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Potent and selective caspase-2 inhibitor prevents MDM-2 cleavage in reversine-treated colon cancer cells

Cell Death & Differentiation (2019) | Download Citation


Most caspases can be positioned unambiguously within the regulated cell death networks of apoptosis and pyroptosis, but the role of caspase-2, a highly conserved protease within the family, remains enigmatic. This is mainly due to lack of selective chemical and biochemical tools for the investigation of this protease. In this study, we used our hybrid combinatorial substrate library (HyCoSuL) approach to broadly profile caspase-2 substrate specificity using peptide scanning libraries. This screen uncovered previously unknown caspase-2 peptidyl substrate preferences, which were further used to develop caspase-2 selective fluorogenic substrates and covalent, irreversible AOMK inhibitors. Finally, we used the champion inhibitor (NH-23-C2) in reversine-treated HCT-116 colon cancer cells to selectively block caspase-2 activity and caspase-2-mediated MDM-2 cleavage. In addition, we showed that NH-23-C2 does not block caspase-3 or caspase-8, which makes it a powerful chemical tool to dissect the true role of caspase-2 in various biological setups.

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This project received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 661187 (to MP) and was supported by the National Science Centre grant 2011/03/B/ST5/01048 in Poland (to MD), the “TEAM/2017-4/32” project, which is carried out within the TEAM programme of the Foundation for Polish Science cofinanced by the European Union under the European Regional Development Fund (to MD), and the NIH grant R01 GM099040 to (GSS). Sanford Burnham Prebys NCI Cancer Center Support Grant P30 CA030199.

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  1. NCI Designated Cancer Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037, USA

    • Marcin Poreba
    • , Katarzyna Groborz
    • , Scott J. Snipas
    • , Guy S. Salvesen
    •  & Marcin Drag
  2. Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology, Wyb. Wyspianskiego 27, 50-370, Wroclaw, Poland

    • Marcin Poreba
    • , Wioletta Rut
    • , Katarzyna Groborz
    •  & Marcin Drag


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MP and MD conceived the project; MP, WR, GS, and MD designed and MP, WR, and KG performed the experiments; SJS contributed new reagents and tools. MP drafted the manuscript and all authors edited the manuscript. All authors reviewed the results and approved the final version of the manuscript.

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The authors declare that they have no conflict of interest.

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Correspondence to Marcin Poreba or Marcin Drag.

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