Membrane lipidomics for the discovery of new antiparasitic drug targets

Review

Membrane lipidomics for the discovery of new antiparasitic drug targets Eric Mare´chal1*, Mickae¨l Riou2*, Dominique Kerboeuf3*, Fre´de´ric Beugnet4*, Pierre Chaminade5* and Philippe M. Loiseau6* 1

Laboratoire de Physiologie Cellulaire Ve´ge´tale, UMR 5168, CNRS-CEA-INRA-Universite´ Joseph Fourier, Institut de Recherches en Sciences et Technologies pour le Vivant, CEA Grenoble, 17 rue des Martyrs, Grenoble, France 2 INRA, UE 1277 Plateforme d’Infectiologie Expe´rimentale (PFIE), 37380 Nouzilly, France 3 INRA, UR 1282 d’Infectiologie Animale et Sante´ Publique (IASP), 37380 Nouzilly, France 4 Me´rial, 29 avenue Tony Garnier, 69007, Lyon, France 5 Universite´ Paris-Sud, Groupe de Chimie Analytique de Paris Sud, EA4041, Faculte´ de Pharmacie, 5 rue J-B Cle´ment, 92296 Chaˆtenay-Malabry, France 6 Universite´ Paris-Sud, Groupe Chimiothe´rapie Antiparasitaire, UMR 8076 CNRS, Faculte´ de Pharmacie, 5 rue Jean-Baptiste Cle´ment, 92296 Chaˆtenay-Malabry, France

Advances in lipid separation methods and mass spectrometry technologies allow the fine characterization of the lipidome of parasites, ranging from unicellular protists to worms, which cause threatening infections in vertebrates, including humans. Specific lipid structures or lipid metabolic pathways can inspire the development of novel antiparasitic drugs. Changes in the lipid balance in membranes of parasites can also provide clues on the dynamics of drugs and some mechanisms of drug resistance. This review highlights recent trends in parasite lipidomics, combined with functional analyses, for the discovery of novel targets and the development of novel drugs. Why target the membrane lipidome of parasites and how? The life cycles of parasites imply numerous and rapid cellular divisions to invasively proliferate in their hosts and compensate losses when going from one host to another. In infected vertebrates, the populations of Kinetoplastida or Apicomplexa unicellular parasites are nearly innumerable. A Leishmania population can count more than 100 millions of parasites in a patient’s spleen and circulating blood [1], and Plasmodium falciparum can reach population levels of hundreds of millions [2] to hundreds of trillions [3] in human patients with normal hematocrit. In the case of parasitic helminths, such as Trematoda circulating in blood vessels and colonizing the liver, pancreas or lung, female worms can produce from 300 eggs/day for Schistosoma mansoni [4] to thousands for Schistosoma japonicum. Intestinal Nematoda such as roundworms (Ascaris, Haemonchus), whipworms (Trichuris) and hookworms (Necator) produce 3000 to 200 000 eggs/day and per female [5]. One issue raised by this dynamic proliferation of parasites is the very rapid propagation of mutants resisting a drug. Therefore, resistance triggered by chemotherapy

*

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Corresponding author: Mare´chal, E. ([email protected]). CAP: Consortium anti-Parasitaire/Chemotherapy against Parasites.

Glossary Atmospheric pressure chemical ionization (APCI): technique used in mass spectrometry (MS) to produce ions at atmospheric pressure. APCI is adapted to the ionization of non-polar (e.g. triacylglycerol) to very polar molecules, with a molecular weight ranging from around 50 to 1000 kDa. Atmospheric pressure photo ionization (APPI): technique used in MS to produce ions after exposure of analytes to UV light photons. APPI is adapted to the ionization of non-polar (e.g. triacylglycerol) to polar molecules, with a molecular weight ranging from around 100 to 2000 kDa. Biomimetic membrane: in vitro reconstitution of a biological membrane, based on the self-organization of polar lipids in mono- or bilayers. Electrospray ionization (ESI) and nanoESI: technique used in MS to produce ions, generating ions in a gas phase without fragmentation of the sprayed sample. ESI is adapted to the ionization of polar to very polar molecules, with a molecular weight ranging from around 100 to 100 000 kDa. ESI is currently the most widely used ionization technique in lipidomics. Disadvantages include the cost and a phenomenon known as ion suppression in the case of lowabundance species in complex mixtures. Absolute quantification of lipids requires class and mass independent internal standards. Miniaturized ESI, known as nanoelectrospray or nanoESI, allows the automated analysis of very large samples sets. Gas chromatography (GC) and GC-MS: method separating and analyzing compounds that can be vaporized without decomposition, based on their selective retention in the matrix of specific columns. GC coupled to flame ionization detection (GC-FID) allows the detection of micrograms to lower nanograms of vaporizable lipids. GC is very widely used for the determination of fatty acid profiles in glycerolipids. GC can be coupled to a mass spectrometer (GC-MS). Glycolipid: a glycerolipid or a phospholipid, with a polar head containing a sugar. High-performance TLC (HPTLC): automated thin layer chromatography (TLC) system allowing the parallel analysis of a large number of samples, with higher throughput and more sensitive detection of lipids. Most recent HPTLC lines include devices that automatically couple TLC with MS (TLC-MS). KEGG: database of metabolic pathways developed by the Kyoto Encyclopedia of Genes and Genomes consortium. Langmuir monolayers: type of biomimetic membrane made by the selfassembly of lipids in a monolayer at the air–water interface. Lipid extraction: procedure by which lipids are extracted from a mixture, based on their specific solubility in organic solvents. Usually after addition of solvents, a biphasic organic–water system is obtained and lipids are purified in the organic phase. Lipid profile: proportions of each lipid class detected and quantified in a sample. A lipid profile is usually shown as a histogram. Lipid raft: membrane microdomains characterized by a specific combination of glycosphingolipids, sterols and protein receptors, more ordered and tightly packed than the surrounding bilayer and involved in important biological functions. In the plasma membrane of parasites, lipid rafts are believed to be important for pathogenic processes. Liquid chromatography (LC) and LC-MS: method separating and analyzing compounds in a liquid solution, based on their selective retention in the matrix

1471-4922/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2011.07.002 Trends in Parasitology, November 2011, Vol. 27, No. 11

