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An in vitro Förster resonance energy transfer-based high-throughput screening assay identifies inhibitors of SUMOylation E2 Ubc9

Abstract

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.

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Fig. 1
Fig. 2: Assay optimization and HTS campaign.
Fig. 3: Surface plasmon resonance analysis shows that WNN0605-F008 and WNN1062-D002 bound to Ubc9 with different kinetics.
Fig. 4: Inhibition of SUMOylation by WNN0605-F008.
Fig. 5: Structure similarity check and Ubc9 binding screening.
Fig. 6: Binding modes and substrate preference of confirmed hits.

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Acknowledgements

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).

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MWW and JYL conceived the idea. MWW, JYL, DHY, YZW, and GW designed the study. YZW, XL, GW, VM and QTZ performed the experiments. YZW, QTZ, JYL, and MWW analyzed the data and wrote the paper.

Corresponding authors

Correspondence to De-hua Yang or Jia-yu Liao or Ming-wei Wang.

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The authors declare no competing interests.

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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

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Keywords

  • SUMOylation inhibitor
  • WNN0605-F008
  • high-throughput screening
  • Ubc9
  • PIAS1
  • Förster resonance energy transfer
  • surface plasmon resonance

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