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-(dimethylamino)pyridine (5 mg) to a mixture of Boc-Gln-OH (2.46 g, 10 mmol) in THF (50 mL) in 100 mL round bottom flask with a stir bar and equipped with a reflux condenser. Heat at reflux for 10 h while stirring until a clear solution is formed.</li>
		<li>Remove the solvent under reduced pressure with a rotary evaporator, add EtOAc (200 mL) and transfer it to a separatory funnel. Wash the organic layer with H2O (50 mL) and brine (50 mL) and dry it over Na2SO4.</li>
		<li>Filter the solution through a short pad of silica gel (5 cm diameter and 5 cm height) and eluate with a further volume (200 mL) of EtOAc.</li>
		<li>Evaporate the solvent and dry the obtained colorless solid in vacuo.</li>
	</ol>
	</li>
	<li>Preparation of tert-butyl N-(1-methyl-2,6-dioxo-3-piperidyl)carbamate (compound 2)
	<ol>
		<li>Combine compound 1 (2.28 g, 10 mmol) with milled potassium carbonate (2.76 g, 20 mmol) and DMF (25 mL) in a 100 mL round bottom flask. Add iodomethane (1.42 g, 0.62 mL, 10 mmol) drop-wise using a syringe and equip the flask with a punctured rubber septum. Place the reaction vessel into an ultrasonication bath for 2 h.</li>
		<li>Dilute the&#160;reaction mixture with EtOAc (100 mL) and transfer it to a separatory funnel. Wash the organic layer with 1 N NaOH (2x 25 mL), H2O (25 mL), and brine (25 mL), and dry it over Na2SO4.</li>
		<li>Filter and evaporate the solvent. Purify the product by column chromatography over silica gel (6 cm column diameter and 20 cm height) using petroleum ether/EtOAc (2:1).</li>
	</ol>
	</li>
	<li>Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (compound 3)
	<ol>
		<li>Combine 3-fluorophthalic anhydride (1.25 g, 7.5 mmol), glutarimide 1 (1.14 g, 5 mmol) and a solution of sodium acetate (0.50 g, 6.0 mmol) in glacial acetic acid (20 mL) in a 50 mL round bottom flask with a stir bar and equipped with a reflux condenser. Heat the mixture at 120 &#176;C for 6 h.</li>
		<li>After cooling, pour the purple mixture onto H2O (100 mL) and stir for 10 min. Collect the formed solid by filtration, wash with H2O (3 &#215; 5 mL) and petroleum ether (3 &#215; 5 mL) and dry in vacuo.</li>
	</ol>
	</li>
	<li>Preparation of 4-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 4)
	<ol>
		<li>Combine 3-fluorophthalic anhydride (1.25 g, 7.5 mmol), glutarimide 2 (1.21 g, 5 mmol) and a solution of sodium acetate (0.50 g, 6.0 mmol) in glacial acetic acid (20 mL) in a 100 mL round bottom flask with a stir bar and equipped with a reflux condenser. Heat the mixture at 120 &#176;C for 6 h.</li>
		<li>After cooling, pour the purple mixture onto H2O (100 mL) and stir for 10 min. Collect the formed solid by filtration, wash with H2O (3 &#215; 5 mL) and petroleum ether (3 &#215; 5 mL) and dry in vacuo.</li>
	</ol>
	</li>
	<li>Preparation of tert-butyl N-[2-[2-[2-[[2-(2,6-dioxo-3-piperidyl)-1,3-dioxo-isoindolin-4-yl]amino]ethoxy]ethoxy]ethyl]carbamate (compound 7)
	<ol>
		<li>Charge a 50 mL round bottom flask with tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (5, 0.41 g, 1.65 mmol), compound 3 (0.41 g, 1.50 mmol), dry DMF (10 mL) and DIPEA (0.39 g, 0.51 mL, 3.0 mmol). Equip with a stir bar and a reflux condenser. Heat under argon atmosphere at 90 &#176;C for 10 h.</li>
		<li>After cooling to room temperature, pour the dark green mixture onto H2O (100 mL) and extract with EtOAc (3x 50 mL) in a separatory funnel. Wash the combined organic layers with H2O (50 mL) and brine (50 mL), dry over Na2SO4, filter, and concentrate in vacuo.</li>
		<li>Purify the crude product by column chromatography over silica gel (3 cm column diameter and 60 cm height) using a gradient of petroleum ether/EtOAc (1:1 to 1:2).</li>
	</ol>
	</li>
	<li>Preparation of homodimer (compound 8)
	<ol>
		<li>Combine the &#945;,&#969;-diamine linker 6 (0.22 g, 0.22 mL, 1.50 mmol), DIPEA (1.05 mL, 6.00 mmol) and a solution of 3 (0.83 g, 3.00 mmol) in dry DMSO (20 mL) in a 50 mL round bottom flask with a stir bar and equipped with a reflux condenser. Heat under argon atmosphere at 90 &#176;C for 18 h.</li>