Review of specific columns. High-pressure liquid chromatography (HPLC) allows the separation of lipids solubilized in organic solvents and can be coupled to a mass spectrometer (LC-MS). Mass spectrometry (MS): analytical technique that measures the mass-tocharge ratio of charged (ionized) molecules. A mass spectrometer is composed of an ion source, a mass analyzer and an ion detector. The mass analyzer can be a quadrupole (Q), a time-of-flight analyzer (TOF) or an ion trap. TOF has a very high mass accuracy. MS data are eventually represented by a spectrum composed of peaks corresponding to mass-to-charge ratios (m/z). MS peaks are specific for fragments of ionized lipid molecules. MS sensitivity allows the detection of picograms to fentograms of lipids. Matrix-assisted laser desorption/ionization (MALDI) and MALDI-TOF: versatile technique used in MS to produce ions, allowing the analysis of biomolecules which tend to be fragile and fragment when ionized by other ionization methods. MALDI is often used to ionize biomolecules upstream a time-of-flight analyzer (MALDI-TOF). Mass-to-charge ratio (m/z): charged particles move in electric and magnetic fields following a path which is strictly dependent on their mass-to-charge ratio. MS peaks therefore correspond to the m/z quantities of the charged fragments hitting the detector. Neutral fragments cannot be detected. When the fragment is positively charged, this quantity is positive and vice versa. When containing only one charge, the m/z quantity corresponds to the mass of the fragment. MetaCyc: database of metabolic pathways developed by the BioCyc consortium. Neutral loss scan: a tandem MS mode used when a molecular ion, produced after fragmentation of a lipid class, loses a specific uncharged fragment. Because uncharged fragments cannot be detected, the specific loss of the corresponding neutral fragment is recorded. The lipid class can thus be characterized in a complex mixture by selecting only the initial molecular ions that lose this specific neutral fragment. Phosphatidylserine species can, for instance, be detected by constant neutral loss scans of m/z 87. Phospholipid: a glycerolipid or a sphingolipid, which has a polar head containing a phosphate. Polar head group: hydrophilic moiety of polar lipids. Polar lipid: general term defining lipids having a hydrophobic moiety and a hydrophilic polar head. Polar lipids make up the bulk of biological membranes. Precursor ion scan: a tandem MS mode used when a molecular ion, produced after fragmentation of a lipid class, loses a specific charged fragment, which is then easily detected. The lipid class can thus be characterized in a complex mixture by selecting only the initial molecular ions that give this specific charged fragment. In negative ion mode, the dissociation of lipids induced in the collision chamber of a mass spectrometer can thus allow the detection of phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerol and phosphatidylcholine by precursor ion scans of m/z –153, –196, –241, –153 and +184. Quadrupole mass analyzer (QMS) and triple quadrupole (QQQ): a QMS is a part of a mass spectrometer, consisting of four parallel circular rods, filtering sample ions based on their mass-to-charge ratio (m/z). Ions are thus separated based on the stability of their trajectories in the oscillating electric fields applied to the rods. Owing to the linear trajectory of ions, a QMS can be aligned in series of quadrupoles in so-called triple quadrupoles (QQQ). In a QQQ, the first QMS allows an initial analysis of ions (based on their m/z ratio) and a selection of ions of interest for subsequent fragmentation and refine analysis. The second quadrupole is then used as a collision chamber generating fragments of the ions filtered in the first QMS. The final refined MS analysis is eventually achieved in the third QMS. QQQ are tandem MS instruments that are very useful for the analysis of complex lipid mixtures. Raft-like structures (RLS): membrane microdomains in which structure and composition is similar to that of lipid rafts. Tandem MS: also known as MS/MS, tandem MS involves multiple steps of MS selection. Between each step, a fragmentation and selection of molecular ions occurs. Tandem MS can be carried out ‘in space’ when the two MS steps are physically separated in specific instruments (triple quadrupoles or QQQ, or a quadrupole and a time-of-flight analyzer in QTOF instruments). Tandem MS can be carried out ‘in time’ in ion trap analyzers. Different modes of analyses are classically achieved with tandem MS, including the precursor ion scan and neutral loss scan modes. Thin layer chromatography (TLC): method of separating and analyzing molecules such as lipids on a sheet of glass, plastic or aluminum foil, coated with a thin layer of absorbent material, usually silica gel. Typically inexpensive and very well established, this technique has a low resolution in direct semiquantification using lipid specific dyes; however, it allows the detection of low micrograms of lipids when coupled to GC, and in most recent techniques, it is efficient in the picogram range, when automatically coupled to MS. Time-of-flight (TOF) and quadrupole time-of-flight (QTOF) mass analyzers: a TOF is part of a mass spectrometer, in which the mass-to-charge ratio (m/z) of ions is determined after their acceleration by an electric field of known strength and record of the time it takes the ion to reach a detector. TOF MS has a very high mass accuracy. A QTOF is a tandem MS instrument in which an initial quadrupole filters sample ions based on their mass-to-charge ratio (m/z). After fragmentation, the filtered ions are then separated in a TOF analyzer.

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spreads rapidly, resulting in a seemingly never-ending challenge for medical and veterinary research. Efforts to develop novel drugs must consequently be sustained. Additionally, membrane expansion for cell division and differentiation, for the elaboration of extracellular barriers, requires a huge resource of lipids. Impairment of lipid biosynthesis thus appears as a potential strategy of choice to fight against parasites. Is the membrane lipidome then targetable for therapeutic purposes? In its simplest definition, a drug target is a component of the parasite, of which impairment is lethal or prevents the ability of a pathogen to proceed in its infectious cycle. Basically, one searches for a vital enzyme, metabolic pathway or dynamic process, absent from the host or sufficiently different, so that developed drugs have little to no adverse effects [6]. The membrane lipidome is constituted of three major categories of lipids, that is glycerolipids, sphingolipids and sterols (Figure 1). To make the bulk of a membrane in a eukaryotic cell, few sterol structures and no more than eight classes of glycerolipids are required, mainly phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG) and mitochondrial diphosphatidylglycerol (DPG). Differential phosphorylation of the inositol polar head (see Glossary) generates seven distinct phosphoinositides, which are quantitatively minor, but essential determinants of the identity of membrane compartments of the endomembrane system. In addition, some membranes, and particularly the plasma membrane, contain sphingolipids and sterol-rich domains, known as rafts, and high proportions of glycolipids with polar heads harboring species-specific glycosylated moieties. To identify specific targets, a first objective of the lipidome mining is thus to identify novel lipid structures and demonstrate that blocking their synthesis provokes severe effects. Search of unique glycolipids has thus been very prolific. Ubiquitous lipids, such as PC, would naively appear as unlikely targets; nevertheless, PC biosynthesis is precisely one of the current promising targets for a novel series of antimalarials [7]. A second objective of membrane lipidome mining is to link lipid profiles with metabolic pathways, because identical lipid structures can be synthesized by distinct pathways. Eventually, lipids are not restricted to membranes; they can serve to store carbon or energy, such as triacylglycerol, or act as signal molecules, such as diacylglycerol, phosphatidic acid or oxygenated fatty acids. Lipidome mining should therefore link membrane lipid profiles with any non-lipid metabolic or signal transduction processes that would be indirectly related to the membrane lipidome and be essential for the life cycle of a parasite. Once a biosynthetic pathway has been validated as an antiparasitic target, the next question is whether it is susceptible to any drugs. Some enzymes involved in synthesizing or modifying lipids are soluble proteins, but most are membrane bound. These enzymes can be surrounded by biological membranes which the drug would need to be able to cross. For instance, the fatty acid synthase of type II (FASII), discussed below in the case of Apicomplexa, is located inside an organelle known as the apicoplast, in the cytosol of the parasites. A drug candidate targeting a 497

