SUMOylation is one of the posttranslational modifications that mediate cellular activities such as transcription, DNA repair, and signal transduction and is involved in the cell cycle. However, only a limited number of small molecule inhibitors have been identified to study its role in cellular processes. Here, we report a Förster resonance energy transfer (FRET) high-throughput screening assay based on the interaction between E2 Ubc9 and E3 PIAS1. Of the 3200 compounds screened, 34 (1.1%) showed higher than 50% inhibition and 4 displayed dose–response inhibitory effects. By combining this method with a label-free surface plasmon resonance (SPR) assay, false positives were excluded leading to discovering WNN0605-F008 and WNN1062-D002 that bound to Ubc9 with KD values of 1.93 ± 0.62 and 5.24 ± 3.73 μM, respectively. We examined the effect of the two compounds on SUMO2-mediated SUMOylation of RanGAP1, only WNN0605-F008 significantly inhibited RanGAP1 SUMOylation, whereas WNN1062-D002 did not show any inhibition. These compounds, with novel chemical scaffolds, may serve as the initial material for developing new SUMOylation inhibitors.
Subscribe to Journal
Get full journal access for 1 year
only $33.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Guo C, Henley JM. Wrestling with stress: roles of protein sumoylation and desumoylation in cell stress response. IUBMB Life. 2014;66:71–7.
Ulrich HD. Ubiquitin and SUMO in DNA repair at a glance. J Cell Sci. 2012;125:249–54.
Vertegaal AC, Srikumar T, Lee C, Osula O, Subramonian D, Zhang XD, et al. A proteomic study of SUMO-2 target proteins. J Biol Chem. 2004;279:33791–8.
Li T, Evdokimov E, Shen RF, Chao CC, Tekle E, Wang T, et al. Sumoylation of heterogeneous nuclear ribonucleoproteins, zinc finger proteins, and nuclear pore complex proteins: a proteomic analysis. Proc Natl Acad Sci USA. 2004;101:8551–6.
Geiss-Friedlander R, Melchior F. Concepts in sumoylation: a decade on. Nat Rev Mol Cell Biol. 2007;8:947–56.
Johnson ES. Protein modification by SUMO. Annu Rev Biochem. 2004;73:355–82.
Seeler JS, Dejean A. SUMO and the robustness of cancer. Nat Rev Cancer. 2017;17:184–97.
Krumova P, Weishaupt JH. Sumoylation in neurodegenerative diseases. Cell Mol Life Sci. 2013;70:2123–38.
Mendler L, Braun T, Muller S. The ubiquitin-like SUMO system and heart function: from development to disease. Circ Res. 2016;118:132–44.
Licciardello MP, Kubicek S. Pharmacological treats for SUMO addicts. Pharmacol Res. 2016;107:390–7.
Mo YY, Moschos SJ. Targeting Ubc9 for cancer therapy. Expert Opin Ther Targets. 2005;9:1203–16.
Moschos SJ, Jukic DM, Athanassiou C, Bhargava R, Dacic S, Wang X, et al. Expression analysis of Ubc9, the single small ubiquitin-like modifier (SUMO) E2 conjugating enzyme, in normal and malignant tissues. Hum Pathol. 2010;41:1286–98.
McDoniels-Silvers AL, Nimri CF, Stoner GD, Lubet RA, You M. Differential gene expression in human lung adenocarcinomas and squamous cell carcinomas. Clin Cancer Res. 2002;8:1127–38.
Tomasi ML, Tomasi I, Ramani K, Pascale RM, Xu J, Giordano P, et al. S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers. Hepatology. 2012;56:982–93.
Moschos SJ, Smith AP, Mandic M, Athanassiou C, Watson-Hurst K, Jukic DM, et al. SAGE and antibody array analysis of melanoma-infiltrated lymph nodes: identification of Ubc9 as an important molecule in advanced-stage melanomas. Oncogene. 2007;26:4216–25.
