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.
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.
Systems Toxicology and Health Risk Assessment Group, CSIR-Indian Institute of Toxicology Research, Lucknow-226001, Uttar Pradesh, India.
Deepak
D
Gurbani
Vandna
V
Kukshal
Julian
J
Laubenthal
Ashutosh
A
Kumar
Alok
A
Pandey
Sarita
S
Tripathi
Ashish
A
Arora
Swatantra K
SK
Jain
Ravishankar
R
Ramachandran
Diana
D
Anderson
Alok
A
Dhawan
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors*; D000951:Antigens, Neoplasm / Q000737:chemistry; D002460:Cell Line; D002853:Chromatography, Liquid; D004250:DNA Topoisomerases, Type II / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008958:Models, Molecular; D011809:Quinones / Q000494:pharmacology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology*
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D011809:Quinones; D059005:Topoisomerase II Inhibitors; D000251:Adenosine Triphosphatases; D004250:DNA Topoisomerases, Type II