Ether–lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether–lipid analogues in Leishmania
Introduction
Leishmaniasis is a tropical and subtropical disease that effects at least 12 million people and causes considerable morbidity and mortality [1]. Clinical treatment is almost entirely dependent upon the use of the pentavalent antimonial drugs such as sodium stibogluconate (Pentostam) and N-methylglucamine antimonate (Glucantime). Both antimonial drugs require high dose regimens and long treatment courses using parenteral administration making them unreliable for wide-scale use [2]. Furthermore, resistance to antimonial drugs has been reported in the fatal form of the disease visceral leishmaniasis [3].
Inhibitors of sterol and phospholipid biosynthesis in kinetoplastid parasites such as Trypanosoma cruzi and different species of Leishmania have been shown to possess potent and selective activity as chemotherapeutic agent's [4]. In addition, ether–lipid (alkylphospholipid) analogues have also attracted much recent attention (reviewed in [5]). These analogues have been shown by a number of groups to possess potent in vitro and in vivo anti-leishmanial activity [6], [7], [8], [9], [10], [11], [12], [13].
In addition, when used orally these analogues exhibited not only outstanding anti-leishmanial activity in mice, especially when compared to Pentostam, but also demonstrated few side effects at therapeutically active dosages [14]. Alkyl-phospholipid analogues were also shown to be effective against T. cruzi and Trypanosoma brucei subspecies [12], [15]. Most recently a topical formulation of Miltefosine (Miltex™) has been used highly successfully to treat experimental cutaneous leishmaniasis [16].
Ether–lipid analogues are also a novel class of anti-cancer agents that possess both in vivo and in vitro anti-neoplastic activity [17], [18], [19], [20], [21], [22]. They exert their anti-proliferative activity, not via classical cytostatic effects, but rather by interfering with phosphoinositol metabolism and signal transduction pathways. In addition, they have also been shown to have pro-differentiation activity and can induce programmed cell death.
Since the late 1970's Leishmania have been known to contain high levels of ether-phospholipids [23], [24], [25], [26]. For example, most of the abundant and seemingly essential glycolipids present on the surface of the parasites are attached to the membrane by alkyl-glycerolipids [27]. Previous studies by Lux et al. [28] have suggested that the anti-leishmanial mechanism of action of alkyl-phospholipid analogues would seem to have several putative targets. These targets included perturbation of alkyl–lipid metabolism and the biosynthesis of alkyl-anchored glycolipids (e.g. LPG) and glycoproteins (e.g. GP63). Here we extend these studies and describe the effects of ether–lipid analogues upon key enzymes involved in acyl- and alkylglycero-phosphocholine metabolism in Leishmania mexicana, including DHAP acyltransferase, acyl-CoA: sn-l-acyl-2-lyso-glycero-3-phosphocholine and sn-l-alkyl-2-lyso-glycero-3-phosphocholine acyltransferases.
Section snippets
Parasites
Leishmania mexicana mexicana promastigotes (MHOWBZ/84/BEL46 and MNYC/BZ/62/M379) were grown in vitro in a semi-defined medium (SDM) supplemented with 10% heat-inactivated fetal calf serum (HI-FCS) as described before [29].
Treatment of ether–lipid analogues
Ether–lipid analogues were stored as 10 mg ml−1 stock solutions in methanol and diluted in SDM containing 10% HI-FCS and 1% dimethyl sulfoxide (DMSO) to give a range of final drug concentrations from 50 to 0.1 μM and drug free control media contained comparable final
Effects of Miltefosine and Edelfosine on promastigotes in culture
Although ether-lipid analogues, such as Miltefosine and Edelfosine, have been well documented as being cytotoxic to Leishmania spp. using in vitro proliferation assays [5], a highly sensitive metabolic assay has not been described. We therefore tested the effects of Miltefosine and Edelfosine, on L. mexicana promastigotes using the Alamar Blue™ viability test. After 72 h of drug treatment in our in vitro culture system both drugs were found to be highly cytotoxic in a concentration dependent
Discussion
From the luminary work of three groups Beach et al. [23]; Hermann and Gercken [24], [25] and Wassef et al. [26] the relatively high abundance of ether–lipids, in leishmanial species is well established. Alkyl-lipids were shown to be an important class of membrane lipid and were predominantly found to have ethanolamine, as the phospho-base group. Other bases, such as choline, were either very low or not detected as major end products of leishmanial ether–lipid biosynthesis. However, later the
Acknowledgments
We thank Frank Menke, Dominique Cottem and Joris van Roy for technical assistance. This investigation received support from ASTA Medica AG, The National Bank of Belgium (Grant 3.4511.96) and the ‘Fonds National de Recherche Scientifique’. N.H. was the recipient of the Michel de Visscher Post-Doctoral Fellowship from the Christian de Duve Institute of Cellular Pathology (Belgium) and received financial support from ‘Conselho Nacional de Desenvolvimento Cientifico e Tecnológico-CNPq’ and
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Present address: Instituto de Microbiologia da Universidade Federal do Rio de Janerio (UFRJ), Centro de Ciências da Saúde Bloco I, Cidade Universitária, Iiha do Fundão, Rio de Janeiro-RJ, 21944-970, Brazil.