The feasibility of drug targeting is demonstrated in in vitro cultures of the human malarial ..... Allred, D. R., Sterling, C. and Morse, P. (1983). Mol. Biochem.
Bioscience Reports, Vol. 7, No. 6, 1987
Review
New Permeability Pathways Induced by the Malarial Parasite in the Membrane of its Host Erythrocyte" Potential Routes for Targeting of Drugs into Infected Cells Hagai Ginsburg and Wilfred D. Stein Received May 6, 1987 KEY WORDS: malaria; Plasmodium falciparum; erythrocyte permeability; drug targeting.
Malarial parasites propagate asexually inside the erythrocytes of their vertebrate host. Six hours after invasion, the permeability of the host cell membrane to anions and small nonelectrolytes starts to increase and reaches its peak as the parasite matures. This increased permeability differs from the native transport systems of the normal erythrocyte in its solute selectivity pattern, its enthalpy of activation and its susceptibility to inhibitors, suggesting the appearance of new transport pathways. A biophysical analysis of the permeability data indicates that the selectivity barrier discriminates between permeants according to their hydrogen bonding capacity and has solubilization properties compared to those of iso-butanol. The new permeability pathways could result from structural defects caused in the host cell membrane by the insertion of parasite-derived polypeptides. It is suggested that the unique transport properties of the new pathways be used to target drugs into infected cells, to affect the parasite either directly or through the modulation of the intraerythrocytic environment. The feasibility of drug targeting is demonstrated in in vitro cultures of the human malarial parasite Plasmodium falciparum.
INTRODUCTION The causal agent of human malaria is one or several of the four Plasmodium species P. vivax, P. ovale, P. malariae and P. falciparum, the latter being the most lethal of the Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
455 0144-8463/87/0600-0455505.00/09 1987PlenumPublishingCorporation
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four. The parasites are transmitted to man through a bite of the female anopheline mosquito in the form of sporozoites. The latter invade the liver cells where they multiply (exoerythrocytic schizogony) and eventually exit to the blood stream and invade the red blood cells. The cycle of development in the erythrocytes (erythrocytic schizogony) is responsible for the clinical manifestations of the disease and the destruction of the host cells. Most of the drugs currently used for clinical treatment and for suppressive prophylaxis are blood schizontocides, i.e. directed against the blood stages of the parasite. The pharmacological usefulness of these drugs is based either on a specific action as in the case of inhibitors of the parasite's dihydrofolate reductase, or on some differential susceptibility of the parasite, as in the case of the quinolinecontaining drugs. In the 1950s these drugs were considered so good that research in malaria chemotherapy declined. But this optimism was short-lived and, during the last 20 years, drug resistance has increased to alarming levels (Bunang and Harinasuta, 1986). Thus, malaria is considered at the present time as the most widespread infectious disease, affecting 3-4 x 108 people, causing the death of 2-3 x 1 0 6 of them per year and expanding at a rate of 107/year. The total population at risk is estimated at 2.2 billion. The appearance of chloroquine-resistant P. faleiparum in South East Asia in the early 1960s prompted the US Army Medical Research and Development Command to launch a programme of search for new drugs, the underlying rationale of the search being the improvement of the formulation, use and delivery of existing blood schizontocides. The results of a tremendous effort were rather disappointing: only two new drugs, mefloquine and halofantrine, have emerged--and parasites resistant to them have already been detected. Only recently have totally novel drugs been developed, aimed once again, at specific metabolic targets in the parasite (Rieckmann, 1983). In parallel, the last decade has witnessed a marked surge in basic malaria research. Substantial knowledge is being accumulated on the biochemistry and the physiology of the parasite and its interaction with its host erythrocyte. The acquired knowledge may hopefully serve to devise new therapeutic approaches which could lead to the ultimate eradication of malaria. It is the purpose of this essay to summarize briefly one such aspect, the effect of parasite growth on the permeability properties of its host cell membrane. We hope to demonstrate that these properties can be used for targeting of drugs into infected cells, allowing novel approaches towards the chemotherapy of malaria.
