Autorzy
Skontaktuj Się z Nami

Zaloguj się

Aby wyświetlić tę treść, wymagana jest subskrypcja JoVE. Zaloguj się lub rozpocznij bezpłatny okres próbny.

W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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...

Dyskusje

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...

Ujawnienia

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

Podziękowania

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.)

Materiały

NameCompanyCatalog NumberComments
1,1'-CarbonyldiimidazoleTCI chemicalsC0119
2,2′-(Ethylenedioxy)-bis(ethylamine)Sigma-Aldrich385506Compound 6
2-MercaptoethanolSigma-AldrichM6250
3-Fluorophthalic anhydride, 98 %Alfa AesarA12275
4-Dimethylaminopyridine, 99 %Acros148270250Toxic
Acrylamidstammlösung/ Bisacrylamid (30%/0,8%)Carl Roth3029.1
Aiolos (D1C1E) mABCell signaling15103S
Anti-CRBN antibody produced in rabbitSigmaHPA045910
Anti-rabbit IgG HRP-linked antibodySigma7074S
Ammonium PersulfateRoth9592.2
Boc-Gln-OHTCI chemicalsB1649
Bovine Serum AlbuminSigma-AldrichA7906-100G
CellTiter-Glo Luminescent Cell Viability AssayPromegaG7571
ChemiDoc XRS+Bio-Rad1708265
DMF, anhydrous, 99.8 %Acros348435000Extra Dry over Molecular Sieve
DMSO, anhydrous, 99.7 %Acros348445000Extra Dry over Molecular Sieve
GlycineSigma-Aldrich15523-1L-R
Goat anti-mouse (HRP conjugated)Santa Cruz biotechnologysc-2005
Halt Protease & Phosphatase Inhibitor Single-use Cocktail (100X)Thermo Scientific1861280
Ikaros (D6N9Y) MabCell signaling14859S
ImmobilonP Transfer Membrane (0,45µm)MerckIPVH000010
Iodomethane, 99 %Sigma-AldrichI8507Highly toxic
MethanolSigma-Aldrich32213-2.5L
Mg132SelleckchemS2619
Mini Trans-Blot electrophoretic transfer cellBio-Rad1703930
Mini-PROTEAN Tetra Vertical Electrophoresis CellBio-Rad1658004
MLN4942biomol (cayman)Cay15217-1
Monoclonal Anti-α-Tubulin antibody produced in mouse (B512)SigmaT5168
N-Ethyldiisopropylamine, 99 %Alfa AesarA11801
Nonfat dried milk powderPanReac AppliChemA0830,0500
Nunc F96 MicroWell White Polystyrene PlateThermo Scientific136101
NuPAGE LDS Sample Buffer (4X)Thermo ScientificNP0008
Pierce BCA Protein Assay kitThermo Scientific23225
PomalidomideSelleckchemS1567
RestoreTM Western Blot Stripping BufferThermo Scientific46430
sodium dodecyl sulfateCarl Roth183.1
Sodium ChlorideSigma-AldrichA9539-500g
TEMEDCarl Roth2367.3
tert-Butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamateSigma-Aldrich89761Compound 5
TricinCarl Roth6977.4
Trizma baseSigma-AldrichT1503-1kg
Tween-20Sigma-AldrichP7949-500ml
WesternBright ECL sprayAdvanstaK-12049-D50

