title: Substituted purine analogues define a novel structural class of catalytic topoisomerase II inhibitors.
abstract: By screening 1,990 compounds from the National Cancer Institute diversity set library against human topoisomerase IIalpha, we identified a novel catalytic topoisomerase II inhibitor NSC35866, a S6-substituted analogue of thioguanine. In addition to inhibiting the DNA strand passage reaction of human topoisomerase IIalpha, NSC35866 also inhibited its ATPase reaction. NSC35866 primarily inhibited DNA-stimulated ATPase activity, whereas DNA-independent ATPase activity was less sensitive to inhibition. We compared the mode of topoisomerase II ATPase inhibition induced by NSC35866 with that of 12 other substituted purine analogues of different chemical classes. The ability of thiopurines with free SH functionalities to inhibit topoisomerase II ATPase activity was completely abolished by DTT, suggesting that these thiopurines inhibit topoisomerase II ATPase activity by covalently modifying free cysteine residues. In contrast, NSC35866 as well as two O6-substituted guanine analogues, O6-benzylguanine and NU2058, could inhibit topoisomerase II ATPase activity in the presence of DTT, indicating that they have a different mechanism of inhibition. NSC35866 did not increase the level of topoisomerase II covalent cleavable complexes with DNA, indicating that it is a catalytic inhibitor and not a poison. NSC35866 was also capable of inducing a salt-stable complex of topoisomerase II on closed circular DNA. In accordance with these biochemical data, NSC35866 could antagonize etoposide-induced cytotoxicity and DNA breaks in human and murine cancer cells, confirming that NSC35866 also functions as a catalytic topoisomerase II inhibitor in cells.
authors.affiliation: Department of Pathology, Diagnostic Centre, National University Hospital, Copenhagen, Denmark. lhjensen@rh.dk
authors.forename: Lars H
authors.initials: LH
authors.lastname: Jensen
authors.forename: Annemette V
authors.initials: AV
authors.lastname: Thougaard
authors.forename: Morten
authors.initials: M
authors.lastname: Grauslund
authors.forename: Birgitte
authors.initials: B
authors.lastname: Søkilde
authors.forename: Elisabeth V
authors.initials: EV
authors.lastname: Carstensen
authors.forename: Henrik K
authors.initials: HK
authors.lastname: Dvinge
authors.forename: Dominic A
authors.initials: DA
authors.lastname: Scudiero
authors.forename: Peter B
authors.initials: PB
authors.lastname: Jensen
authors.forename: Robert H
authors.initials: RH
authors.lastname: Shoemaker
authors.forename: Maxwell
authors.initials: M
authors.lastname: Sehested
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism; D018288:Carcinoma, Small Cell / Q000188:drug therapy / Q000201:enzymology; D002384:Catalysis; D045744:Cell Line, Tumor; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004273:DNA, Neoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006657:Histones / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000201:enzymology; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011687:Purines / Q000494:pharmacology*; D013866:Thioguanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
chemical_list: D000951:Antigens, Neoplasm; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006657:Histones; C504879:NSC35866; D011687:Purines; D059005:Topoisomerase II Inhibitors; D004250:DNA Topoisomerases, Type II; D013866:Thioguanine; D015739:Nocodazole