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Issue 12, 2019
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Label-free target identification reveals oxidative DNA damage as the mechanism of a selective cytotoxic agent

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Abstract

Phenotypic screening can not only identify promising first-in-class drug candidates, but can also reveal potential therapeutic targets or neomorphic functions of known proteins. In this study, we identified target proteins of SB2001, a cytotoxic agent that acts specifically against HeLa human cervical cancer cells. Because SB2001 lacks chemical modification sites, label-free target identification methods including thermal stability shift-based fluorescence difference in two-dimensional gel electrophoresis (TS-FITGE) and thermal proteome profiling (TPP) were applied to characterize its mechanism of action. Owing to their differences, the two label-free target identification methods uncovered complementary target candidates. Candidates from both methods were prioritized according to their selective lethality upon the knockdown of those genes in HeLa cells, compared to CaSki cells which were used as a negative control cell line from the human cervix. LTA4H was identified only by TS-FITGE, but not by TPP, because only one isoform was stabilized by SB2001. Furthermore, it was implied that a non-canonical function of LTA4H was involved in the SB2001 activity. MTH1 was identified by both TS-FITGE and TPP, and SB2001 inhibited the function of MTH1 in hydrolyzing oxidized nucleotides. Compared to CaSki cells, HeLa cells displayed downregulated DNA mismatch repair pathways, which made HeLa cells more susceptible to the oxidative stress caused by SB2001, resulting in increased 8-oxoG concentrations, DNA damage, and subsequent cell death.

Graphical abstract: Label-free target identification reveals oxidative DNA damage as the mechanism of a selective cytotoxic agent

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Publication details

The article was received on 07 Dec 2018, accepted on 09 Feb 2019 and first published on 19 Feb 2019


Article type: Edge Article
DOI: 10.1039/C8SC05465G
Citation: Chem. Sci., 2019,10, 3449-3458
  • Open access: Creative Commons BY-NC license
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    Label-free target identification reveals oxidative DNA damage as the mechanism of a selective cytotoxic agent

    H. Park and S. B. Park, Chem. Sci., 2019, 10, 3449
    DOI: 10.1039/C8SC05465G

    This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material and it is not used for commercial purposes.

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