Induction of calf thymus topoisomerase II-mediated DNA breakage by the antibacterial isothiazoloquinolones A-65281 and A-65282

Abstract

A number of quinolones and related antibacterial compounds were screened for activity against calf thymus topoisomerase II by using the P4 unknotting and DNA breakage assays. Several compounds from different structural classes which inhibited DNA unknotting with 50% inhibitory concentrations ranging from 8 to 25 ,ug/ml were identified. Two experimental isothiazoloquinolones from this group, designated A-65281 and A-65282, were also found to induce considerable DNA breakage mediated by calf thymus topoisomerase II, with 32P-end-labeled pBR322 as the substrate. These compounds were nearly as potent as teniposide, with DNA breakage activity evident at concentrations as low as 4 ,ug/ml. However, some differences in DNA cleavage patterns from those with teniposide were evident. These studies have thus identified a new class of agents which have activity against both bacterial and eukaryotic type H topoisomerases. The implications of these data for the selectivity of topoisomerase-directed compounds and the potential toxicity of such compounds developed as antibacterial agents are discussed. topoisomerase II preparations (3,23,25,34,35). A similar pattern of effects has been demonstrated for the quinolone antibacterial agents. Treatment of bacteria with the quinolone prototype, nalidixic acid, results in extensive chromosomal damage (7, 11). Drug-induced DNA breakage can also be catalyzed by purified DNA gyrase (8,10,36). In related studies it was recently demonstrated that additional quinolone-binding sites appear upon the formation of gyrase-DNA complexes and that occupancy of these sites correlates with the occurrence of DNA breakage (30).In general, the agents directed against type II topoisomerases are specific for either bacterial or eukaryotic enzymes. However, in view of the mechanistic similarities and the sequence homologies shared by the bacterial and eukaryotic enzymes, the possibility exists that some agents interact with both classes. Indeed, several antibacterial quinolones which inhibit the P4 DNA-unknotting activity of various eukaryotic type II topoisomerases have been identified (1,15,17). Also, ciprofloxacin and several experimental fluroroquinolones induce topoisomerase II-mediated DNA breakage (1,12,24). These findings raised the possibility that additional classes of compounds that interact with both prokaryotic and eukaryotic enzymes will be identified. Additionally, they underscored the need to carefully assess the cross-reactivity of any topoisomerase-directed antibacterial agent against eukaryotic topoisomerases. Studies to address this important question and their implications for potential quinolone toxicity have been discussed in a recent review (13).We report here the identification of two closely related antibacterial isothiazoloquinolones, designated as A-65281 and A-65282, which are potent inhibitors of the P4-unknotting reaction catalyzed by calf thymus topoisomerase II. Moreover, these compounds are potent inducers of DNA breakage by calf thymus topoisomerase II. These data thus

Drug features that contribute to the activity of quinolones against mammalian topoisomerase II and cultured cells: correlation between enhancement of enzyme-mediated DNA cleavage in vitro and cytotoxic potential

“…Substitution of the aromatic C-7 4'-hydroxyphenyl ring by either an aliphatic 4'-hydroxypiperidine group 472) (35,42) and is supported by the fact that most quinolones with activity against the eukaryotic type II enzyme contain a C-7 ring with a hydrogen-bonding group either at the 4'-position (3,19,(39)(40)(41) or as a substituent off the 4'-position (18,31,32,41). A second and intriguing possibility is that the C-7 4'-hydroxyphenyl ring mimics the active-site tyrosine of topoisomerase II (26,29) …”

“…1) and CP-67,015, also were reported to stimulate DNA cleavage mediated by the eukaryotic type II enzyme (3). Since that initial report, a number of novel quinolones with activity against topoisomerase II, eukaryotic cells, or mammalian tumors have been described (8,9,18,19,31,32,37,(39)(40)(41). In contrast to clinically relevant quinolone antimicrobial agents, many of these latter compounds contain an aromatic substituent at the C-7 position (3,9,18,31,32,39,40).…”

CP-115,953 stimulates cytokine production by lymphocytes

“…However, maximally enhanced IL-2 production by CP-115,953 and ciprofloxacin is achieved at 25 and 282 M, respectively (Fig. 4 and 7) (29). Thus, a correlation between topoisomerase II inhibition and IL-2 production does not exist.…”

“…Fluoroquinolones in clinical use have, in general, been found to be only weak inhibitors of the eukaryotic topoisomerase II in in vitro assays exploring relaxation or catenation of supercoiled double-stranded DNA (19,25,26). However, a number of new fluoroquinolone derivatives with enhanced activity against eukaryotic topoisomerase II exhibited by their strong inhibitory effects on eukaryotic DNA replication have been characterized, and their structure-activity relationships have been determined (3,9,12,13,17,19,29,43,47,52). CP-115,953 (6,8-difluoro-7-[4Ј-hydroxyphenyl]-1-cyclopropyl-4-quinolone-3-carboxylic acid) is one of the most investigated quinolones with an increased effect upon eukaryotic topoisomerases (Fig.…”

Novel 1-8-bridged chiral quinolones with activity against topoisomerase II: stereospecificity of the eukaryotic enzyme

“…Recently, a number of quinolones with greatly increased potencies (relative to ofloxacin) toward eukaryotic systems have been reported (2,4,16,17,28,29, 3941). The most active of these novel compounds possess aromatic substituents at the C-7 position (2,16,28,29,39,40).…”