Mo YY, Yu Y, Ee PL, Beck WT. Overexpression of a dominant-negative mutant Ubc9 is associated with increased sensitivity to anticancer drugs. Cancer Res. 2004;64:2793–8.
Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, et al. Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci USA. 1998;95:10626–31.
Johnson ES, Gupta AA. An E3-like factor that promotes SUMO conjugation to the yeast septins. Cell. 2001;106:735–44.
Kahyo T, Nishida T, Yasuda H. Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol Cell. 2001;8:713–8.
Kotaja N, Karvonen U, Janne OA, Palvimo JJ. PIAS proteins modulate transcription factors by functioning as SUMO-1 ligases. Mol Cell Biol. 2002;22:5222–34.
Rabellino A, Melegari M, Tompkins VS, Chen W, Van Ness BG, Teruya-Feldstein J, et al. PIAS1 promotes lymphomagenesis through MYC upregulation. Cell Rep. 2016;15:2266–78.
Rabellino A, Andreani C, Scaglioni PP. The role of PIAS SUMO E3-ligases in cancer. Cancer Res. 2017;77:1542–7.
Liao JY, Song Y, Liu Y. A new trend to determine biochemical parameters by quantitative FRET assays. Acta Pharmacol Sin. 2015;36:1408–15.
Song Y, Madahar V, Liao J. Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions. Ann Biomed Eng. 2011;39:1224–34.
Liu Y, Song Y, Madahar V, Liao J. Quantitative förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease. Anal Biochem. 2012;422:14–21.
Song Y, Liao J. An in vitro förster resonance energy transfer-based high-throughput screening assay for inhibitors of protein-protein interactions in Sumoylation pathway. Assay Drug Dev Technol. 2012;10:336–43.
Wiryawan H, Dan K, Etuale M, Shen Y, Liao J. Determination of SUMO1 and ATP affinity for the SUMO E1 by quantitative FRET technology. Biotechnol Bioeng. 2015;112:652–8.
Nguyen AW, Daugherty PS. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol. 2005;23:355–60.
Fivash M, Towler EM, Fisher RJ. Biacore for macromolecular interaction. Curr Opin Biotechnol. 1998;9:97–101.
Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4:67–73.
Baell JB, Holloway GA. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem. 2010;53:2719–40.
Kim YS, Nagy K, Keyser S, Schneekloth JS Jr. An electrophoretic mobility shift assay identifies a mechanistically unique inhibitor of protein sumoylation. Chem Biol. 2013;20:604–13.
Vauquelin G. Effects of target binding kinetics on in vivo drug efficacy: koff, kon and rebinding. Br J Pharmacol. 2016;173:2319–34.
Matunis MJ, Coutavas E, Blobel G. A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol. 1996;135:1457–70.
Mahajan R, Gerace L, Melchior F. Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association. J Cell Biol. 1998;140:259–70.
Mahajan R, Delphin C, Guan T, Gerace L, Melchior F. A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell. 1997;88:97–107.
Tempe D, Piechaczyk M, Bossis G. SUMO under stress. Biochem Soc Trans. 2008;36:874–8.
Saitoh H, Hinchey J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem. 2000;275:6252–8.
Tatham MH, Jaffray E, Vaughan OA, Desterro JM, Botting CH, Naismith JH, et al. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem. 2001;276:35368–74.
Fukuda I, Ito A, Hirai G, Nishimura S, Kawasaki H, Saitoh H, et al. Ginkgolic acid inhibits protein sumoylation by blocking formation of the E1-SUMO intermediate. Chem Biol. 2009;16:133–40.
Hewitt WM, Lountos GT, Zlotkowski K, Dahlhauser SD, Saunders LB, Needle D, et al. Insights into the allosteric inhibition of the SUMO E2 enzyme Ubc9. Angew Chem Int Ed Engl. 2016;55:5703–7.
Hirohama M, Kumar A, Fukuda I, Matsuoka S, Igarashi Y, Saitoh H, et al. Spectomycin B1 as a novel sumoylation inhibitor that directly binds to SUMO E2. ACS Chem Biol. 2013;8:2635–42.
