以波马利多米德为基础的人种-PROTACs对E3泛基金的化学灭活
Summary
这项工作描述了一种基于波马利多米德的合成和表征,即一种基于双功能的同质性PROTAC,作为诱导E3泛性联苯基雷布龙(CRBN)的泛化和降解的新方法,该物质是沙利度胺类比靶的目标。
Abstract
免疫调节药物 (IMiDs) 沙利度胺及其类似物, 利利多米德和波马利多米德, 所有FDA批准的药物用于治疗多发性骨髓瘤, 诱导淋巴转录因子伊卡罗斯 (IKZF1) 和 Aiolos 的泛化和降解(IKZF3)通过脑蛋白酶(CRBN)E3泛蛋白联苯,用于蛋白酶降解。IMiDs最近被用于生成双功能蛋白解靶向嵌合物(PROTACs),以针对CRBN E3附和酶的泛化和蛋白酶降解的其他蛋白质。我们设计并合成了基于波马利多米德的同质功能PROTC,并分析了它们诱导CRBN自导无化和降解的能力。在这里,CRBN同时充当E3泛基丁和靶子。同质-PROTAC化合物8降解CRBN具有高效力,对IKZF1和IKZF3仅产生最小剩余影响。化合物8的CRBN失活对细胞活力和不同多发性骨髓瘤细胞系的增殖没有影响。这种同质性PROTAC可以废除多发性骨髓瘤细胞中IMiD的作用。因此,我们的同质性波马利多米德化合物可能有助于识别CRBN的内源基质和生理功能,并研究IMiD的分子机制。
Introduction
免疫调节药物 (IMiDs) 沙利度胺及其类似物, 利利多米德和波马利多米德, 所有批准用于治疗多发性骨髓瘤, 结合 E3 泛素结合脑素 (CRBN), 基质适配器的 cullinin4A-RING E3 泛素结合(CRL4CRBN)1,2,3.IMiD 的结合增强了 CRL4CRBN与淋巴转录因子 Ikaros (IKZF1) 和 Aiolos (IKZF3) 的亲和力,导致其泛化和降解(图 1)4,5 ,6,7,8. 由于 IKZF1 和 IKZF3 对多发性骨髓瘤细胞至关重要,因此它们的失活会导致生长抑制。SALL4最近被发现作为CRBN的另一种IMiD诱导的新基质,可能是导致致畸和所谓的康特甘灾难在1950年代由沙利多米德9,10造成的。相反,卡辛激酶1+(CK1+)是CRBN的一种利利多米德特异性基质,与染色体5q缺失11的骨髓增生综合征的治疗效果有关。
小分子靶向特定蛋白质降解的能力是现代药物开发中令人振奋的一个暗示。虽然沙利度胺及其类似物的机制是在人类首次使用后发现的,但所谓的Proteolyis Targeting Chimeras (PROTACs) 被设计为专门针对感兴趣的蛋白质 (POI)(图2)12,13,14,15,16,17,18。PROTACs 是异体功能分子,由 POI 的特定配体组成,通过链接器连接到 E3 泛性联苯的配体,如 CRBN 或 von-Hippel-Lindau (VHL)18、19、20、 21,22.PROTACs诱导形成瞬态三元复合物,将POI定向到E3泛基质连体酶,导致其泛化和蛋白酶降解。与常规抑制剂相比,PROTACs的主要优点是,与POI结合就足够了,而不是其抑制作用,因此PROTACs可能针对更广泛的蛋白质,包括那些被认为无法药物的蛋白质,转录因子15.此外,嵌合分子具有催化作用,因此具有高效力。在泛基辛转移到POI后,三元复合物分离,可用于形成新的复合物。因此,非常低的PROTAC浓度足以降解靶蛋白23。
在这里,我们描述了一个波马利多米德-波马利多米德共聚物(化合物8)的合成,它招募CRBN来降低自身24。E3 泛基苯甲二苯CRBN 同时充当招募者和目标(图 3)。为了验证我们的数据,我们还合成了负绑定控制(化合物9)。 我们的数据证实,新合成的同质性PROTAC是CRBN降解的特异性,对其他蛋白质的影响最小。
Protocol
1. PROTAC分子的制备 注意:使用前请查阅所有相关材料安全数据表 (MSDS)。这些合成中使用的几种化学品具有毒性和致癌性。请使用所有适当的安全操作和个人防护设备。 制备三丁基N-(2,6-二恶英-3-烟酸)碳水化合物(化合物1) 在带搅拌棒并配有反流冷凝器的100 mL圆形底瓶中,在100 mL圆形瓶中加入1,1′-碳二甲酰胺(1.95克,12毫摩尔)和4-(二甲基氨基)…
Representative Results
在这里,我们描述了基于同质的波马利多米德PROTAC用于CRBN降解的设计、合成和生物评价。我们的PROTAC与两个CRBN分子同时相互作用,形成三元复合物,诱导CRBN自泛化和原体降解,仅对波马利多米德诱导的新基质IKZF1或IKZF3产生最小剩余影响。 在以前公布的一系列基于波马利多米德的PROTAC分子24中,化合物8…
Discussion
此处描述的CRBN此类同质-PROTAC的设计依赖于波马利多米德与CRBN的具体亲和力,该设计已成功应用于众多异质功能PROTAC,并使得PROTAC 8的发展成为高度选择性CRBN降解剂。我们的分子的特异性已经通过蛋白体分析24得到证实。对于基因介导的淘汰,排除和验证副作用是具有挑战性的和耗时的。此外,化学诱导的敲除是可逆的,快速和直接适用于广泛的细胞和组织类?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了德国福森斯格明舍夫特(Emmy-Noether计划Kr-3886/2-1和SFB-1074到J.K.)的支持;FOR2372 到 M.G.)
Materials
| 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 |
References
- Ito, T., et al. Identification of a primary target of thalidomide teratogenicity. Science. 327 (5971), 1345-1350 (2010).
- 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).
- Fischer, E. S., et al. Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature. 512 (7512), 49-53 (2014).
- 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).
- Kronke, J., Hurst, S. N., Ebert, B. L. Lenalidomide induces degradation of IKZF1 and IKZF3. Oncoimmunology. 3 (7), e941742 (2014).