		<li>After cooling to room temperature, pour the dark green mixture onto H2O (100 mL) and extract with EtOAc (3x 50 mL) in a separatory funnel. Wash the combined organic layers with H2O (50 mL) and brine (50 mL), dry over Na2SO4, filter and concentrate in vacuo.</li>
		<li>Purify the crude product by column chromatography over silica gel (3 cm column diameter and 50 cm height) using a gradient of petroleum ether/EtOAc (1:2) to EtOAc.</li>
	</ol>
	</li>
	<li>Preparation of heterodimer (compound 9)
	<ol>
		<li>Dissolve compound 7 (0.83 g, 1.65 mmol) in dry CH2Cl2 (10 mL). Add trifluoroacetic acid (10 mL) and stir the yellow mixture at 40 &#176;C for 2 h in a closed 50 mL round bottom flask.</li>
		<li>Remove the volatiles and coevaporate with CH2Cl2 (4x 5 mL). Dry the residue in vacuo for 10 h.</li>
		<li>Redissolve the material in dry DMF (20 mL). Add compound 4 (0.44 g, 1.50 mmol) and DIPEA (0.78 g, 1.05 mL, 6.00 mmol) and equip the flask with a reflux condenser. Heat under argon atmosphere at 90 &#176;C for 10 h.</li>
		<li>After cooling to room temperature, pour the dark green mixture onto H2O (100 mL) and extract with EtOAc (3x 50 mL) in a separatory funnel. Wash the combined organic layers with saturated NaHCO3 (50 mL), H2O (50 mL), 10% KHSO4 (50 mL), H2O (50 mL), and brine (50 mL), dry over Na2SO4, filter and concentrate in vacuo.</li>
		<li>Purify the crude product by column chromatography over silica gel (3 cm column diameter and 50 cm height) using a gradient of petroleum ether/EtOAc (1:2) to EtOAc.</li>
		<li>Elucidate and verify molecule structure (Figure 5A compound 8, 5B compound 9) by 1H NMR and 13C NMR spectra in DMSO-d6 on a nuclear magnetic resonance (NMR) spectrometer. Check that the purity of both compounds is higher than 97% by means of liquid chromatography-mass spectrometry (LC-MS), applying a diode array detection (DAD) at 220&#8211;500 nm.</li>
	</ol>
	</li>
</ol>
2. Functional validation of PROTAC molecules
<ol>
	<li>Western blot analysis of CRBN degradation by PROTACs 
	NOTE: The effects of compound 8 and compound 9 on CRBN protein level were tested by western blot analysis. In addition, the impact on IKZF1 and IKZF3 levels could also be confirmed (Figure 6).
	<ol>
		<li>Sample preparation
		<ol>
			<li>Dissolve compounds 8 and 9, lenalidomide (Len), pomalidomide (Pom), MG132, and MLN-4924 in DMSO at a concentration of 10 mM, aliquot and store at -80 &#176;C until further usage.</li>
			<li>Seed 1 x 106 MM1S cells in a 6-well plate with 2.5 mL media and treat cells with 100 nM or 1 &#181;M compound 8 or 9 for 24 h.</li>
			<li>Harvest cells after treatment and centrifuge at 700 x g, 5 min, 4 &#176;C. Wash cell pellet with cold 1x PBS to remove remaining media, centrifuge at 700 x g, 5 min, 4 &#176;C, and discard supernatant. Repeat this step once.</li>
			<li>Lyse cells in lysis buffer (25 mM Tris HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol, 1x Protease &#38; Phosphatase Inhibitor Cocktail) for 10 min on ice, centrifuge at 320 x g for 10 min, 4 &#176;C. Harvest supernatant and determine protein concentration by a bicinchoninic acid protein assay (BCA assay) according to the manufacturer&#8217;s protocol.</li>
			<li>Denature proteins (15&#8211;30 &#181;g/sample) with 1x LDS loading buffer (5% 2-mercaptoethanol) and boil 10 min, 75 &#176;C.</li>
		</ol>
		</li>
		<li>SDS-PAGE
		<ol>
			<li>Fix gel sandwich with a 10% separating gel [4 mL 3x gel buffer (3 M Tris/HCl, 0.3% (w/v) sodium dodecyl sulfate (SDS), pH 8.45), 4 mL acrylamide 30%, 2.52 mL glycerol 50%, 1.395 mL H2O, 75 &#181;L 11% ammonium persulfate (APS), and 9.75 &#181;L TEMED] and a 4% stacking gel [1.992 mL 3x gel buffer, 0.792 mL 30% acrylamide, 3.168 mL H2O, 36 &#181;L 11% APS, and 6 &#181;L TEMED] in an electrode assembly unit. Remove combs, flush wells with cathode buffer (100 mM Tris/HCl, 100 mM tricine, 0.1% (w/v) SDS), and load samples.</li>
