Abstract
The evolutionally conserved and abundant post-translational modifier ubiquitin (Ub) is involved in a vast number of cellular processes. Imbalanced ubiquitination is associated with a range of diseases. Consequently, components of the ubiquitylation machinery, such as deubiquitinating enzymes (DUBs) that control the removal of Ub, are emerging as therapeutic targets. Here, we describe a robust assay suitable for small-molecule inhibitor screening. This assay has the potential to drive the development of small-molecule compounds that can selectively target DUBs.
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References
Komander D, Rape M (2012) The ubiquitin code. Ann Rev Biochem 81. https://doi.org/10.1146/annurev-biochem-060310-170328
Harrigan JA, Jacq X, Martin NM, Jackson SP (2018) Deubiquitylating enzymes and drug discovery: emerging opportunities. Nat Rev Drug Discov 17. https://doi.org/10.1038/nrd.2017.152
Lange SM, Armstrong LA, Kulathu Y (2022) Deubiquitinases: from mechanisms to their inhibition by small molecules. Mol Cell 82. https://doi.org/10.1016/j.molcel.2021.10.027
Kooij R, Liu S, Sapmaz A et al (2020) Small-molecule activity-based probe for monitoring ubiquitin C-terminal hydrolase L1 (UCHL1) activity in live cells and zebrafish embryos. J Am Chem Soc 142. https://doi.org/10.1021/jacs.0c07726
Panyain N, Godinat A, Lanyon-Hogg T et al (2020) Discovery of a potent and selective covalent inhibitor and activity-based probe for the deubiquitylating enzyme UCHL1, with antifibrotic activity. J Am Chem Soc 142. https://doi.org/10.1021/jacs.0c04527
Schauer NJ, Magin RS, Liu X et al (2020) Advances in discovering deubiquitinating enzyme (DUB) inhibitors. J Med Chem 63. https://doi.org/10.1021/acs.jmedchem.9b01138
Hassiepen U, Eidhoff U, Meder G et al (2007) A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate. Anal Biochem 371. https://doi.org/10.1016/j.ab.2007.07.034
Hameed DS, Sapmaz A, Burggraaff L et al (2019) Development of ubiquitin-based probe for metalloprotease deubiquitinases. Angewandte Chemie International Edition 58. https://doi.org/10.1002/anie.201906790
Wang X, D’Arcy P, Caulfield TR et al (2015) Synthesis and evaluation of derivatives of the proteasome deubiquitinase inhibitor b-AP15. Chem Biol Drug Design 86. https://doi.org/10.1111/cbdd.12571
Lamberto I, Liu X, Seo H-S et al (2017) Structure-guided development of a potent and selective non-covalent active-site inhibitor of USP7. Cell Chem Biol 24. https://doi.org/10.1016/j.chembiol.2017.09.003
Zhang J-H, Chung TDY, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. SLAS Discov 4. https://doi.org/10.1177/108705719900400206
Borodovsky A, Ovaa H, Meester WJN et al (2005) Small-molecule inhibitors and probes for ubiquitin- and ubiquitin-like-specific proteases. ChemBioChem 6. https://doi.org/10.1002/cbic.200400236
Cho J, Park J, Kim EE, Song EJ (2020) Assay systems for profiling deubiquitinating activity. Int J Mol Sci 21. https://doi.org/10.3390/ijms21165638
Ekkebus R, van Kasteren SI, Kulathu Y et al (2013) On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc 135. https://doi.org/10.1021/ja309802n
de Jong A, Merkx R, Berlin I et al (2012) Ubiquitin-based probes prepared by total synthesis to profile the activity of deubiquitinating enzymes. ChemBioChem 13. https://doi.org/10.1002/cbic.201200497
Varca AC, Casalena D, Chan WC et al (2021) Identification and validation of selective deubiquitinase inhibitors. Cell Chem Biol 28. https://doi.org/10.1016/j.chembiol.2021.05.012
Lipinski CA, Lombardo F, Dominy BW, Feeney PJ (2001) Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings 1PII of original article: S0169-409X(96)00423-1. The article was originally published in Advanced Drug Delivery Reviews 23 (1997) 3–25. 1. Adv Drug Deliv Rev 46. https://doi.org/10.1016/S0169-409X(00)00129-0
Acknowledgements
This work was supported by the EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN Grant No. 875510) and NWO (VIDI Grant VI. 213.110 to M.P.C.M).
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Scherpe, S., Sapmaz, A., Mulder, M.P.C. (2023). Plate-Based Screening for DUB Inhibitors. In: Merk, D., Chaikuad, A. (eds) Chemogenomics. Methods in Molecular Biology, vol 2706. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3397-7_7
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DOI: https://doi.org/10.1007/978-1-0716-3397-7_7
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