Review

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Glycerolipids CH2OOCR1 R2COO

Main precursors:

CH2OOCR1

CH2OOCR1

O NH

R2COO C H

H

C

R2COO C

N O

CH2 - P - P -CH2 -

CH2- P

H

CH2OH

OH OH

Phosphatidic acid (PA)

CH2 OOC(18:2) (18:2)COO

C

-

C

(20:3)COO

CH2 - P

CH2

CH2 + N

CH2 + N H 3

CH3

Phosphatidylcholine (PC)

(18:3)COO

H -

CH2

CH3

C

-

Diacylglycerol (DAG)

C

(16:0)COO

H

CH2 - P

CH2 OOC(18:3)

CH2 OOC(18:3)

CH2 OOC(18:3)

CH2 OOC(18:0)

H

CH2 - P

CH2 - P

H3C

CH2 OOC(18:2) (18:2)COO

H

Cytidine diphosphate diacylglycerol (CDP-DAG)

C

(16:0)COO

H

C H CH2 - P - H 2C

CH2 - P -OH2 C

-

H

inositol

CH2 CH-COO + N H3

C

H C CH2 - P - H2 C

OH

HOH2C (16:0)COO

C

OH

H

CH2 OOC(18:3)

Phosphatidylethanolamine (PE)

Phosphatidylserine (PS)

Phosphatidylinositol (PI)

Phosphatidylglycerol (PG)

Sphingolipids

Diphosphatidylglycerol (DPG)

Sterols

Main precursors: OH

Main precursors:

OH

NH3+

Sphingoid base (e.g. sphingosine) Squalene OH

OH H3C

RONH

H3C CH3

Ceramide = sphingosine + amide-acyl (R)

CH3 H

CH3 OH

P

CH2 CH2 N+

CH3 CH3

H

HO

CH3

CH3

Sterols (e.g. ergoserol occuring in Leishmania)

RONH

Phosphocholine ceramide

OH

P

CH2 CH2

NH3+

Key: RONH

: Fatty acid

Phosphoethanol ceramide CH2OH O

OH

P

: Phosphate : Phospholipids (glycerolipids and sphingolipids)

β-galactose

O

RONH

Cerebroside (e.g. monogalactosylcerebroside) TRENDS in Parasitology

Figure 1. Major classes of lipids of the membrane lipidome. The three main classes of membrane lipids (i.e. glycerolipids, sphingolipids and sterols) are shown with their polar heads on the right. Acyls are shown in grey boxes. The main glycerolipids are assembled by esterification of the sn-1 and sn-2 positions of a glycerol backbone with acyls and a polar head at position sn-3. These glycerolipids are neosynthesized from three precursors (PA, DAG and CDP-DAG). In some cases, fatty acid chains can be linked via ether or vinyl ether linkages. Examples of acyl structures with various numbers of carbon and double bonds are indicated. Sphingolipids are assembled by linkage of a fatty acid by an amide bond on a long-chain base, also called sphingoid base, such as sphingosine. Simple structures are shown but a variety of unique

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Review soluble subunit of FASII, such as haloxyfop targeting acetyl-CoA carboxylase, cerulenin targeting FabF, thiolactomycin targeting FabF and FabH or triclosan developed to target FabI would have to cross the plasma membrane of the host cell, the parasitophorous vacuole membrane, the plasma membrane of the parasite and the four membranes surrounding the apicoplast. As a result, in targeting the lipidome the chemical properties of the screened or designed drug candidates must be considered, which enable the compound to circulate into hydrophilic and lipophilic environments. In this review, we introduce lipidomic strategies currently applied to a large variety of parasites. Unique lipid structures and biosynthetic pathways are pointed out that are promising targets for novel drugs. Eventually, the question of the ability of a drug to find a target embedded in a membrane and how lipid composition can alter the efficacy of developed drugs is addressed. Technologies to characterize the lipidome of parasites The preparation of parasite samples for lipidomic analyses Lipids are small organic compounds characterized by their hydrophobicity and molecular diversity. Lipidomics addresses their comprehensive analysis [8–14], requiring appropriate separation and analytical methods. The first requirement for the analysis of the parasite lipidome is to avoid contamination by lipids of the host. Pioneering lipidomic analyses were based on the comparison of uninfected and infected host cells [15]. Owing to the technological progress in sensitivity, it is now possible to analyze samples obtained after refined purification of parasites, despite a low yield [16,17]. Recent advances include the analyses of parasite membrane organelles [18], subdomains such as rafts [17,19] or minor lipid classes such as glycosylphosphatidylinositols [20]. The presence of peripheral structures protecting the parasites, such as cysts, eggshells and cuticles [21,22], require special procedures for lipid extraction. Extraction of lipids from nematodes thus combines freezing and mechanical grinding before treatment by solvents. The homogenates can then be analyzed by one or a combination of the following methods, usually performed in specific technological platforms. Sensitive analyses based on mass spectrometry Lipidomic analyses heavily rely on the advances in mass spectrometry (MS), either by coupling MS with another separation method such as gas chromatography (GC-MS) or liquid chromatography (LC-MS) or by aligning mass spectrometers (tandem MS) [23]. The detection and quantification of all the lipids in a series of samples is known as lipid profiling and can thus be performed with high throughput using state-of-the-art, but still expensive, MS instruments [24]. The identification of molecular species that would vary between different sample groups is achieved by multivariate statistics to determine groupspecific signals. Leishmania lipid profiling has thus been