Odniesienia

  1. Ito, T., et al. Identification of a primary target of thalidomide teratogenicity. Science. 327 (5971), 1345-1350 (2010).
  2. Lopez-Girona, A., et al. Cereblon is a direct protein target for immunomodulatory and antiproliferative activities of lenalidomide and pomalidomide. Leukemia. 26 (11), 2326-2335 (2012).
  3. Fischer, E. S., et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature. 512 (7512), 49-53 (2014).
  4. Gandhi, A. K., et al. 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). British Journal of Haematology. 164 (6), 811-821 (2014).
  5. Kronke, J., Hurst, S. N., Ebert, B. L. Lenalidomide induces degradation of IKZF1 and IKZF3. Oncoimmunology. 3 (7), e941742 (2014).
  6. Lu, G., et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 343 (6168), 305-309 (2014).
  7. Zhu, Y. X., Kortuem, K. M., Stewart, A. K. Molecular mechanism of action of immune-modulatory drugs thalidomide, lenalidomide and pomalidomide in multiple myeloma. Leukemia & Lymphona. 54 (4), 683-687 (2013).
  8. Chamberlain, P. P., et al. Structure of the human Cereblon-DDB1-lenalidomide complex reveals basis for responsiveness to thalidomide analogs. Nature Structural & Molecular Biology. 21 (9), 803-809 (2014).
  9. Donovan, K. A., et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. eLife. 7, (2018).
  10. Matyskiela, M. E., et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nature Chemical Biology. 14 (10), 981-987 (2018).
  11. Kronke, J., et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature. 523 (7559), 183-188 (2015).
  12. Sakamoto, K. M., et al. Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proceedings of the National Academy of Sciences of the United States of America. 98 (15), 8554-8559 (2001).
  13. Sakamoto, K. M., et al. Development of Protacs to target cancer-promoting proteins for ubiquitination and degradation. Molecular & Cellular Proteomics. 2 (12), 1350-1358 (2003).
  14. Schneekloth, J. S., et al. Chemical genetic control of protein levels: selective in vivo targeted degradation. Journal of the American Chemical Society. 126 (12), 3748-3754 (2004).
  15. Gu, S., Cui, D., Chen, X., Xiong, X., Zhao, Y. PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery. Bioessays. 40 (4), e1700247 (2018).
  16. Collins, I., Wang, H., Caldwell, J. J., Chopra, R. Chemical approaches to targeted protein degradation through modulation of the ubiquitin-proteasome pathway. Biochemical Journal. 474 (7), 1127-1147 (2017).
  17. Neklesa, T. K., Winkler, J. D., Crews, C. M. Targeted protein degradation by PROTACs. Pharmacology & Therapeutics. 174, 138-144 (2017).
  18. Winter, G. E., et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science. 348 (6241), 1376-1381 (2015).
  19. Maniaci, C., et al. Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation. Nature Communications. 8 (1), 830 (2017).
  20. Crew, A. P., et al. Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1. Journal of Medicinal Chemistry. , (2017).
  21. Lu, J., et al. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chemistry & Biology. 22 (6), 755-763 (2015).
  22. Steinebach, C., et al. PROTAC-mediated crosstalk between E3 ligases. Chemical Communications. 55 (12), 1821-1824 (2019).
  23. Tinworth, C. P., Lithgow, H., Churcher, I. Small molecule-mediated protein knockdown as a new approach to drug discovery. Medchemcomm. 7 (12), 2206-2216 (2016).
  24. Steinebach, C., et al. Homo-PROTACs for the Chemical Knockdown of Cereblon. ACS Chemical Biology. 13 (9), 2771-2782 (2018).
  25. Ambrozak, A., et al. Synthesis and Antiangiogenic Properties of Tetrafluorophthalimido and Tetrafluorobenzamido Barbituric Acids. ChemMedChem. 11 (23), 2621-2629 (2016).
  26. Zhou, B., et al. Discovery of a Small-Molecule Degrader of Bromodomain and Extra-Terminal (BET) Proteins with Picomolar Cellular Potencies and Capable of Achieving Tumor Regression. Journal of Medicinal Chemistry. , (2017).
  27. Zhang, C., et al. Proteolysis Targeting Chimeras (PROTACs) of Anaplastic Lymphoma Kinase (ALK). European Journal of Medicinal Chemistry. 151, 304-314 (2018).
  28. Runcie, A. C., Chan, K. H., Zengerle, M., Ciulli, A. Chemical genetics approaches for selective intervention in epigenetics. Current Opinion in Chemical Biology. 33, 186-194 (2016).
  29. Kronke, J., et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 343 (6168), 301-305 (2014).
  30. Eichner, R., et al. Immunomodulatory drugs disrupt the cereblon-CD147-MCT1 axis to exert antitumor activity and teratogenicity. Nature Medicine. 22 (7), 735-743 (2016).
  31. Zhu, Y. X., et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood. 118 (18), 4771-4779 (2011).
  32. Kortum, K. M., et al. Targeted sequencing of refractory myeloma reveals a high incidence of mutations in CRBN and Ras pathway genes. Blood. 128 (9), 1226-1233 (2016).
  33. Gil, M., et al. Cereblon deficiency confers resistance against polymicrobial sepsis by the activation of AMP activated protein kinase and heme-oxygenase-1. Biochemical and Biophysical Research Communications. 495 (1), 976-981 (2018).
  34. Kim, H. K., et al. Cereblon in health and disease. Pflügers Archiv: European Journal of Physiology. 468 (8), 1299-1309 (2016).
  35. Lee, K. M., et al. Disruption of the cereblon gene enhances hepatic AMPK activity and prevents high-fat diet-induced obesity and insulin resistance in mice. Diabetes. 62 (6), 1855-1864 (2013).

Przedruki i uprawnienia

Zapytaj o uprawnienia na użycie tekstu lub obrazów z tego artykułu JoVE

Zapytaj o uprawnienia

Przeglądaj więcej artyków

CereblonE3 Ubiquitin LigasePomalidomidePROTACsChemical InactivationMolecular MechanismThalidomide AnalogsProtein DegradersSynthesisSodium AcetateGlutarimide3 fluorophthalic AnhydrideDimethyl SulfoxideAnti tumor ActivityClinical Implications

This article has been published

Video Coming Soon

JoVE Logo

Prywatność

Warunki Korzystania

Zasady

Skontaktuj Się z Nami

Poleć Bibliotece

BIULETYNY JoVE

Badania

Edukacja

Autorzy

Bibliotekarze

O JoVE

Copyright © 2025 MyJoVE Corporation. Wszelkie prawa zastrzeżone