<td></td>
		</tr>
		<tr>
			<td>MLN4942</td>
			<td>biomol (cayman)</td>
			<td>Cay15217-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal Anti-&#945;-Tubulin antibody produced in mouse (B512)</td>
			<td>Sigma</td>
			<td>T5168</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Ethyldiisopropylamine, 99 %</td>
			<td>Alfa Aesar</td>
			<td>A11801</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat dried milk powder</td>
			<td>PanReac AppliChem</td>
			<td>A0830,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nunc F96 MicroWell White Polystyrene Plate</td>
			<td>Thermo Scientific</td>
			<td>136101</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE LDS Sample Buffer (4X)</td>
			<td>Thermo Scientific</td>
			<td>NP0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pomalidomide</td>
			<td>Selleckchem</td>
			<td>S1567</td>
			<td></td>
		</tr>
		<tr>
			<td>RestoreTM Western Blot Stripping Buffer</td>
			<td>Thermo Scientific</td>
			<td>46430</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate</td>
			<td>Carl Roth</td>
			<td>183.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9539-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>tert-Butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate</td>
			<td>Sigma-Aldrich</td>
			<td>89761</td>
			<td>Compound 5</td>
		</tr>
		<tr>
			<td>Tricin</td>
			<td>Carl Roth</td>
			<td>6977.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P7949-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>WesternBright ECL spray</td>
			<td>Advansta</td>
			<td>K-12049-D50</td>
			<td></td>
		</tr>
	</tbody>
</table>

chemical inactivation of the e3 ubiquitin ligase cereblon by pomalidomide-based homo-protacs

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN&#8216;s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

Here we described the design, synthesis and biological evaluation of a homodimeric pomalidomide-based PROTAC for the degradation of CRBN. Our PROTAC interacts simultaneously with two CRBN molecules and forms ternary complexes that induces self-ubiquitination and proteasomal degradation of CRBN with only minimal remaining effects on pomalidomide-induced neo-substrates IKZF1 or IKZF3.
Out of a series of previously published pomali...

Watch this Scientific Journal Video about Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs at JoVE.com

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple ...

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