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achieved with a very high-resolution and mass accuracy [24–26]. To characterize selected sets of lipid classes or subclasses (e.g. phospholipidomics [27], sphingolipidomics [28]), tandem MS with a triple quadrupole is the method of choice, because after collision of analyzed molecules the loss of a polar-head fragment can generate a specific ion used as a diagnostic signature of a complete lipid class or subclass, which can then be profiled in precursor ion scan mode. Alternatively, neutral loss scanning permits the detection of lipids that have lost the same fragment following collision. Although GC-MS is still used for fatty acids or sterols, LC-MS or direct infusion of crude lipid extracts into the spectrometer interface has become prominent in recent studies [29]. The most common ionization techniques are electrospray ionization (ESI) [30] and atmospheric pressure chemical ionization (APCI), although MALDI-TOF (matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry) is emerging in the lipidomic field [31]. Shotgun lipidomics [32] is carried out by directly infusing crude lipid extracts in the ESI source. Separation of lipid classes is then based on intrinsic electrical properties. Shotgun lipidomics is inherently biased towards the most abundant and easily ionized lipids [26]. The recent atmospheric pressure photo ionization (APPI) source was found of particular interest for non-polar lipids [33]. The efficiency of LC separation prior to the introduction in the MS ion source plays an important role in the detection of minor lipid species. The blood plasma lipidome was thus assessed by ultra high pressure LC coupled with high-resolution fast-scanning MS, generating informationrich profiles [34]. This technique will undoubtedly be crucial for the analyses of parasites proliferating in blood vessels. High-performance thin-layer chromatography for screens and metabolic labeling of parasites To complement MS, thin layer chromatography (TLC) remains a robust technique to analyze lipids of known structures and screen for phenotypic variations [35]. Automated high-performance TLC (HPTLC) [36], combined with the use of radiolabeled precursors, is a straightforward method to analyze lipid syntheses inside parasitic cells, for a large array of functional studies [37–41], from in vitro to in vivo conditions [40]. Radioactive precursors may thus allow the identification of essential lipid biosynthetic pathways that could be targeted. For example, radiolabeled fatty acids (FAs) have been used to show FA scavenging by Toxoplasma gondii [41], whereas radiolabeled acetate could be incorporated into T. gondii acyllipids indicating the possibility of de novo FA biosynthesis [39]. Distinct conclusions can hence be drawn based on experimental design. Accuracy of precursors for de novo biosynthesis of lipids should be selected with caution. Acetate has, for instance, been widely used as a precursor for FA de novo synthesis in apicomplexan parasites [39,40] or apicomplexa-infected rodents [40]. In apicomplexans,

glycosylated polar heads occurs in parasites. Sterols derive from the squalene structure. A variety of sterols exist, including compounds linked to fatty acids in the case of sterol acyl-esters. Phospholipids contain a phosphated polar head and can be glycerol or sphingolipids. Abbreviations: cytidine diphosphate-diacylglycerol (CDP-DAG); diacylglycerol (DAG); phosphatidic acid (PA).

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Review the FA synthase is located inside an organelle called the apicoplast, but in this organelle, acetate cannot be activated into acetyl-CoA, and no acetyl-CoA transporter is currently known in the apicoplast. It is now considered that phosphoenolpyruvate would be a more accurate precursor and that this metabolite should be used in future studies of this essential process in apicomplexans [42]. HPTLC is thus a rapid and efficient method to follow lipid synthetic fluxes, but results strongly depend on initial hypotheses, and the drawn conclusion should be supported by complementary lines of evidence. Once a metabolic hypothesis has been fully validated, HPTLC is also a method of choice to screen for mutations or drugs impairing the lipidomic fluxes. Imaging lipids and lipid domains in parasites The subcellular localization of lipids can be analyzed by three methods: (i) in Toxoplasma, fluorescent compounds, in the form of fluorophore-conjugated lipids, have been used to mimic lipids diverted from host cells [41]. It is, however, difficult to assess whether the observed relocation of fluorescence reflects an in vivo process. (ii) The second method is based on fluorescent proteins harboring lipid-binding domains [43,44]. Phosphatidylinositol 3-monophosphate was studied in Toxoplasma using such a probe [45]. It should be noted that the overexpression of a protein with a lipidbinding domain might result in changes in vivo, resulting in altered interactions between the studied lipid and its natural receptors. (iii) The third method is based on immunostaining with specific antibodies, such as those raised against glycolipid polar heads [17]. It is then important to assess the specificity of the antibody and take into account the possible aggregation of the visualized lipids that might be triggered by the antibody. Imaging the lipid domains is also essential because these lipid structures, enriched in sterols, sphingolipids and specific proteins, are suspected to play crucial roles in pathogenesis and drug resistance. Large proportions of active P-glycoproteins (Pgp), responsible for drug efflux, have been localized in raft-like structures (RLS) in nematodes by confocal microscopy, and a relationship has been established among RLS–Pgp complexes, xenobiotic transport and drug resistance [46]. Mining the lipidome of parasites to search for unique lipid structures In the search for parasite-specific lipids, the analyses of glycolipids has been one of the most prolific, pointing out glycosylglycerolipids such as phosphatidylinositols (GPIs) [47,48] or glycosylsphingolipids such as inositolphosphoryl ceramide (IPC) [49] and sulfoglycosphingolipids [50]. Glycero- or sphingolipids terminated by non-mammalian a-Gal(1!6)b-Gal in Apicomplexans [17,38] or b-Gal(1!6) b-Gal in Trematoda parasites can thus inspire novel chemotherapeutic strategies as long as the precise structures of the specific lipids and their biosynthetic pathways can be eventually elucidated [49]. In the shorter term, glycolipids are excellent candidates for immunotherapeutic strategies [48,51]. Glycolipids seem ideal structures to search for parasite-specific lipids. With the gain in sensitivity of analytic methods, future research will have to focus on 500