			<li>Fill anode buffer (100 mM Tris/HCl, pH 8.9) into the tank. Load protein sample from step 2.1.1.4 and run SDS-PAGE at 70 V, 20 min, followed by 115 V, 150 min at constant voltage.</li>
		</ol>
		</li>
		<li>Immunoblotting and detection of CRBN, IKZF1 and IKZF3
		<ol>
			<li>Activate PVDF membrane (0.45 &#181;m) in 100% methanol for 1 min. Equilibrate membrane and separating gel in 1x transfer buffer [10x transfer buffer (192 mM glycine, 25 mM Tris-base/HCl, 900 mL H2O), 20% methanol, 0.1% SDS, pH 8.3].</li>
			<li>Assemble blotting cassette according to manufacturer&#8217;s protocol. Transfer gel at 180 mA for 90 min.</li>
			<li>Wash membrane 3x in 1x TBS-T (25 mM Tris/HCl, 150 mM NaCl, pH 7.6, 0.1% Tween 20) for 5&#8211;10 min each at room temperature. Block membrane in 5% nonfat-dried milk (NFDM), TBS-T for 1 h at room temperature. Wash membrane 3x in 1X TBS-T for 5&#8211;10 min each at room temperature.</li>
			<li>Incubate membrane with primary antibody for CRBN (1:500 in 5% BSA, TBS-T) with gentle shaking at 4 &#176;C, overnight.</li>
			<li>Wash membrane 3x in 1X TBS-T for 5&#8211;10 min each at room temperature. Incubate membrane with anti-mouse (1:10.000 in 5% NFDM, TBS-T) or anti-rabbit (1:5.000 in 5% NFDM, TBS-T) secondary antibody coupled to horseradish peroxidase HRP (1 h at room temperature.)</li>
			<li>Wash membrane 2x in 1X TBS-T for 5&#8211;10 min each at room temperature. Repeat this step twice with 1x TBS.</li>
			<li>Incubate membrane for 2 min with HRP substrate solution according to manufacturer&#8217;s protocols and detect chemiluminescence in a chemiluminescence detection device.</li>
			<li>Wash membrane 1x in 1X TBS for 5&#8211;10 min each at room temperature. For release of antibodies, strip membrane in commercially available stripping buffer for 15 min. Wash membrane 3x in 1X TBS for 5&#8211;10 min each at room temperature.</li>
			<li>Reblock membrane in 5% nonfat-dried milk, TBS-T for 1 h at room temperature. Wash membrane 3x in 1x TBS-T for 5&#8211;10 min each at room temperature and reprobe with IKZF1, IKZF3 or tubulin according to step 2.1.3.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Competition experiments with MG132, MLN4942 or pomalidomide 
	NOTE: To confirm whether CRBN is degraded via the ubiquitin-proteasome pathway, we performed competition experiments with the proteasome inhibitor MG132 and a neddylation activating enzyme (NAE) inhibitor MLN4942 (Figure 7).
	<ol>
		<li>Seed 1 x 106 MM1S cells per well in a 6-well plate. Pretreat cells with 10 &#181;M MG132, 10 &#181;M MLN4942, or lenalidomide (100x), and incubate 1 h at 37 &#176;C, 5% CO2.</li>
		<li>Add 100 nM compound 8 for 3 h at 37 &#176;C, 5% CO2.</li>
		<li>Harvest cells for western blot according to step 2.1.1.</li>
	</ol>
	</li>
	<li>Cell viability assays in multiple myeloma cell lines 
	NOTE: This assay is used to test the impact on cell viability and additionally, antagonize the effect of IMiDs on multiple myeloma cells by pretreatment of the cells with compound 8 (Figure 8, Figure 9A,B).
	<ol>
		<li>Seed 5 x 104 MM1S cells per well in a 96-well plate in biological triplicates for viability assay. For western blot analysis, seed 1 x 106 MM1S cells per well in a 6-well plate in biological triplicates.</li>
		<li>Treat cells with DMSO or 100 nM, 1 &#181;M, or 10 &#181;M compound 8, compound 9 or pomalidomide and incubate for 24 h, 48 h, or 96 h at 37 &#176;C, 5% CO2. For rescue experiments, treat cells with 100 nM compound 8 for 3 h, before or after addition of 1 &#181;M pomalidomide and incubate for 96 h.</li>
		<li>Measure 96-well plate luminescence with a luminescent cell viability assay, according to the manufacturer&#8217;s protocol on a plate reader or harvest cells from the 6-well plate for western blot analysis.</li>
	</ol>
	</li>
</ol>

