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Antimicrobial Agents and Chemotherapy, August 1999, p. 2066-2068, Vol. 43, No. 8
Division of Clinical Pharmacology, Department
of Medicine, Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205-2185
Received 9 March 1999/Returned for modification 4 May 1999/Accepted 27 May 1999
Six fluoroquinolones presently in clinical use and four
investigational tetracyclic fluoroquinolones were tested for in vitro activity against bloodstream-form Trypanosoma brucei
brucei. All compounds had measurable activity, but the
tetracyclic analogs were most potent, with 50% effective
concentrations in the low micromolar range. In general, trypanosomes
were more susceptible than L1210 leukemia cells. Consistent with the
notion that they target type II topoisomerase in trypanosomes, the
fluoroquinolones promote the formation of protein-DNA covalent complexes.
African trypanosomes are the
parasitic protozoa that cause sleeping sickness, a disease that has
undergone a dramatic and devastating resurgence in recent years
(28). Untreated, sleeping sickness is fatal, and the
treatment options presently available are increasingly limited
(14, 19, 21). This unfortunate situation also pertains to
diseases caused by closely related pathogens, the American trypanosome
and Leishmania spp. The need for new molecular targets on
which to base future treatment strategies is clear and immediate.
Promising targets for new broad-spectrum antitrypanosomal drugs are the
DNA topoisomerases (3, 7). Poisoning of these enzymes is the
molecular mechanism of action for clinically useful antitumor and
antibacterial agents (9, 11). These compounds act by
stabilizing intracellular DNA-topoisomerase complexes. Upon the
addition of alkali or strong denaturants, the enzyme may be recovered
covalently attached to its DNA substrate (18). Inhibitor-induced covalent complexes have been used to demonstrate the
existence of both mitochondrial and nuclear topoisomerases in
trypanosomes (25, 27). A number of investigators have
reported that fluoroquinolones (which target type II topoisomerases in prokaryotes) have activity in vitro or in vivo against trypanosomes or
Leishmania (1, 10, 12, 22-24, 32). However, none
of these studies provides experimental evidence that the observed antiparasitic effect is a result of topoisomerase poisoning. We evaluated an array of fluoroquinolones against Trypanosoma brucei brucei in vitro to determine whether they are cytotoxic and
whether they promote the formation of protein-DNA complexes.
Fluoroquinolones.
Norfloxacin was obtained from Merck Sharp & Dohme (West Point, Pa.), enoxacin from Parke-Davis, Pharmaceutical
Research Division, Warner-Lambert Company (Ann Arbor, Mich.),
ciprofloxacin from Miles, Inc. (West Haven, Conn.), pefloxacin from
Rhone-Poulenc Rorer, (Mexico City, Mexico), fleroxacin from Hoffmann-La
Roche Inc. (Nutley, N.J.), and ofloxacin from R.W. Johnson
Pharmaceutical Research Institute (Raritan, N.J.). KB-5246, KB-5290,
KB-6600, and KB-6625 were supplied by Kanebo, Ltd. (Osaka, Japan)
(16, 17, 30). VM26 was a kind gift from Leroy Lui (Robert
Wood Johnson Medical School, Piscataway, N.J.). Stock solutions were prepared as follows: ciprofloxacin, pefloxacin, fleroxacin, and KB-5246
were dissolved in sterile water; norfloxacin, enoxacin, ofloxacin,
KB-5290, KB-6600, and KB-6625 in 100 mM NaOH; and VM26 in dimethyl sulfoxide.
Assays.
Bloodstream-form T. brucei (MiTat 1.2, strain 427) organisms were grown axenically (8) in phenol
red-free medium, as we described previously (2). L1210 (ATCC
CCL-219) mouse leukemia cells were maintained in phenol red-free RPMI
1640 supplemented with 15% heat-inactivated fetal bovine serum (Life
Technologies). Ten concentrations of each fluoroquinolone were assayed
in quadruplicate. Exponentially growing cells were incubated with or
without fluoroquinolone for 20 h, then lysed and incubated for 3 to 6 h with p-nitrophenyl phosphate. Acid phosphatase
activity was determined, and 50% effective concentrations
(EC50) were obtained (2, 4). Covalent
protein-DNA complexes were assayed by the potassium sodium dodecyl
sulfate (KSDS) method as described previously (3, 25), with
the modification that cultured trypanosomes were labeled with
[methyl-3H]thymidine (NET027E; 20 Ci/mmol; New
England Nuclear, Boston, Mass.) at 8.3 µM in the medium.
Antitrypanosomal activities of fluoroquinolones.