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other sources of molecular diversity, in particular at the level of the hydrophobic moiety of membrane lipids, such as the fatty acid (which length can vary from 12 to more than 26 carbon atoms, be even or odd numbered and be modified by addition of double bonds, oxygen atoms, cycles, etc.), the long-chain base of glycerolipids and sphingolipids (which length can also vary), and sterols, which have not been systematically characterized up to now. Mining lipidome metabolism to search for unique lipid biosynthetic pathways Limits of bioinformatic resources to mine lipid metabolic pathways of parasites Parasite lipid homeostasis results from four processes: (i) diversion from host; (ii) de novo syntheses; (iii) lipid conversions; and (iv) trafficking (example in Figure 2 for the glycerolipidome). Regardless of the difficulty in annotating genomes of parasites [52], it is crucial to have access to the most complete metabolic maps to select probable targets. Unfortunately, databases such as KEGG [53] and MetaCyc [54] are far from being exploitable. KEGG provides global metabolic schemes, including metabolites, enzymes and links to drugs [53]. KEGG metabolic maps are available for 14 Apicomplexa, one pathogenic Ciliate, five Kinetoplastida, one Trematoda and one pathogenic Nematoda. Comparing maps of different organisms highlights differences that should be helpful to find target candidates. However, maps are designed based on enzymatic reactions and, for instance, the map for FA synthesis is identical for FA synthase of type I (FASI, a multiprotein complex) in the cytosol of mammalian cells and of type II (FASII, dissociated enzymes) in the apicoplast of Apicomplexa. Furthermore, FA synthesis in mammals combines the cytosolic FASI producing 16- to 18-carbon acyls, with mitochondrial FASII components producing 8-carbon precursors of lipoate. In the case of T. gondii containing both a cytosolic FASI and an apicoplast FASII, only the latter is shown. The molecular diversity of FAs (carbon length and desaturations) is not shown. Trafficking is not represented, and a lipid generated in a given subcellular membrane can be mapped together with an enzyme localized in another membrane. In contrast to KEGG, the MetaCyc view of metabolism is fragmented, reflecting knowledge gaps, and allowing the design of pathways that are unique to some phyla [54], including seven Apicomplexa, three pathogenic Ciliates and one pathogenic Amoebozoa. Similar to KEGG, membrane compartmentalization is missing and molecular diversity of FAs (carbon-chain length, unsaturation level, oxidation, etc.) is not shown. Alternative databases taking into account the uniqueness of the metabolism of parasites are thus badly needed. The best, if not sole example, is the Malaria Parasite Metabolic Pathways database [55]. The quality is high, missing data are documented and enzyme subcellular localizations are shown. Nevertheless, similar to KEGG or MetaCyc, the in silico search of potential targets is difficult in the context of lipid metabolism, because the molecular diversity of lipids, compartmentalization of pathways and existence of alternative routes to generate identical structures cannot be easily compared. Experimental metabolomic studies are therefore necessary.

Review

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FFA or glycerolipids from host

Glycerol or glycerolipids from host Glycerol transporter? PLA?

PLA? FA transporter? Fatty acids FA scavenging from host? * and / or * Neosynthesis by a FASI? and / or * Neosynthesis by a FASII? and / or Neosynthesis by a FAE?

acyl(R1COO) – CoA/ACP

Pool of acyl-CoA/ACP

(DGDG) Plastid lipids?

**

PLD?

AT2?

**

*** Polar head-precursor transporter(s)?

DAG

PC

Polar head precursor(s) from host

Endomembrane phospholipids

PA

(SQDG)

Glycerolipids from host

PA transporter?

P

Scavenging from host?

(MGDG)

AT1?

Scavenging from host?

LPA

acyl(R1COO) – CoA/ACP

Glycerolipids from host DAG transporter?

PLC?

?

G3P

CDP – DAG

MT?

PE

PS

PG

PI Mitochondria lipids

***

DPG

Scavenging from host? Direct import? Phospholipids from host TRENDS in Parasitology

Figure 2. Targeting the membrane glycerolipidome of a unicellular parasite. Summary of the redundant pathways that a parasitic cell could harbor to synthesize a category of lipids needed to build its cellular membranes, here the acyl-glycerolipids. The metabolism of glycerolipids is highly compartmentalized and if blocked at one point, redundant routes might be used by the parasite to bypass the interrupted pathway. Redundant pathways combine scavenging of more or less elaborated precursors, de novo syntheses and lipid conversions. The scavenging processes allow precursors to be imported into the parasite, probably due to transporters, generally after the hydrolysis of host lipids by specific enzymes, the lipases, into free FAs, glycerol, diacylglycerol or phosphatidic acid. FAs can also be obtained by neosynthesis due to fatty acid synthases or elongases. Phospholipids or phospholipid polar heads could also be diverted from the host. All these products feed several key pools to provide four main groups of lipids (FAs, plastids lipids, endomembrane phospholipids and mitochondrial lipids). These pools include acyl-CoA or acyl-ACP that can be combined to glycerol3-phosphate by the action of two acyltransferases thus generating phosphatidic acid. Phosphatidic acid and diacylglycerol are the precursors for all diacyl-glycerolipids, either by the DAG or by the CDP-DAG pathways, producing phospholipids (PS, PC, PE, PI, PG, DPG), glycolipids (MGDG, DGDG) or sulfoquinovosyldiacylglycerol found in some plastid-containing cells. Once a specific feature has been detected in the lipidome of a parasite, it is thus essential to characterize the corresponding metabolic process and assess that this process can be targeted and that the parasite will not bypass it, when chemically impaired. (*) FA scavenging from host has been demonstrated in Apicomplexa, but parasites such as Plasmodium falciparum and Toxoplasma gondii also contain a FASII system that could also neosynthesize FAs; the presence of a FASI system in T. gondii could be a third route to generate FAs. (**) Acyl transferases of the apicoplast might be potential targets for drugs to fight against malaria. (***) Import of polar head precursors and methyltransferases converting ethanolamine into choline are currently used as targets to develop novel antimalarials (see text). Abbreviations: acyl carrier protein (ACP); acyl-coenzyme A (Acyl-CoA); acyltransferases (AT1 and AT2); cytidine diphosphate-diacylglycerol (CDP-DAG); diacylglycerol (DAG); diphosphatidylglycerol (DPG); fatty acid (FA); fatty acid elongases (FAE); fatty acid synthases I or II (FASI or FASII); free fatty acid (FFA); galactosyldiacylglycerol, mono and di- (MGDG, DGDG); glycerol-3-phosphate (G3P); lysophosphatidic acid (LPA); methyl-transferases (MT); phosphatidic acid (PA); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylglycerol (PG); phosphatidylinositol (PI); phosphatidylserine (PS); phospholipase A (PLA); phospholipase C (PLC); phospholipase D (PLD); sulfoquinovosyldiacylglycerol (SQDG).