Komiya M, Ito A, Endo M, Hiruma D, Hattori M, Saitoh H, et al. A genetic screen to discover sumoylated proteins in living mammalian cells. Sci Rep. 2017;7:17450–7.
Vertegaal AC, Andersen JS, Ogg SC, Hay RT, Mann M, Lamond AI, et al. Distinct and overlapping sets of SUMO-1 and SUMO-2 target proteins revealed by quantitative proteomics. Mol Cell Proteom. 2006;5:2298–310.
Brandt M, Szewczuk LM, Zhang H, Hong X, McCormick PM, Lewis TS, et al. Development of a high-throughput screen to detect inhibitors of TRPS1 sumoylation. Assay Drug Dev Technol. 2013;11:308–25.
Saitoh N, Uchimura Y, Tachibana T, Sugahara S, Saitoh H, Nakao M, et al. In situ sumoylation analysis reveals a modulatory role of RanBP2 in the nuclear rim and PML bodies. Exp Cell Res. 2006;312:1418–30.
Zlotkowski K, Hewitt WM, Sinniah RS, Tropea JE, Needle D, Lountos GT, et al. A small-molecule microarray approach for the identification of E2 enzyme inhibitors in ubiquitin-like conjugation pathways. SLAS Discov. 2017;22:760–6.
Liu B, Mink S, Wong KA, Stein N, Getman C, Dempsey PW, et al. PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity. Nat Immunol. 2004;5:891–8.
Sampson DA, Wang M, Matunis MJ. The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification. J Biol Chem. 2001;276:21664–9.
Yunus AA, Lima CD. Lysine activation and functional analysis of E2-mediated conjugation in the SUMO pathway. Nat Struct Mol Biol. 2006;13:491–9.
Mascle XH, Lussier-Price M, Cappadocia L, Estephan P, Raiola L, Omichinski JG, et al. Identification of a non-covalent ternary complex formed by PIAS1, SUMO1, and UBC9 proteins involved in transcriptional regulation. J Biol Chem. 2013;288:36312–27.
Erlanson DA. Introduction to fragment-based drug discovery. Top Curr Chem. 2012;317:1–32.
Desterro JM, Rodriguez MS, Kemp GD, Hay RT. Identification of the enzyme required for activation of the small ubiquitin-like protein SUMO-1. J Biol Chem. 1999;274:10618–24.
Desterro JM, Thomson J, Hay RT. Ubc9 conjugates SUMO but not ubiquitin. FEBS Lett. 1997;417:297–300.
Rodriguez MS, Dargemont C, Hay RT. SUMO-1 conjugation in vivo requires both a consensus modification motif and nuclear targeting. J Biol Chem. 2001;276:12654–9.
Hay RT. SUMO: a history of modification. Mol Cell. 2005;18:1–12.
We are indebted to Ji Wu, Zhong-lian Cao, Jiu-qing Tan and Ying-yan Jiang for technical assistance. This work was partially supported by the National Natural Science Foundation of China grants 81872915 (MWW), 81573479 (DHY), 81773792 (DHY), and 21704064 (QTZ), The National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (2018ZX09735-001 to MWW and 2018ZX09711002-002-005 to DHY), The National Key R&D Program of China grant 2018YFA0507000 (MWW), and Novo Nordisk-CAS Research Fund grant NNCAS-2017-1-CC (DHY).
The authors declare no competing interests.
About this article
Cite this article
Wang, Yz., Liu, X., Way, G. et al. An in vitro Förster resonance energy transfer-based high-throughput screening assay identifies inhibitors of SUMOylation E2 Ubc9. Acta Pharmacol Sin 41, 1497–1506 (2020). https://doi.org/10.1038/s41401-020-0405-7
- SUMOylation inhibitor
- high-throughput screening
- Förster resonance energy transfer
- surface plasmon resonance