The ubiquitin–proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.
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Jin, J., Li, X., Gygi, S.P. & Harper, J.W. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging. Nature 447, 1135–1138 (2007).
Swatek, K.N. & Komander, D. Ubiquitin modifications. Cell Res. 26, 399–422 (2016).
Komander, D. & Rape, M. The ubiquitin code. Annu. Rev. Biochem. 81, 203–229 (2012).
Richardson, P.G. et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N. Engl. J. Med. 348, 2609–2617 (2003).
Brownell, J.E. et al. Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8–AMP mimetic in situ. Mol. Cell 37, 102–111 (2010).
Huang, X. & Dixit, V.M. Drugging the undruggables: exploring the ubiquitin system for drug development. Cell Res. 26, 484–498 (2016).
Bedford, L., Lowe, J., Dick, L.R., Mayer, R.J. & Brownell, J.E. Ubiquitin-like protein conjugation and the ubiquitin–proteasome system as drug targets. Nat. Rev. Drug Discov. 10, 29–46 (2011).
Anderson, D.J. et al. Targeting the AAA ATPase p97 as an approach to treat cancer through disruption of protein homeostasis. Cancer Cell 28, 653–665 (2015).
Krönke, J. et al. Lenalidomide induces ubiquitination and degradation of CK1α in del(5q) MDS. Nature 523, 183–188 (2015).
McGrath, J.P., Jentsch, S. & Varshavsky, A. UBA1: an essential yeast gene encoding ubiquitin-activating enzyme. EMBO J. 10, 227–236 (1991).
Kulkarni, M. & Smith, H.E. E1 ubiquitin-activating enzyme UBA1 plays multiple roles throughout C. elegans development. PLoS Genet. 4, e1000131 (2008).
Misra, M. et al. Dissecting the specificity of adenosyl sulfamate inhibitors targeting the ubiquitin-activating enzyme. Structure 25, 1120–1129 (2017).
Amidon, B.S. et al. Indole-substituted pyrrolopyrimidinyl inhibitors of Uba6. US Patent 9593121B2. (2017).
Soucy, T.A. et al. An inhibitor of NEDD8-activating enzyme as a new approach to treat cancer. Nature 458, 732–736 (2009).
Hershko, A. The ubiquitin system for protein degradation and some of its roles in the control of the cell division cycle. Cell Death Differ. 12, 1191–1197 (2005).
Gu, J.J. et al. MLN2238, a proteasome inhibitor, induces caspase-dependent cell death, cell cycle arrest, and potentiates the cytotoxic activity of chemotherapy agents in rituximab-chemotherapy-sensitive or rituximab-chemotherapy-resistant B cell lymphoma preclinical models. Anticancer Drugs 24, 1030–1038 (2013).
Milhollen, M.A. et al. Inhibition of NEDD8-activating enzyme induces re-replication and apoptosis in human tumor cells consistent with de-regulating CDT1 turnover. Cancer Res. 71, 3042–3051 (2011).
Ulrich, H.D. Ubiquitin and SUMO in DNA repair at a glance. J. Cell Sci. 125, 249–254 (2012).
Christianson, J.C. & Ye, Y. Cleaning up in the endoplasmic reticulum: ubiquitin in charge. Nat. Struct. Mol. Biol. 21, 325–335 (2014).
Moudry, P. et al. Ubiquitin-activating enzyme UBA1 is required for cellular response to DNA damage. Cell Cycle 11, 1573–1582 (2012).
Ungermannova, D. et al. Identification and mechanistic studies of a novel ubiquitin E1 inhibitor. J. Biomol. Screen. 17, 421–434 (2012).
Collins, A.R. The comet assay for DNA damage and repair: principles, applications and limitations. Mol. Biotechnol. 26, 249–261 (2004).
Vlachostergios, P.J., Patrikidou, A., Daliani, D.D. & Papandreou, C.N. The ubiquitin–proteasome system in cancer, a major player in DNA repair. Part 2: transcriptional regulation. J. Cell. Mol. Med. 13 9B, 3019–3031 (2009).