Absolute requirement of experimental validation: the lessons of acyl-lipid drug development in Apicomplexa The discovery of a plant-like plastid FASII in Apicomplexans such as Plasmodium or Toxoplasma [56] has stimulated an intense search for possible drugs among herbicides and antibiotics, such as thiolactomycin [56,57] or triclosan [40,58]. However, ambiguous and non-reproducible in vitro and in vivo results raised questions on the target validity. It was thus shown by genetic knockout that FASII could be dispensable in the blood stages of Plasmodium [59] and that off-targets were probably responsible for measured drug effects [60,61]. By contrast, the metabolism of a ubiquitous glycerolipid (i.e. phosphatidylcholine) in Plasmodium has been dissected by various biochemical, bioinformatic and genetic approaches [7,62,63] and proved to be an ideal target for novel drugs [7,64], even inspiring the design of drugs for other parasites in the Kinetoplas-

tida phylum [65]. Genomic clues should therefore be completed by genetic and biochemical evidence. Relative proportions of ubiquitous phospholipids such as PC and PE also determine the general properties of drugs in membranes, a point addressed in the following section. The question of drug dynamics in the membrane environment Drug dynamics analyzed in membrane models The interaction of a drug with the parasite plasma membrane is a prerequisite for its action. Accordingly, it is essential to investigate whether the specific properties of the parasite limiting membranes can alter the drug efficiency. Vesicles can be prepared from parasite plasma membranes to measure salt permeability variations that might be provoked by a drug to assess the possibility of 501

Review drug-induced pore formation [66]. Drug–membrane interactions can, however, be dissected more precisely using biomimetic membrane models, including Langmuir monolayers that are spontaneously formed at the air–water interface or immobilized artificial membranes in chromatographic systems. A drug–lipid interaction can, for instance, be studied using a monolayer composed of a single lipid species [67]. However, based on a comprehensive lipidomic profiling, a biomimetic lipid monolayer reflecting a plasma membrane or a lipid raft composition can be analyzed, with a particular focus on differences between wild type and drug-resistant parasites [67]. Amphotericin B was the first antiparasitic drug for which the interaction with lipids, and particularly sterols, was investigated using the Langmuir film [67]. The amphotericin B-induced membrane pore is responsible for ion leakage, contributing to Leishmania and fungi death. Modeling studies of the amphotericin B–sterol channel, comparing relative affinity of amphotericin B for cholesterol and ergosterol, have shown a better stability and a higher diameter of the pore when amphotericin B bound to ergosterol, present in fungi and Leishmania membranes and absent in mammals [68,69]. Miltefosine is an antileishmanial lipid-like drug for which an affinity for sterols was demonstrated by the Langmuir monolayer technique, suggesting that membrane lipid rafts could be a miltefosine reservoir [70]. A miltefosine analog, edelfosine, was studied on the Langmuir monolayer, in combination with amphotericin B, and this drug combination resulted in a strong interaction between drugs, explaining a decrease in the antileishmanial activity of this combination [71]. The Langmuir monolayer technique was also useful in demonstrating that sitamaquine, an antileishmanial drug in development for which no transporter was found [72]; the drug accumulated in Leishmania donovani through passive diffusion, relying on an electrical gradient [73]. Drug dynamics analyzed in living parasites Nematodes are complex organisms that possess very thick, specific protective barriers (eggshells and cuticles) for which the exact biochemical composition is unknown and cannot be reconstructed. In addition to model membranes, the use of living cells or living nematodes represents a primary and essential approach in understanding the role of lipids in drug dynamics in these parasites. All parasite membranes and barriers analyzed thus far in nematodes include sterols. In vertebrates, cholesterol is the main component of membranes and is known to modulate the activity of pumps such as Pgp which are thought to be involved in drug efflux. Sterols could thus be used to modulate resistance to antiparasitic drugs. The role of cholesterol can be studied in living cells/ organisms due to cholesterol/sterol acceptors such as methyl-b-cyclodextrin which remove cholesterol from membranes. The adaptation of this technique to nematodes showed that sterols could also alter eggshells and membranes, consequently changing the activity of Pgp and the susceptibility to drugs in nematodes [22,35,74] or unicellular parasites [70,75]. Depletion of cholesterol in Leishmania or nematodes consequently increases drug 502

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resistance while cholesterol loading decreases it [22,75]. The decrease in cholesterol concentration was associated with the highest drug efflux. Moreover, a significant difference in sterol concentration was found between nematodes susceptible or resistant to anthelmintics, the latter having a naturally lower sterol concentration [22,35]. The interplay between cholesterol and phospholipids is also crucial for drug dynamics. Phospholipids classes are the same in nematodes and in vertebrates. Regardless of the fact that FAs within the same classes of lipids can vary greatly between the phyla, a significant relationship between lipid class composition and drug resistance was nevertheless found to depend on the equilibrium between free cholesterol and phospholipids with PC, PE and phosphatidic acid having the most significant impacts [35]. Manipulating lipid composition of nematodes and elucidating sterol pathways and the balance with phospholipids can thus help improve drug efficiency and the design of more potent antiparasitic strategies. Results obtained with several nematode species, parasites of plants or animals, showed that plant oils can be antiparasitic or adjuvants to antiparasitic drugs [74], supporting this perspective for future drug developments. Concluding remarks The spectacular technological advances in high sensitivity MS, HPTLC and high-resolution imaging has accelerated the structural and functional characterization of the lipidome of parasites, ranging from unicellular protists to worms, which cause threatening diseases in vertebrates, including humans. The search for specific lipid structures has proven efficient to determine unique glycosylated polar heads in parasite membrane lipids, and the current challenge is to expand this search to identify specific substructures in hydrophobic moieties of membrane lipids such as acyls, long-chain bases and sterols. The next step is the assessment that the specific feature detected in the parasite lipidome is indeed vital for the pathogen. When validated by metabolic and functional studies, the specificity of the parasite lipidome can then inspire the development of novel drugs. The definition of precise lipidomic profiles also allow the reconstitution of biomimetic systems to study the dynamics and bioavailability of developed drugs in parasite limiting membranes, giving clues concerning the resistance that could arise from membrane lipid remodeling. Future developments will therefore strongly depend on the progresses in sensitivity and throughput of lipidomic technologies made available to the scientific community, mainly by the access to specific technological platforms. References 1 Maurya, R. et al. (2010) Evaluation of blood agar microtiter plates for culturing Leishmania parasites to titrate parasite burden in spleen and peripheral blood of patients with visceral leishmaniasis. J. Clin. Microbiol. 48, 1932–1934 2 Dietz, K. et al. (2006) Mathematical model of the first wave of Plasmodium falciparum asexual parasitemia in non-immune and vaccinated individuals. Am. J. Trop. Med. Hyg. 75, 46–55 3 Geraghty, E.M. et al. (2007) Overwhelming parasitemia with Plasmodium falciparum infection in a patient receiving infliximab therapy for rheumatoid arthritis. Clin. Infect. Dis. 44, e82–e84 4 Loverde, P.T. and Chen, L. (1991) Schistosome female reproductive development. Parasitol. Today 7, 303–308