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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. 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=Cereblon+in+health+and+disease.">Cereblon in health and disease.</a> Pfl&#252;gers Archiv: European Journal of Physiology. 468 (8), 1299-1309 (2016).</li><li>Lee, 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=Disruption+of+the+cereblon+gene+enhances+hepatic+AMPK+activity+and+prevents+high-fat+diet-induced+obesity+and+insulin+resistance+in+mice.">Disruption of the cereblon gene enhances hepatic AMPK activity and prevents high-fat diet-induced obesity and insulin resistance in mice.</a> Diabetes. 62 (6), 1855-1864 (2013).</li></ol>

The authors do not declare a potential financial conflict of interest.

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 chemically induced knockdown is reversible, rapid and directly applicable to a wide spectrum of cells and tissue types28.
The IMiDs thalidomide, lenalidomide, and pomalidomide have become a mainstay in the treatment of multiple myeloma, B-cell lymphomas and myelodysplastic syndrome. IMiDs mediate their activity by modulating the specificity of the CRBN-CRL4 E3 ligase to degrade the neo-substrates IKZF1, IKZF3, or CK1&#945;6,11,29. In addition, IMiDs have been shown to abrogate the chaperone function of CRBN on two other proteins, MCT-1 and BSG, that are also important for multiple myeloma growth30. The degradation of CRBN by the homo-bifunctional PROTAC was well tolerated by most multiple myeloma cell lines tested, implying that CRBN inactivation alone is not sufficient to cause killing of multiple myeloma cells. In contrast, pre-treatment with compound 8 abrogated the effects of IMiDs on IKZF1/3 degradation and rescued multiple myeloma cells from lenalidomide and pomalidomide. This is in line with genetic inactivation of CRBN and deleterious CRBN mutations found in lenalidomide-resistant multiple myeloma patients and highlights the essential role of CRBN in the mechanism of IMiDs31,32. The homo-PROTAC 8 can therefore be a useful tool to mimic a state of IMiD resistance. Other effects of IMiDs that are not fully understood yet like inhibition of angiogenicity or TNF&#945; release may derive from an inhibition of CRBN function and our homo-PROTAC are suitable tools to investigate the inactivation of CRBN further. In addition, the chemically induced knockdown of CRBN by compound 8 may help to identify new endogenous substrates of CRBN and elucidate the physiological functions of CRBN. Given that our compound 8 had no effects on cancer cell line proliferation, CRBN inhibition alone has no anti-tumor activity. However, CRBN degraders may be clinically applicable in diseases other than cancer. In this regard, CRBN inactivation was recently shown to confer resistance to sepsis and to prevent high-fat-diet-induced obesity in mice 33,34,35.
In conclusion, we generated and validated the first chemical inhibitor of CRBN that can serve as a useful tool for future biomedical investigations on CRBN-related signaling and molecular mechanism of thalidomide and its analogs.