We tested six
commercially available fluoroquinolones and four experimental
tetracyclic fluoroquinolones for antitrypanosomal activity against
axenically cultured bloodstream-form T. brucei (Table
1). Each of these compounds was
cytotoxic, not cytostatic, in the assay. All ten had measurable
activity, with EC50 that varied some 100-fold. Against
trypanosomes, norfloxacin had an EC50 in the micromolar
range, which is somewhat improved by N substitution at X8
(enoxacin) or by substituting a cyclopropyl group at R1
(ciprofloxacin). Conversely, activity was somewhat reduced by addition
of an N-methyl group to the R7 piperazine side
group (pefloxacin) or, more markedly, by addition of fluorine to the
R1 ethyl and X8 sites (fleroxacin). Activity
was similarly diminished by bridging the X8 and
R1 sites to form a tricyclic compound (ofloxacin).
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Antitrypanosomal Activity of
Fluoroquinolones
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TABLE 1.
Antitrypanosomal activities of
tested fluoroquinolones
Selective toxicity. For all compounds but pefloxacin, toxicity was as much as 2.6-fold greater for trypanosomes than for L1210 mammalian cells (Table 1). Though modest, this margin of difference is encouraging and may be even greater between parasites and nonmalignant mammalian cells. L1210 cells are nonadherent and have a rapid doubling time of about 14 h, ideal characteristics for our assay system. However, the topoisomerase II content of such cells is usually higher than that of nonmalignant cells (6, 15). Because the degree of toxicity is related to the number of complexes that are formed (20, 29), use of topoisomerase II-rich L1210 cells likely leads to overestimation of the toxicity to normal host tissues.
The tetracyclic fluoroquinolones exhibited twofold-greater toxicity toward T. brucei than toward L1210 cells. Variants of the piperazine or pyrrolidine group at R8 (nucleus C [Table 1]) may further improve this selective toxicity. For example, in a series of sparfloxacin analogs, trans-3,5-dimethylpiperazine at R7 (nucleus A [Table 1]) was more than 50 times more active than its cis isomer at inducing mammalian topoisomerase II-mediated DNA breaks (13). However, the two isomers were equally active in stimulating gyrase-mediated cleavage. Screening of tetracyclic fluoroquinolones with 3,5-dimethylpiperazine or other piperazinyl or pyrrolidinyl moieties at R8 may allow discovery of an analog that is similarly selective between mammalian and trypanosome topoisomerase II.Protein-DNA complexes.
Fluoroquinolone-promoted protein-DNA
complexes are detectable in African trypanosomes (Table
2), and there is a tendency for complex
formation to correlate with antiparasitic activity. The tetracyclic
compounds, which are most cytotoxic, trapped 4 to 11% of labeled DNA,
whereas the less potent fluoroquinolones trapped only 1 to 3% of the
label, effects that are dose related (data not shown). A striking
exception is KB-5246, which has unexpectedly low complex formation
given its antiparasitic potency (Table 2). Although this finding may be
explained in a variety of ways (e.g., compound penetration into cells),
one intriguing possibility is that the cytotoxicity of KB-5246 is
attributable, at least in part, to protein-DNA complex formation with
the topologically highly complex mitochondrial DNA (kDNA) of these
organisms (26). The KSDS assay primarily reports complex
formation that is nuclear in origin, since approximately 96% of the
DNA in T. brucei is nuclear (5). Supporting
evidence exists for the possibility of distinct nuclear and
mitochondrial topoisomerase II forms in kinetoplastids and other
parasites, and these enzymes may be distinguished from one another by
differing drug susceptibilities (25, 31). Perhaps the
bicyclic fluoroquinolones we tested target a mitochondrial topoisomerase II in T. brucei, whereas the tetracyclic
fluoroquinolones target both mitochondrial and nuclear enzymes.
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ACKNOWLEDGMENTS |
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We thank Goro Tsukamotomo of Kanebo Ltd. New Drug Research Laboratories for his interest in this work and for providing the tetracyclic fluoroquinolones. Lourdes Juan Lopez of Rhone-Poulenc Rorer, S.A. de C.V., kindly and effectively expedited our request for pefloxacin. We thank Annette Bodley, Donna Klinedinst, and Suji Xie for helpful scientific discussions and technical assistance.
This work was supported by Public Health Service grant AI28855 from the National Institutes of Health, by the Burroughs Wellcome Fund, by the Swiss National Research Foundation (C.B.), and by the Janggen-Poehn Foundation (C.B.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Clinical Pharmacology, Department of Medicine, Johns Hopkins University School of Medicine, 301 Hunterian Building, 725 North Wolfe St., Baltimore, MD 21205-2185. Phone: (410) 955-1888. Fax: (410) 955-2634.
Present address: Swiss Tropical Institute, Department of Medical
Parasitology and Infection Biology, 4002 Basel, Switzerland.
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Antimicrobial Agents and Chemotherapy, August 1999, p. 2066-2068, Vol. 43, No. 8
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[Abstract] [Full Text]
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