Review 5 Bethony, J. et al. (2006) Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 367, 1521–1532 6 Saidani, N. et al. (2009) Potential and limits of in silico target discovery – case study of the search for new antimalarial chemotherapeutic targets. Infect. Genet. Evol. 9, 359–367 7 Ben Mamoun, C. et al. (2010) Targeting the lipid metabolic pathways for the treatment of malaria. Drug Dev. Res. 71, 44–55 8 Shevchenko, A. and Simons, K. (2010) Lipidomics: coming to grips with lipid diversity. Nat. Rev. Mol. Cell Biol. 11, 593–598 9 Dennis, E.A. (2009) Lipidomics joins the omics evolution. Proc. Natl. Acad. Sci. U.S.A. 106, 2089–2090 10 Kita, Y. et al. (2006) Lipidomics approaches for a comprehensive analysis of fatty acids and metabolites by LC/ESI-MS. Prostaglandins Other Lipid Mediat. 79, 150–151 11 Wenk, M.R. (2005) The emerging field of lipidomics. Nat. Rev. Drug Discov. 4, 594–610 12 McKee, D.L. (2003) Lipidomics. Scientist 17, 20 13 Lagarde, M. et al. (2003) Lipidomics is emerging. Biochim. Biophys. Acta 1634, 61 14 Feng, L. et al. (2003) Lipidomics: probing lipid-protein interactions using immobilized lipids. FASEB J. 17, A993–A994 15 Hsiao, L.L. et al. (1991) Modification of host cell membrane lipid composition by the intra-erythrocytic human malaria parasite Plasmodium falciparum. Biochem. J. 274, 121–132 16 Welti, R. et al. (2007) Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids. Biochemistry 46, 13882–13890 17 Botte, C. et al. (2008) Subcellular localization and dynamics of a digalactolipid-like epitope in Toxoplasma gondii. J. Lipid Res. 49, 746–762 18 Besteiro, S. et al. (2009) Export of a Toxoplasma gondii rhoptry neck protein complex at the host cell membrane to form the moving junction during invasion. PLoS Pathog. 5, e1000309 19 Azzouz, N. et al. (2006) Toxoplasma gondii grown in human cells uses GalNAc-containing glycosylphosphatidylinositol precursors to anchor surface antigens while the immunogenic Glc-GalNAc-containing precursors remain free at the parasite cell surface. Int. J. Biochem. Cell Biol. 38, 1914–1925 20 Azzouz, N. et al. (2005) Removal of phospholipid contaminants through precipitation of glycosylphosphatidylinositols. Anal. Biochem. 343, 152–158 21 Riou, M. et al. (2005) Immunolocalisation of an ABC transporter, P-glycoprotein, in the eggshells and cuticles of free-living and parasitic stages of Haemonchus contortus. Parasitol. Res. 96, 142–148 22 Riou, M. et al. (2003) Modulation of the multidrug resistance (MDR) system in the nematode Haemonchus contortus by changing cholesterol content: effects on resistance to anthelmintics. J. Antimicrob. Chemother. 52, 180–187 23 Navas-Iglesias, N. et al. (2009) From lipids analysis towards lipidomics, a new challenge for the analytical chemistry of the 21st century. Part II: analytical lipidomics. Trends Anal. Chem. 28, 393–403 24 Schwudke, D. et al. (2007) Top-down lipidomic screens by multivariate analysis of high-resolution survey mass spectra. Anal. Chem. 79, 4083–4093 25 Zheng, L. et al. (2010) Profiling of lipids in Leishmania donovani using hydrophilic interaction chromatography in combination with Fourier transform mass spectrometry. Rapid Commun. Mass Spectrom. 24, 2074–2082 26 Griffiths, W.J. and Wang, Y. (2009) Mass spectrometry: from proteomics to metabolomics and lipidomics. Chem. Soc. Rev. 38, 1882–1896 27 Willmann, J. et al. (2008) Hyphenated tools for phospholipidomics. J. Biomol. Tech. 19, 211–216 28 Haynes, C.A. et al. (2009) Sphingolipidomics: methods for the comprehensive analysis of sphingolipids. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877, 2696–2708 29 Roberts, L.D. et al. (2008) A matter of fat: an introduction to lipidomic profiling methods. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 871, 174–181 30 Lehtonen, M. et al. (2011) Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 879, 677–694 31 Schiller, J. et al. (2007) MALDI-TOF MS in lipidomics. Front. Biosci. 12, 2568–2579

