Mechanism of Inhibition of the ATPase Domain of Human Topoisomerase II alpha by 1,4-Benzoquinone, 1,2-Naphthoquinone, 1,4-Naphthoquinone, and 9,10-Phenanthroquinone

作者：Gurbani Deepak; Kukshal Vandna; Laubenthal Julian; Kumar Ashutosh; Pandey Alok; Tripathi Sarita; Arora Ashish; Jain Swatantra K; Ramachandran Ravishankar; Anderson Diana; Dhawan Alok^*

来源：Toxicological Sciences, 2012, 126(2): 372-390.

DOI：10.1093/toxsci/kfr345

• quinones
• human topoisomerase II alpha
• in silico modeling
• ATPase inhibition
• BEAS-2B human lung cells
• DNA double-strand breaks

The inhibition of human topoisomerase II alpha (Hu-TopoII alpha), 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-TopoII alpha 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-TopoII alpha. Biochemical inhibition assays with the purified ATPase domain of Hu-TopoII alpha revealed that 1,4-BQ is the most potent inhibitor followed by 1,4-NQ %26gt; 1,2-NQ %26gt; 9,10-PQ %26gt; 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 %26gt; 1,2-NQ %26gt; 9,10-PQ %26gt; 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-TopoII alpha-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-TopoII alpha. Furthermore, the increase in gamma-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-TopoII alpha. 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-TopoII alpha-mediated clastogenic and leukemogenic events.

• 出版日期2012-4