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&#039;-Carbonyldiimidazole</td><td>TCI chemicals</td><td>C0119</td><td></td></tr><tr><td>2,2&amp;prime;-(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&amp;ouml;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 &amp;amp; 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&amp;micro;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-&amp;alpha;-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 pomalidomide-based PROTAC molecules24, compound 8 was particularly efficient in the chemical-induced degradation of CRBN. Its synthesis can be accomplished as follows (Figure 4). A 1,1'-carbonyldiimidazole-promoted condensation of Boc-protected l-glutamine leads to the cyclized imide 1. The N-methylated analog 2 is accessible through alkylation with methyl iodide. Both building blocks (1 and 2) are transformed, after N-deprotection under acidic conditions, to phthalimide derivatives (3 and 4) in the course of a ring-opening/recyclization reaction using 3-fluorophthalic anhydride. Thalidomide analogs, in general, are susceptible to hydrolytic decomposition and should only be used in the next step after sufficient drying. Compound 3 is susceptible to an aromatic nucleophilic substitution with primary aliphatic amines25; this conversion was found to proceed efficiently only when dry solvents are used. The design of a true homodimeric product implies the linker connection of two identical functional substructures and the application of symmetrical linker. The linker which is part of PROTAC 8 represents an N-to-N, polyethylene-based linear chain. The corresponding α,ω-diamine 6 leads to the desired final compound 8 when reacted with building block 3 in molar ration of 1:2 in DMSO at 90 °C. Among other analytical data24, the structure of 8 was verified by NMR spectra (Figure 5A). Compound 9, designed as a suitable negative control, has an only minimal, but critical structural deviations, compared to the active homo-PROTAC 8. It is known that N-methylation within the glutarimide portion abolishes CRBN binding26,27. One pomalidomide portion of the negative control compound 9 bears an N-methyl residue. It can be prepared by a first nucleophilic substitution of 3 with the N-monoprotected linker building block 5, followed by cleavage of the Boc protecting group and a subsequent coupling to intermediate 4. Owing to its asymmetrical structure, some of the corresponding carbons showed distinct 13C NMR signals (Figure 5B).
The homo-PROTAC 8 was observed to be highly potent, leading to an almost complete proteasomal degradation of CRBN. The interpretation of CRBN, IKZF1 and IKZF3 protein levels in multiple myeloma cells were confirmed by western blot analysis (Figure 6, Figure 7, Figure 9B), a semi-quantitative standard method, where the change in protein expression can be detected easily. The antibodies used in this paper are of good quality and the method is an optimized standard procedure in our lab.
In addition, degradation of CRBN by compound 8 did not affect cell viability and conferred resistance to IMiDs (Figure 8, Figure 9A), which is in line with CRISPR/Cas9-mediated knockout of CRBN by sgRNAs24. The luminescence signal in the cell viability assay was based on ATP release, which can be interpreted as dead cell count. This method can be easily performed in a short time with a high number of samples. An alternative method for the measurement of viable/dead cells is an Annexin V/ 7-AAD staining by flow cytometry.
<img alt="Thalidomide mechanism diagram; CRBN ligase triggers ubiquitin-induced degradation; multiple myeloma." class="xfigimg" src="/files/ftp_upload/59472/59472fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: The E3 ubiquitin ligase CRBN is the main target of IMiDs. Immunomodulatory drugs bind to CRBN and recruit several neo-substrates for proteasomal degradation. IMiD-induced degradation of the lymphoid transcription factors IKZF1 and IKZF3 is responsible for the effects on multiple myeloma cells and some of the immunomodulatory properties. Casein kinase 1α is selectively degraded by lenalidomide but not the other IMiDs and contributes to the activity of lenalidomide in myelodysplastic syndrome with loss of chromosome 5q. SALL4 was recently discovered as a common target of all IMiDs that is likely linked to the teratogenicity induced by thalidomide and its analogs. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="PROTAC mechanism diagram; POI, linker, IMiD bind CRBN, leading to proteasomal degradation process." class="xfigimg" src="/files/ftp_upload/59472/59472fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: PROTACs degrade the protein of interest (POI). PROTACs are heterobifunctional molecules, where a linker connects a ubiquitin ligase ligand to a POI ligand. By the formation of ternary complexes, a ubiquitin ligase, such as CRBN, then ubiquitinates the POI, resulting in its proteasomal degradation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chemically induced CRBN knockdown; proteasomal degradation process; molecular diagram." class="xfigimg" src="/files/ftp_upload/59472/59472fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Bifunctional homo-PROTAC for the degradation of the E3 ubiquitin ligase CRBN. In a pomalidomide-based homo-PROTAC, two ubiquitin ligase binders are connected to induce cross-ubiquitination of CRBN resulting in a chemically induced knockdown of CRBN. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Chemical synthesis diagram; fluorophthalic reactions; chemical structures, synthesis steps, reagents." class="xfigimg" src="/files/ftp_upload/59472/59472fig4.jpg" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4: Synthesis of homodimer 8 and heterodimer 9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="NMR spectra diagrams; chemical shift analysis for structural elucidation, spectrum A and B comparison." class="xfigimg" src="/files/ftp_upload/59472/59472fig5.jpg" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5: 1H NMR (top) and 13C NMR (bottom) spectra. Spectra of compound 8 (A) and compound 9 (B) were recorded in DMSO-d6 on an NMR spectrometer. Chemical shifts are given in parts per million (ppm). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Western blot results; CRBN, IKZF1, IKZF3 analysis under DMSO, Pom, compounds; protein expression." class="xfigimg" src="/files/ftp_upload/59472/59472fig6.jpg" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6: Effects of compounds 8 and 9 on CRBN, IKZF1, and IKZF3. Pomalidomide-based homo-PROTAC compound 8 induces CRBN degradation with weak remaining effects of pomalidomide on IKZF1 and IKZF3. In contrast, compound 9 that contains a methyl group on one of the pomalidomide residues has no effect at the indicated concentrations (µM). MM1S cells were treated for 24 h treatment. Effects on CRBN, IKZF1, IKZF3 and tubulin (loading control) were analyzed by western blot. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Western blot results; protein expression analysis; CRBN, IKZF1, IKZF3, Tubulin; drug treatments." class="xfigimg" src="/files/ftp_upload/59472/59472fig7.jpg" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7: CRBN degradation can be blocked by the proteasome inhibitor MG132 or by MLN4942 that blocks ubiquitin ligases indirectly via neddylation inhibition. The multiple myeloma cell line MM1s was pretreated with 10 µM MG132, 10 µM MLN4924 for 1 h before addition of homo-PROTAC compound 8 at 100 nM for 3 h of combined treatment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability bar chart for compounds, 24-96h DMSO comparison, μM concentration, cancer research." class="xfigimg" src="/files/ftp_upload/59472/59472fig8.jpg" data-altupdatedat="1747911167000" data-alt="Figure 8"> 
Figure 8: Cell viability assay in MM1S multiple myeloma cells. Effects of compound 8 and negative binding control compound 9 on cell viability in the pomalidomide sensitive myeloma cell line MM1S after 24 h, 48 h and 96 h treatment. Cell viability was measured after 4 days in triplicates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell viability analysis; bar graph and Western blot; drug treatment effects on protein expression." class="xfigimg" src="/files/ftp_upload/59472/59472fig9.jpg" data-altupdatedat="1747911167000" data-alt="Figure 9"> 
Figure 9: Compound 8 antagonizes the effect of pomalidomide in multiple myeloma cell lines. Cells were pretreated with 100 nM compound 8 for 3 h. Afterwards 1 µM pomalidomide was added. Cell viability was measured after 4 days in triplicates. *** p &lt;0.001 according to Student´s t-test (A). Western blot analysis for CRBN, IKZF1, IKZF3 and tubulin (loading control) after pretreatment of MM1S cells with 100 nM compound 8 for 3 h, before addition of 1 µM pomalidomide (B). Reprinted (adapted) with permission from Steinebach, C. et al. 201824. Copyright 2019 American Chemical Society. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/59472/59472fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