Trends in Parasitology November 2011, Vol. 27, No. 11 32 Han, X. and Gross, R.W. (2005) Shotgun lipidomics: electrospray ionization mass spectrometric analysis and quantitation of cellular lipidomes directly from crude extracts of biological samples. Mass Spectrom. Rev. 24, 367–412 33 Cai, S.S. et al. (2007) Comparison of atmospheric pressure photoionization and atmospheric pressure chemical ionization for normal-phase LC/MS chiral analysis of pharmaceuticals. Anal. Chem. 79, 2491–2498 34 Sandra, K. et al. (2010) Comprehensive blood plasma lipidomics by liquid chromatography/quadrupole time-of-flight mass spectrometry. J. Chromatogr. A 1217, 4087–4099 35 Riou, M. et al. (2007) Relationships between sterol/phospholipid composition and xenobiotic transport in nematodes. Parasitol. Res. 100, 1125–1134 36 Fuchs, B. et al. (2010) Lipid analysis by thin-layer chromatography – a review of the current state. J. Chromatogr. A 1218, 2754–2774 37 Azzouz, N. et al. (2008) Metabolic labeling and structural analysis of glycosylphosphatidylinositols from parasitic protozoa. Methods Mol. Biol. 446, 183–198 38 Marechal, E. et al. (2002) Synthesis of chloroplast galactolipids in apicomplexan parasites. Eukaryot. Cell 1, 653–656 39 Bisanz, C. et al. (2006) Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from apicoplast-generated fatty acids versus scavenging of host cell precursors. Biochem. J. 394, 197–205 40 Surolia, N. and Surolia, A. (2001) Triclosan offers protection against blood stages of malaria by inhibiting enoyl-ACP reductase of Plasmodium falciparum. Nat. Med. 7, 167–173 41 Charron, A.J. and Sibley, L.D. (2002) Host cells: mobilizable lipid resources for the intracellular parasite Toxoplasma gondii. J. Cell Sci. 115, 3049–3059 42 Mazumdar, J. et al. (2006) Apicoplast fatty acid synthesis is essential for organelle biogenesis and parasite survival in Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103, 13192–13197 43 Lemmon, M.A. (2003) Phosphoinositide recognition domains. Traffic 4, 201–213 44 Backer, J.M. (2010) New methods for capturing the mystery lipid, PtdIns5P. Biochem. J. 428, e1–e2 45 Tawk, L. et al. (2010) Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis. PLoS Pathog. 7, e1001286 46 Riou, M. et al. (2010) Drug resistance is affected by colocalization of P-glycoproteins in raft-like structures unexpected in eggshells of the nematode Haemonchus contortus. Biochem. Cell Biol. 88, 459–467 47 Ferguson, M.A. et al. (1994) Glycosyl-phosphatidylinositol molecules of the parasite and the host. Parasitology 108 (Suppl.), S45–S54 48 Debierre-Grockiego, F. et al. (2010) Binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages. J. Biol. Chem. 285, 32744–32750 49 Sonda, S. and Hehl, A.B. (2006) Lipid biology of Apicomplexa: perspectives for new drug targets, particularly for Toxoplasma gondii. Trends Parasitol. 22, 41–47 50 Landoni, M. et al. (2007) Plasmodium falciparum biosynthesizes sulfoglycosphingolipids. Mol. Biochem. Parasitol. 154, 22–29 51 Boutlis, C.S. et al. (2002) Antibodies to Plasmodium falciparum glycosylphosphatidylinositols: inverse association with tolerance of parasitemia in Papua New Guinean children and adults. Infect. Immun. 70, 5052–5057 52 Birkholtz, L.M. et al. (2006) Integration and mining of malaria molecular, functional and pharmacological data: how far are we from a chemogenomic knowledge space? Malar. J. 5, 110 53 Kanehisa, M. et al. (2010) KEGG for representation and analysis of molecular networks involving diseases and drugs. Nucleic Acids Res. 38, D355–D360 54 Caspi, R. et al. (2010) The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res. 38, D473–D479 55 Ginsburg, H. and Tilley, L. (2011) Plasmodium falciparum metabolic pathways (MPMP) project upgraded with a database of subcellular locations of gene products. Trends Parasitol. 27, 285–286 56 Waller, R.F. et al. (2003) A type II pathway for fatty acid biosynthesis presents drug targets in Plasmodium falciparum. Antimicrob. Agents Chemother. 47, 297–301

503

Review 57 Prigge, S.T. et al. (2003) The initiating steps of a type II fatty acid synthase in Plasmodium falciparum are catalyzed by pfACP, pfMCAT, and pfKASIII. Biochemistry 42, 1160–1169 58 McLeod, R. et al. (2001) Triclosan inhibits the growth of Plasmodium falciparum and Toxoplasma gondii by inhibition of apicomplexan Fab I. Int. J. Parasitol. 31, 109–113 59 Yu, M. et al. (2008) The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites. Cell Host Microbe 4, 567–578 60 Baschong, W. et al. (2011) Triclosan is minimally effective in rodent malaria models. Nat. Med. 17, 33–34 34–35 author reply 61 Surolia, A. and Surolia, N. (2011) Triclosan is minimally effective in rodent malaria models – reply. Nat. Med. 17, 34–35 62 Besteiro, S. et al. (2010) Exploring metabolomic approaches to analyse phospholipid biosynthetic pathways in Plasmodium. Parasitology 137, 1343–1356 63 Dechamps, S. et al. (2010) Rodent and nonrodent malaria parasites differ in their phospholipid metabolic pathways. J. Lipid Res. 51, 81–96 64 Caldarelli, S.A. et al. (2010) Exploration of potential prodrug approach of the bis-thiazolium salts T3 and T4 for orally delivered antimalarials. Bioorg. Med. Chem. Lett. 20, 3953–3956 65 Ibrahim, H.M. et al. (2010) Symmetrical choline-derived dications display strong anti-kinetoplastid activity. J. Antimicrob. Chemother. 66, 111–125 66 Cohen, B.E. (1986) Concentration- and time-dependence of amphotericin-B induced permeability changes across ergosterolcontaining liposomes. Biochim. Biophys. Acta 857, 117–122

504

Trends in Parasitology November 2011, Vol. 27, No. 11 67 Rakotomanga, M. et al. (2005) Alteration of fatty acid and sterol metabolism in miltefosine-resistant Leishmania donovani promastigotes and consequences for drug-membrane interactions. Antimicrob. Agents Chemother. 49, 2677–2686 68 Baginski, M. et al. (2002) Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels. Biochim. Biophys. Acta 1567, 63–78 69 Resat, H. and Baginski, M. (2002) Ion passage pathways and thermodynamics of the amphotericin B membrane channel. Eur. Biophys. J. 31, 294–305 70 Saint-Pierre-Chazalet, M. et al. (2009) Membrane sterol depletion impairs miltefosine action in wild-type and miltefosine-resistant Leishmania donovani promastigotes. J. Antimicrob. Chemother. 64, 993–1001 71 Hac-Wydro, K. et al. (2009) Langmuir monolayer study toward combined antileishmanian therapy involving amphotericin B and edelfosine. J. Phys. Chem. B 113, 14239–14246 72 Lopez-Martin, C. et al. (2008) Sitamaquine sensitivity in Leishmania species is not mediated by drug accumulation in acidocalcisomes. Antimicrob. Agents Chemother. 52, 4030–4036 73 Duenas-Romero, A.M. et al. (2007) Interaction of sitamaquine with membrane lipids of Leishmania donovani promastigotes. Biochim. Biophys. Acta 1768, 246–252 74 Kerboeuf, D. et al. (2010) Membrane drug transport in helminths. AntiInfective Agents Med. Chem. 9, 113–129 75 Barratt, G. et al. (2009) Cellular transport and lipid interactions of miltefosine. Curr. Drug Metab. 10, 247–255