- Lu, G., et al. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science. 343 (6168), 305-309 (2014).
- 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).
- 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).
- Donovan, K. A., et al. Thalidomide promotes degradation of SALL4, a transcription factor implicated in Duane Radial Ray syndrome. eLife. 7, (2018).
- Matyskiela, M. E., et al. SALL4 mediates teratogenicity as a thalidomide-dependent cereblon substrate. Nature Chemical Biology. 14 (10), 981-987 (2018).
- Kronke, J., et al. Lenalidomide induces ubiquitination and degradation of CK1alpha in del(5q) MDS. Nature. 523 (7559), 183-188 (2015).
- 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).
- 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).
- 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).
- 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).
- 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).
- Neklesa, T. K., Winkler, J. D., Crews, C. M. Targeted protein degradation by PROTACs. Pharmacology & Therapeutics. 174, 138-144 (2017).
- Winter, G. E., et al. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science. 348 (6241), 1376-1381 (2015).
- 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).
- 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).
- Lu, J., et al. Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chemistry & Biology. 22 (6), 755-763 (2015).
- Steinebach, C., et al. PROTAC-mediated crosstalk between E3 ligases. Chemical Communications. 55 (12), 1821-1824 (2019).
- 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).
- Steinebach, C., et al. Homo-PROTACs for the Chemical Knockdown of Cereblon. ACS Chemical Biology. 13 (9), 2771-2782 (2018).
- Ambrozak, A., et al. Synthesis and Antiangiogenic Properties of Tetrafluorophthalimido and Tetrafluorobenzamido Barbituric Acids. ChemMedChem. 11 (23), 2621-2629 (2016).
- 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).
- Zhang, C., et al. Proteolysis Targeting Chimeras (PROTACs) of Anaplastic Lymphoma Kinase (ALK). European Journal of Medicinal Chemistry. 151, 304-314 (2018).
- 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).
- Kronke, J., et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 343 (6168), 301-305 (2014).
- 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).
- Zhu, Y. X., et al. Cereblon expression is required for the antimyeloma activity of lenalidomide and pomalidomide. Blood. 118 (18), 4771-4779 (2011).
- 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).
- 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).
- Kim, H. K., et al. Cereblon in health and disease. Pflügers Archiv: European Journal of Physiology. 468 (8), 1299-1309 (2016).
- 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).
Cite This Article
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).