chemical-inactivation-e3-ubiquitin-ligase-cereblon-pomalidomide-based

Nanyang Technological University

Research

JoVE Journal

Chemistry

13.1K Views.  University Hospital Ulm. Onze homodimeric PROTACs zijn de eerste chemische remmers van Cereblon en kunnen helpen om het moleculaire mechanisme van thalidomide analogen verder te verduidelijken, evenals om de fysiologische functie van Cereblon te onderzoeken. Deze nieuwe PROTAC-platformtechnologie kan zeer nuttig zijn voor het specifiek targeten van proteïnen die anders als ontraceerbaar worden beschouwd. CRBN remming alleen heeft geen anti-tumor activiteit.CRBN-degraders kunnen echter klinische implicaties he...

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

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

Cancer Research

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Medicine

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Biochemistry

Ubiquitin Chain Analysis by Parallel Reaction Monitoring

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The protocol outlined here takes advantage of the di-glycine (-GG) modification left after the tryptic digestion of ubiquitin incorporated in a chain. By quantifying these topology-characteristic peptides the relative abundance of each ubiquitin chain topology can be determined. The steps required to quantify these peptides by a parallel reaction monitoring experiment are reported taking into consideration the stabilization of ubiquitin chains. Preparation of heavy controls, cell lysis, and digestion are described along with the appropriate mass spectrometer setup and data analysis workflow. An example data set with perturbations in ubiquitin topology is presented, accompanied by examples of how optimization of the protocol can affect results. By following the steps outlined, a user will be able to perform a global assessment of the ubiquitin topology landscape within their biological context.

This method describes the assessment of global changes in ubiquitin chain topology. The assessment is performed by the application of a mass spectrometry-based targeted proteomics approach.

Assessment of the global profile of ubiquitin chain topologies within a proteome is of interest to answer a wide range of biological questions. The ...

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, including cell survival, proliferation, and differentiation. It is a reversible process that consists of a covalent attachment of ubiquitin to the substrate through a cascade reaction of at least three different enzymes, composed of E1 (Ubiquitin-activation enzyme), E2 (Ubiquitin-conjugating enzyme), and E3 (Ubiquitin-ligase enzyme). The E3 complex plays an important role in substrate recognition and ubiquitylation. Here, a protocol is described to evaluate substrate ubiquitylation in mammalian cells using transient co-transfection of a plasmid encoding the selected substrate, an E3 ubiquitin ligase, and a tagged ubiquitin. Before lysis, the transfected cells are treated with the proteasome inhibitor MG132 (carbobenzoxy-leu-leu-leucinal) to avoid substrate proteasomal degradation. Furthermore, the cell extract is submitted to small-scale immunoprecipitation (IP) to purify the polyubiquitylated substrate for subsequent detection by western blotting (WB) using specific antibodies for ubiquitin tag. Hence, a consistent and uncomplicated protocol for ubiquitylation assay in mammalian cells is described to assist scientists in addressing ubiquitylation of specific substrates and E3 ubiquitin ligases.

We provide a detailed protocol for a ubiquitylation assay of a specific substrate and an E3 ubiquitin-ligase in mammalian cells. HEK293T cell lines were used for protein overexpression, the polyubiquitylated substrate was purified from cell lysates by immunoprecipitation, and resolved in SDS-PAGE. Immunoblotting was used to visualize this post-translational modification.

Ubiquitylation is a post-translational modification which occurs in eukaryotic cells that is critical for several biological pathways' regulation, ...

Purification of Ubiquitinated p53 Proteins from Mammalian Cells

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate protein. The ubiquitination process occurs intracellularly in eukaryotes and regulates almost all basic cellular biological processes. Purification of ubiquitinated proteins aids the investigation of the role of ubiquitination in controlling the function of substrate proteins. Here, a step-by-step procedure to purify ubiquitinated proteins in mammalian cells is described with the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified under stringent nondenaturing and denaturing conditions. Total cellular Flag-tagged p53 protein was purified with anti-Flag antibody-conjugated agarose under nondenaturing conditions. Alternatively, total cellular His-tagged ubiquitinated protein was purified using nickel-charged resin under denaturing conditions. Ubiquitinated p53 proteins in the eluates were successfully detected with specific antibodies. Using this procedure, the ubiquitinated forms of a given protein can be efficiently purified from mammalian cells, facilitating studies on the roles of ubiquitination in regulating protein function.

The protocol describes a step-by-step method to purify ubiquitinated proteins from mammalian cells using the p53 tumor suppressor protein as an example. Ubiquitinated p53 proteins were purified from cells under stringent nondenaturing and denaturing conditions.

Ubiquitination is a type of posttranslational modification that regulates not only the stability but also the localization and function of a substrate ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Characterization of Ternary Complex Formation in PROTACs Using Biophysical Assays

The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the &#39;hook effect&#39; on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4BD2 protein (SPR KD = 1 nM, ITC KD = 4 nM) and the VHL complex (SPR KD = 29 nM, ITC KD = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR KD = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR KD= ~ 3 &#181;M). In that case, the binding for CRBN in SPR was not saturable, resulting in a &#34;hook-effect&#34;. Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Author Spotlight: Evaluating Biophysical Assays for Characterizing PROTACS Ternary Complexes 

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Immunology and Infection

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

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

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