Cytotoxic effect of amide derivatives of trifluoromethionine against the enteric protozoan parasite Entamoeba histolytica

https://doi.org/10.1016/j.ijantimicag.2009.08.016Get rights and content

Abstract

Amoebiasis, caused by infection with the enteric protist Entamoeba histolytica, is one of the major parasitic diseases. Although metronidazole and its derivatives are currently employed in therapy, the paucity of effective drugs and potential clinical resistance necessitate the development of a novel drug. Trifluoromethionine (TFM) is a promising lead compound for antiamoebic drugs. To potentiate the antiamoebic effect of TFM, we synthesised various amide derivatives of TFM and evaluated their cytotoxicity. The amide derivatives of TFM were observed to have a superior cytotoxic effect compared with TFM and metronidazole against E. histolytica in vitro. Although TFM showed cytotoxicity following degradation by methionine γ-lyase, the derivatives were degraded by the enzyme less efficiently compared with TFM. We further demonstrated that a representative derivative was hydrolysed by the amoebic cell lysate to first yield TFM, followed by degradation similar to TFM. Hydrolysis was partially inhibited by protease inhibitors. A single subcutaneous or oral administration of TFM and its amide derivatives also effectively prevented the formation of amoebic liver abscess in a rodent model. These data demonstrate the improved effectiveness of TFM derivatives against E. histolytica infection and elucidate the mechanisms underlining the mode of action of these compounds.

Introduction

Amoebiasis is an infectious disease caused by the enteric protozoan parasite Entamoeba histolytica and is the second leading cause of death from parasitic diseases after malaria. Only metronidazole and related compounds are commonly used against invasive intestinal and extraintestinal amoebiasis [1], [2]. Although clinical resistance against metronidazole has not yet been demonstrated, sporadic cases of treatment failure have been reported [1]. In addition, it has been shown that this parasite easily adapts to therapeutic levels of metronidazole in vitro [3]. Resistance to metronidazole is also acquired easily by many bacterial species as well as Giardia intestinalis and Trichomonas vaginalis[1]. Therefore, the development of a novel antiamoebic drug is urgently required.

Pathways present exclusively in microorganisms but missing in humans may potentially represent a rational target for drug development. Entamoeba histolytica has several unique metabolic features. It lacks both the forward and reverse trans-sulphuration pathways that convert methionine to cysteine via cystathionine or vice versa, whilst it possesses l-methionine γ-lyase (MGL) (EC. 4.4.1.11) to decompose methionine, homocysteine and cysteine and produces ammonia, α-keto acid and volatile thiols [4]. MGL is present in only limited lineages of bacteria, parasitic protozoa and plants but is absent in mammals (see in [5]). In E. histolytica, two isoenzymes of MGL (MGL1 and MGL2) with distinct substrate specificities have been identified [5].

Trifluoromethionine (TFM), also know as S-trifluoromethyl-l-homocysteine, a fluorinated analogue of methionine, has been shown to be highly toxic to bacteria, Porphyromonas gingivalis, T. vaginalis and E. histolytica in vitro [6], [7], [8], [9]. It was also reported to treat infections by P. gingivalis and T. vaginalis in a rodent model [7], [8]. In the present study, the in vitro and in vivo efficacy of TFM and its derivatives were examined to gain insight into the structure–activity relationship and to determine the mechanism underlying their mode of action.

Section snippets

Chemical synthesis of trifluoromethionine and its derivatives

The synthetic scheme and structure of TFM and its analogues are shown in Supplementary Fig. 1.

Parasites and cultivation

Trophozoites of E. histolytica HM-1:IMSS cl6 were cultured axenically in BIS medium at 35.5 °C as described previously [10].

In vitro cytotoxicity assay of trifluoromethionine derivatives against Entamoeba histolytica trophozoites and Chinese hamster ovary (CHO) cells

Approximately 5 × 103 amoebae in 280 μL of medium were seeded into each well of a 96-well plate and incubated with various concentrations of TFM derivatives for 48 h. Following incubation, the amoebae were washed with 100 μL of pre-warmed Opti-MEM® I (Invitrogen, Carlsbad, CA) and

Results and discussion

The in vitro cytotoxicities of TFM, 15 TFM derivatives and 4 control compounds lacking fluorine were evaluated by measuring their 50% inhibitory concentration (IC50) values against E. histolytica trophozoites (Table 1). Whilst TFM showed an IC50 of 7.34 μM, the derivatives TFM-01 and TFM-02 had IC50 values of 2.22–2.28 μM. Three difluoroanilide compounds [2,3-difluoroanilide (TFM-03), 2,6-difluoroanilide (TFM-04) and 2,5-difluoroanilide (TFM-05)] also had a comparable 2.5–3.0-fold reduction in

Acknowledgments

The authors thank Rumiko Kosugi (Department of Parasitology, Gunma University Graduate School of Medicine, Gunma, Japan) for technical assistance. They also thank Mr Yuji Kakazu and Mr Akiyoshi Hirayama (Institute for Advanced Biosciences, Keio University, Yamagata, Japan) for technical help with mass spectrometry.

Funding: This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to DS (20590429) and TN (

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Present address: Department of Biochemistry, Rajendra Memorial Research Institute of Medical Sciences, Agam Kuan, Patna 800007, India.

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