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

* These authors contributed equally
Chemistry

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Summary

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.

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Lindner, S., Steinebach, C., Kehm, H., Mangold, M., Gütschow, M., Krönke, J. Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs. J. Vis. Exp. (147), e59472, doi:10.3791/59472 (2019).

Abstract

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‘s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

Introduction

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 1α (CK1α) 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.

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Protocol

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.

  1. Preparation of tert-butyl N-(2,6-dioxo-3-piperidyl)carbamate (compound 1)
    1. Add 1,1'-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.
    2. 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.
    3. 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.
    4. Evaporate the solvent and dry the obtained colorless solid in vacuo.
  2. Preparation of tert-butyl N-(1-methyl-2,6-dioxo-3-piperidyl)carbamate (compound 2)
    1. 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.
    2. Dilute the 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.
    3. 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).
  3. Preparation of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (compound 3)
    1. 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 °C for 6 h.
    2. 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 × 5 mL) and petroleum ether (3 × 5 mL) and dry in vacuo.
  4. Preparation of 4-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (compound 4)
    1. 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 °C for 6 h.
    2. 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 × 5 mL) and petroleum ether (3 × 5 mL) and dry in vacuo.
  5. 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)
    1. 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 °C for 10 h.
    2. 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.
    3. 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).
  6. Preparation of homodimer (compound 8)
    1. Combine the α,ω-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 °C for 18 h.
    2. 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.
    3. 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.
  7. Preparation of heterodimer (compound 9)
    1. 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 °C for 2 h in a closed 50 mL round bottom flask.
    2. Remove the volatiles and coevaporate with CH2Cl2 (4x 5 mL). Dry the residue in vacuo for 10 h.
    3. 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 °C for 10 h.
    4. 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.
    5. 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.
    6. 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 220–500 nm.

2. Functional validation of PROTAC molecules

  1. 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).
    1. Sample preparation
      1. 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 °C until further usage.
      2. Seed 1 x 106 MM1S cells in a 6-well plate with 2.5 mL media and treat cells with 100 nM or 1 µM compound 8 or 9 for 24 h.
      3. Harvest cells after treatment and centrifuge at 700 x g, 5 min, 4 °C. Wash cell pellet with cold 1x PBS to remove remaining media, centrifuge at 700 x g, 5 min, 4 °C, and discard supernatant. Repeat this step once.
      4. 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 & Phosphatase Inhibitor Cocktail) for 10 min on ice, centrifuge at 320 x g for 10 min, 4 °C. Harvest supernatant and determine protein concentration by a bicinchoninic acid protein assay (BCA assay) according to the manufacturer’s protocol.
      5. Denature proteins (15–30 µg/sample) with 1x LDS loading buffer (5% 2-mercaptoethanol) and boil 10 min, 75 °C.
    2. SDS-PAGE
      1. 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 µL 11% ammonium persulfate (APS), and 9.75 µL TEMED] and a 4% stacking gel [1.992 mL 3x gel buffer, 0.792 mL 30% acrylamide, 3.168 mL H2O, 36 µL 11% APS, and 6 µL 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.
      2. 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.
    3. Immunoblotting and detection of CRBN, IKZF1 and IKZF3
      1. Activate PVDF membrane (0.45 µm) 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].
      2. Assemble blotting cassette according to manufacturer’s protocol. Transfer gel at 180 mA for 90 min.
      3. Wash membrane 3x in 1x TBS-T (25 mM Tris/HCl, 150 mM NaCl, pH 7.6, 0.1% Tween 20) for 5–10 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 5–10 min each at room temperature.
      4. Incubate membrane with primary antibody for CRBN (1:500 in 5% BSA, TBS-T) with gentle shaking at 4 °C, overnight.
      5. Wash membrane 3x in 1X TBS-T for 5–10 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.)
      6. Wash membrane 2x in 1X TBS-T for 5–10 min each at room temperature. Repeat this step twice with 1x TBS.
      7. Incubate membrane for 2 min with HRP substrate solution according to manufacturer’s protocols and detect chemiluminescence in a chemiluminescence detection device.
      8. Wash membrane 1x in 1X TBS for 5–10 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 5–10 min each at room temperature.
      9. Reblock membrane in 5% nonfat-dried milk, TBS-T for 1 h at room temperature. Wash membrane 3x in 1x TBS-T for 5–10 min each at room temperature and reprobe with IKZF1, IKZF3 or tubulin according to step 2.1.3.4.
  2. 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).
    1. Seed 1 x 106 MM1S cells per well in a 6-well plate. Pretreat cells with 10 µM MG132, 10 µM MLN4942, or lenalidomide (100x), and incubate 1 h at 37 °C, 5% CO2.
    2. Add 100 nM compound 8 for 3 h at 37 °C, 5% CO2.
    3. Harvest cells for western blot according to step 2.1.1.
  3. 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).
    1. 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.
    2. Treat cells with DMSO or 100 nM, 1 µM, or 10 µM compound 8, compound 9 or pomalidomide and incubate for 24 h, 48 h, or 96 h at 37 °C, 5% CO2. For rescue experiments, treat cells with 100 nM compound 8 for 3 h, before or after addition of 1 µM pomalidomide and incubate for 96 h.
    3. Measure 96-well plate luminescence with a luminescent cell viability assay, according to the manufacturer’s protocol on a plate reader or harvest cells from the 6-well plate for western blot analysis.

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Representative Results

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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Synthesis of homodimer 8 and heterodimer 9. Please click here to view a larger version of this figure.

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). Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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 <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. Please click here to view a larger version of this figure.

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Discussion

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 CK1α6,11,29. 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 TNFα 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.

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Disclosures

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

Acknowledgments

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.)

Materials

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

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References

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