1 Department of Protozoology, Faculty of Tropical Medicine, Mahidol University, Bangkok; 4 Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand; 2 School of Biological Sciences, The University of Auckland, Auckland; 3 Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand
Received 14 July 2002; returned 5 February 2003; revised 11 February 2003; accepted 6 May 2003
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Abstract |
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Keywords: Trichomonas vaginalis, AT-specific drugs, DNA minor groove
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Introduction |
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DNA minor groove binding drugs (MGBs) are small compounds that bind specifically to the minor groove of B-DNA. MGBs can be divided into two classes based on their binding sequence preferences. One class of MGB (e.g. anthramycins) binds preferentially to GC sites by irreversible alkylation at the 2-amino group of guanines, but the majority of such compounds preferentially bind reversibly at AT-rich regions in the minor groove (AT-specific MGBs), without major distortion of the DNA structure.3 These drugs display a broad spectrum of antiviral, antibacterial, antitumour and antiprotozoal activity.4 DNA topoisomerase I may be the pharmacological target of AT-specific minor groove binding ligands.5
The AT-specific MGB class of bisquaternary quinolinium compounds (e.g. 4-[4-[4-[4(N-methylquinolinium)-amino]benzamido] anilino]-N-methylpyridinium, SN 6999; Figure 1) were developed initially as antileukaemic drugs.6 As the genome of T. vaginalis is heavily weighted in favour of AT base pairs (71%),7 we tested a series of compounds related to SN 6999 to determine whether they could exhibit antitrichomonal activity in culture.
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Materials and methods |
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A local strain of T. vaginalis employed in this study was obtained from a patient at Rajavithi Hospital, Bangkok, Thailand.
Axenic culture of T. vaginalis
Trypticase yeast extract maltose (TYM) medium was used for culturing T. vaginalis. A single colony of T. vaginalis was obtained aseptically using the agar plate culture technique8 and was transferred to a screw-capped tube containing 7 mL of TYM medium. One millilitre of inactivated human serum was then added to the medium as nutrient supplement. To prevent bacterial contamination of the culture, 1000 U/mL of penicillin G and 500 µg/mL of streptomycin sulphate were used. Cultures were incubated at 37°C and subcultures were conducted every 23 days.
Drug preparation
Bisquaternary quinolinium compounds were initially dissolved in DMSO at a concentration of 9 mM. Metronidazole was dissolved in sterile distilled water at a concentration of 1 mM. Working drug solutions were freshly prepared before use by diluting with TYM medium to the required concentrations.
Test of AT-specific minor groove binding drugs on T. vaginalis
Standard microtitre plates with 96 flat-bottomed wells were used in the test. Drugs of various concentrations were added to the wells in 50 µL aliquots. A 200 µL suspension containing 1 x 104 T. vaginalis was then placed into each well. All the drug and control studies were carried out in duplicate; a minimum of two independent experiments was conducted for each drug tested. The plates were incubated at 37°C and were examined after 24 h with an inverted microscope (25x magnification) for motile trichomonads. The minimal inhibition concentration (MIC) is defined as the lowest drug concentration in which no motile organism was seen. DMSO at 2% (v/v) concentration had no effect on parasite motility.
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Results and discussion |
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Structureantileukaemic activity relationships of bisquaternary quinolinium compounds have been analysed previously.9 For this activity, there was an absolute requirement for a quaternary centre; the free bases where R1 = H showed no antileukaemic effect, whereas the methyl bisquaternary salt (R1 = Me) was highly inhibitory, and there was relatively little difference between R1 = Me and R1 = Et. Derivatives with electron donor substituents (CH3, OCH3, NO2, NH2) in the quinoline ring provided the most active antileukaemic compounds. Both 6- and 7-aminoquinoline variants were also highly active, and showed less chronic toxicity, with several 6-aminoquinoline variants giving a high percentage of indefinite survivors in L1210 tests.10
Against T. vaginalis, in the R1 = Me series, NH2 substitution at R2 (compound 2) or 5-NH2 at R3 (compound 3) were less effective than the unsubstituted compound 1. The MICs for compounds with substituents (CH3, OCH3, NMe2, NH2) in the quinoline ring providing electron donation to the 4-NH position were also higher than for 1. Several 6- and 7-aminoquinoline variants (compounds 7, 8 and 11), highly active as antileukaemic compounds, also showed low comparative efficacy against T. vaginalis. Again in contrast to the antileukaemic activity, compounds with substituents (6-Cl, 7-Cl, 8-OMe; compounds 9, 10, 13) providing electron withdrawal from the 4-NH position were more active than those with electron-donating groups (although still not as active as the unsubstituted compound 1). Amino group substitution on the anilino ring (R3) in compounds 3 and 7 also decreased the activity against T. vaginalis.
The most active compound, 1, was almost as potent as metronidazole against wild-type T. vaginalis. This compound has also been shown to have relatively low toxicity to human cells (IC50 Jurkat leukaemia cells > 20 µM; R.K. Ralph, unpublished data). Although the MGB compounds were not tested against metronidazole-resistant strains of T. vaginalis, the targets of the two drugs are different. Whether or not this renders cross-resistance unlikely must be tested in future studies. More work needs to be done to decide whether bisquaternary quinolinium compounds can be successfully developed for use in the chemotherapy of trichomoniasis. In particular, the nature of the substitution at R1 should be studied further.
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Acknowledgements |
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Footnotes |
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References |
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