Ether–lipid (alkyl-phospholipid) metabolism and the mechanism of action of ether–lipid analogues in Leishmania

Abstract

Ether–lipid (alkyl-phospholipid) analogues such as Miltefosine possess potent in vitro and in vivo anti-leishmanial activity and these compounds are currently undergoing clinical trials in humans. These analogues are also effective against Trypanosoma cruzi and Trypanosoma brucei subspecies but their mode of action is not known. Leishmania have high levels of ether–lipids and these are mainly found in the glycosylphosphatidylinositol-anchored glycolipids and glycoproteins present on the surface of the parasites. In Leishmania mexicana promastigotes we have studied both the initiating steps for the biosynthesis of ether–lipids, and key remodelling steps. The effect of Miltefosine and Edelfosine, on key enzymes involved in the metabolism of ether–lipids has been studied. The enzymes include dihydroxyacetonephosphate acyltransferase, sn-l-acyl-2-lyso-glycero-3-phosphocholine and sn-l-alkyl-2-lyso-glycero-3-phosphocholine acyltransferases. We confirm that the initiating steps in ether–lipid metabolism in Leishmania are present in glycosomes, and that Miltefosine or Edelfosine did not perturb these enzymes. The metabolism of the latter phosphatidylcholine base intermediates, which may be involved in the remodelling of acyl- and alkyl-glycerophospholipids, was also seemingly associated with glycosomes. Both Miltefosine and Edelfosine inhibited this microbody (glycosomal) located alkyl-specific-acyl-CoA acyltransferase in a dose-dependent manner with an inhibitory concentration of 50 μM. It is suggested therefore that a perturbation of ether–lipid remodelling could be responsible for the anti-leishmanial action of these drugs.

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.