Wildey, G. et al. Pharmacogenomic approach to identify drug sensitivity in small-cell-lung cancer. PLoS One 9, e106784 (2014).
Wilmott, J.S. et al. BRAFV600E protein expression and outcome from BRAF inhibitor treatment in BRAFV600E metastatic melanoma. Br. J. Cancer 108, 924–931 (2013).
Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity. Nature 483, 603–607 (2012).
Huang, J. et al. NEDD8 inhibition overcomes CKS1B-induced drug resistance by upregulation of p21 in multiple myeloma. Clin. Cancer Res. 21, 5532–5542 (2015).
Milhollen, M.A. et al. Treatment-emergent mutations in NAEβ confer resistance to the NEDD8-activating enzyme inhibitor MLN4924. Cancer Cell 21, 388–401 (2012).
Yang, Y. et al. Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 67, 9472–9481 (2007).
Xu, G.W. et al. The ubiquitin-activating enzyme E1 as a therapeutic target for the treatment of leukemia and multiple myeloma. Blood 115, 2251–2259 (2010).
Gilbert, L.A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Wang, T., Wei, J.J., Sabatini, D.M. & Lander, E.S. Genetic screens in human cells using the CRISPR–Cas9 system. Science 343, 80–84 (2014).
Yau, R. & Rape, M. The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586 (2016).
Chen, J.J. et al. Mechanistic studies of substrate-assisted inhibition of ubiquitin-activating enzyme by adenosine sulfamate analogs. J. Biol. Chem. 286, 40867–40877 (2011).
Gavin, J.M. et al. Mechanistic studies on activation of ubiquitin and di-ubiquitin-like protein, FAT10, by ubiquitin-like-modifier-activating enzyme 6, Uba6. J. Biol. Chem. 287, 15512–15522 (2012).
Leslie, A.G. The integration of macromolecular diffraction data. Acta Crystallogr. D Biol. Crystallogr. 62, 48–57 (2006).
Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D Biol. Crystallogr. 62, 72–82 (2006).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Potterton, E., Briggs, P., Turkenburg, M. & Dodson, E. A graphical user interface to the CCP4 program suite. Acta Crystallogr. D Biol. Crystallogr. 59, 1131–1137 (2003).
Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D Biol. Crystallogr. 53, 240–255 (1997).
Manfredi, M.G. et al. Antitumor activity of MLN8054, an orally active small-molecule inhibitor of Aurora A kinase. Proc. Natl. Acad. Sci. USA 104, 4106–4111 (2007).
Pocock, S.J. & Simon, R. Sequential treatment assignment with balancing for prognostic factors in the controlled clinical trial. Biometrics 31, 103–115 (1975).
Duffey, M.O. et al. Discovery of a potent and orally bioavailable benzolactam-derived inhibitor of Polo-like kinase 1 (MLN0905). J. Med. Chem. 55, 197–208 (2012).
National Research Council. Guide for the Care and Use of Laboratory Animals 8th edn. (The National Academies Press, 2011).
The authors would like to thank W. Harper (Harvard Medical School) for the UBA6-knockout and control MEFs, A. Berger for critical review of the manuscript, and E. Koenig and P. Shah for genomic data analysis. We would also like to thank J. Afroze, I. Bharathan, J. Gaulin, M. Girad, C. McIntyre, F. Soucy, T.T. Wong and Y. Ye for performing the chemical synthesis of TAK-243. All activities were completed and funded through Takeda Pharmaceuticals Inc.
All of the authors were employees of Takeda Pharmaceuticals at the time of these studies.
Supplementary Figures 1–16 & Supplementary Tables 1, 3 (PDF 5164 kb)
Life Sciences Reporting Summary (PDF 208 kb)
Selectivity profiling of TAK-243 against kinases, cellular receptors (Novascreen) and carbonic anhydrases. (XLSX 30 kb)
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Hyer, M., Milhollen, M., Ciavarri, J. et al. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment. Nat Med 24, 186–193 (2018). https://doi.org/10.1038/nm.4474
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