Mechanism of inhibition of the ATpase domain of human topoisomerase IIα by 1,4-benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, and 9,10-phenanthroquinone

Deepak Gurbani, Vandna Kukshal, Julian Laubenthal, Ashutosh Kumar, Alok Pandey, Sarita Tripathi, Ashish Arora, Swatantra K. Jain, Ravishankar Ramachandran, Diana Anderson, Alok Dhawan

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21 Scopus citations

Abstract

The inhibition of human topoisomerase IIα (Hu-TopoIIα), a major enzyme involved in maintaining DNA topology, repair, and chromosome condensation/decondensation results in loss of genomic integrity. In the present study, the inhibition of ATPase domain of Hu-TopoIIα as a possible mechanism of genotoxicity of 1,4-benzoquinone (BQ), hydroquinone (HQ), naphthoquinone (1,2-NQ and 1,4-NQ), and 9,10-phenanthroquinone (9,10-PQ) was investigated. In silico modeling predicted that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ could interact with Ser-148, Ser-149, Asn-150, and Asn-91 residues of the ATPase domain of Hu-TopoIIα. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoIIα revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ. Ligand-binding studies using isothermal titration calorimetry revealed that 1,4-BQ, HQ, 1,4-NQ, 1,2-NQ, and 9,10-PQ enter into four sequentially binding site models inside the domain. 1,4-BQ exhibited the strongest binding, followed by 1,4-NQ > 1,2-NQ > 9,10-PQ > HQ, as revealed by their average K d values. The cellular fate of such inhibition was further evidenced by an increase in the number of Hu-TopoIIα-DNA cleavage complexes in the human lung epithelial cells (BEAS-2B) using trapped in agarose DNA immunostaining (TARDIS) assay, which utilizes antibody specific for Hu-TopoIIα. Furthermore, the increase in γ-H2A.X levels quantitated by flow cytometry and visualized by immunofluorescence microscopy illustrated that accumulation of DNA double-strand breaks inside the cells can be attributed to the inhibition of Hu-TopoIIα. These findings collectively suggest that 1,4-BQ, 1,2-NQ, 1,4-NQ, and 9,10-PQ inhibit the ATPase domain and potentially result in Hu-TopoIIα-mediated clastogenic and leukemogenic events.

Original languageEnglish (US)
Pages (from-to)372-390
Number of pages19
JournalToxicological Sciences
Volume126
Issue number2
DOIs
StatePublished - Apr 2 2012
Externally publishedYes

Keywords

  • ATPase inhibition
  • BEAS-2B human lung cells
  • DNA double-strand breaks
  • Human topoisomerase II alpha
  • In silico modeling
  • Quinones

ASJC Scopus subject areas

  • Toxicology

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    Gurbani, D., Kukshal, V., Laubenthal, J., Kumar, A., Pandey, A., Tripathi, S., Arora, A., Jain, S. K., Ramachandran, R., Anderson, D., & Dhawan, A. (2012). Mechanism of inhibition of the ATpase domain of human topoisomerase IIα by 1,4-benzoquinone, 1,2-naphthoquinone, 1,4-naphthoquinone, and 9,10-phenanthroquinone. Toxicological Sciences, 126(2), 372-390. https://doi.org/10.1093/toxsci/kfr345