Elsevier

Biochemical Pharmacology

Volume 60, Issue 9, 1 November 2000, Pages 1367-1379
Biochemical Pharmacology

Novel mechanisms of DNA topoisomerase II inhibition by pyranonaphthoquinone derivatives—eleutherin, α lapachone, and β lapachone∗

∗ A preliminary account of this work was presented at the AACR-NCI-EORTC Conference held in Washington, DC, November 16–19, 1999.
https://doi.org/10.1016/S0006-2952(00)00437-8Get rights and content

Abstract

Pyranonaphthoquinones have diverse biological activities against Gram-positive bacteria, fungi, and mycoplasms, and, recently, there has also been an increasing interest in their anti-cancer activity. This study includes three derivatives: eleutherin (compound 1), β lapachone (compound 2), and its structural isomer, α lapachone (compound 3). The mechanism of topoisomerase II inhibition by the three derivatives was examined systematically with respect to the steps of the catalytic cycle of the enzyme. Etoposide, the prototypical enzyme poison, was used as a control and in combination with compounds 1–3 to localize their mechanism of action. The study revealed that eleutherin (1) and β lapachone (2) inhibited topoisomerase II by inducing religation and dissociation of the enzyme from DNA in the presence of ATP. Whereas compound 2 was an “irreversible” inhibitor of topoisomerase II, compound 1 merely slowed the catalytic cycle of the enzyme. α Lapachone (3), on the other hand, inhibited initial non-covalent binding of topoisomerase II to DNA and, in addition, induced religation of DNA breaks (even in pre-established ternary complexes) before dissociating the enzyme from DNA. Compound 3 was an “irreversible” inhibitor of topoisomerase II. The diverse and unique mechanisms of topoisomerase II inhibition by pyranonaphthoquinone derivatives reveal novel ways to target the enzyme with potential for anti-cancer drug design.

Section snippets

Materials

pBR322 was prepared by standard methods. Topoisomerase II (p170 isoform) was purchased from TopoGEN, Inc. HindIII-digested pBR322 was labeled with [α-32P]dCTP (3000 Ci/mmol, ICN Radiochemicals) using T4 DNA polymerase (GIBCO-BRL). Eleutherin (compound 1) was isolated from the bulb of E. americana, and was a gift from Dr. Y. Imakura (Naruto University of Education) [5]. β Lapachone and α lapachone were prepared according to methods published by Hooker [14]. Etoposide was provided by the Natural

Catalytic inhibition of topoisomerase II

Relaxation of supercoiled pBR322 by either topoisomerase I or II was measured in the presence of the three pyranonaphthoquinone derivatives to evaluate catalytic inhibition and DNA unwinding activities. Compounds 1-3 did not inhibit topoisomerase I, nor did they possess unwinding activities, suggesting that compounds 1-3 did not bind to DNA (data not shown). The compounds, however, were selective inhibitors of topoisomerase II. Figure 2 shows inhibition of the enzyme analyzed in a gel

Discussion

Several structurally diverse natural and synthetic compounds inhibit topoisomerase II at different steps of the catalytic cycle. A few such examples of catalytic inhibitors include aclarubicin and acridones (which inhibit the initial enzyme–DNA interactions 16, 20), merbarone (which inhibits enzyme-mediated DNA cleavage [21]), staurosporine (which inhibits strand passage by competing at the ATP binding site [22]), novobiocin (which inhibits ATP hydrolysis and prevents enzyme turnover [23]), and

Acknowledgements

This work was performed by P. K. in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

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