PARASITE-INDUCED PERMEABILIZATION OF HOST CELL MEMBRANE
A single malarial parasite invades an erythrocyte in which it multiplies asexually to produce 16-32 progeny within 48-72 hours. Such multiplication obviously requires an intensive metabolism which has to take place in a host cell with very limited metabolic activity of its own. One of the most conspicuous consequences of parasite enslavement of the host cell is the demonstrable permeabilization of the host cell membrane. Permeabilization allows for the increased transcellular traffic of substrates
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and catabolites, which serves to cope with yet another aspect of parasitism, namely the diminished synthetic ability of the parasite (Sherman, 1979). Rodent and avian erythrocytes, infected with their specific parasite species, are highly permeable to carbohydrates and amino acids (Homewood and Neame, 1974; Neame and Homewood, 1975; Sherman and Tanigoshi, 1974a,b). The high permeability of hexitols of human erythrocytes infected with P. falciparum was later utilized to lyse selectively cells infected with mature parasites--by suspending them in isotonic solutions of the permeants, thereby establishing a method for the synchronization of the parasite cell cycle in culture (Lambros and Vanderberg, 1979). The lysis of infected cells is possible because they retain their relatively low permeability to the major cationic constituents of the cell sap (Dunn, 1969; Bookchin et al., 1980), in the face of their increased permeability to hexitols. Permeabilization starts about 6 hours after invasion, increases with, and depends on, parasite raaturation (Kutner et al., 1985); hence ceils harbouring mature parasites are selectively lysed. Solute selectivity remains essentially unaltered in the face of a more than tenfold increase in permeability during parasite maturation (Elford et al., 1985; Ginsburg et al., 1986c). Classical inhibitors of the native transport systems of the erythrocyte have no effect on anion (Kutner et al., 1982, 1983) or carbohydrate (Ginsburg et al., 1983) transport through the parasite-induced pathways but both were susceptible to the surface dipole modifier phloretin. In addition, the temperature dependence of transport was considerably smaller than that of the native systems (e.g. an enthalpy of activation of 11 kcal per mole as opposed to 20-33 kcal/mole) and s]howed no breaks in the Arrhenius plot (Ginsburg et al., 1983; Kutner et al., 1983, 1985) as opposed to the native systems which show a break in the range of 10-20~ (Knauf, 1979). The solute selectivity of the new pathways was totally different from that of the native systems (Ginsburg et al., 1985; Kutner et al., 1983, 1985). Most importantly, this pattern of permeabilization of the host cell membrane is such as to allow a steady traffic of small metabolites needed for the parasite, without affecting greatly the metabolism of the host cell and without affecting its osmotic integrity. Were the parasitized erythrocyte to be substantially permeabilized also to cations, it would not be able to regulate its volume and would lyse before the parasite had completed its cell cycle. BIOLOGICAL IMPORTANCE OF THE NEW PATHWAYS We have reasoned that the new pathways are established in order to allow for the intense metabolic needs o~ the parasite. An inspection of a few examples seems worthwhile. Glucose is the major substrate for energy production for both host cell and parasite. Consideration of the transport capacity of the human erythrocyte glucose carrier, vis a vis the glucose consumption of the parasitized cell, indicates that the native glucose transporter can provide for all the needs of the infected cell. However, this is not the case in rodent red blood cells where the native glucose transport is 100 fold slower and hence, the host cell membrane must be permeabilized in order to meet the excessive needs of the invader. Lactate is the major end-product of the energy metabolism of host cell and
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parasite alike. Comparison of the basal lactate permeability of normal erythrocytes (Deuticke et a l., 1978) with the rate of lactate production by infected cells (70-120 times higher than that of uninfected cells (Pfaller et al., 1982)), indicates that without the contribution of the new pathways, lactate could quickly accumulate in the infected cell to inhibitory levels. While this may be true for human erythrocytes infected with P. falciparum, it may be irrelevant to P. berghei-infected rat cells, where the basic permeability to lactate is 100 fold higher than that of human erythrocytes. The parasite can obtain most of the amino acids it requires for protein synthesis through the digestion of host cell cytosol. However, some acids must be supplied from the extracellular medium. Thus, it has been shown that the cultivation of the parasite in vitro depends on the presence in the growth medium of isoleucine, cysteine, tyrosine, glutamate, glutamine, methionine and proline (Divo et al., 1985). Isoleucine is presumably needed as a result of its scarcity in host cell cytosol, while cysteine, glutamate and glutamine are probably required during the reductive metabolism of the parasite, which uses glutamate (for the production of NADPH through glutamate dehydrogenase) and glutathione (Roth et al., 1986). Here again, the basal permeability of the uninfected cell (A1-Saleh and Wheller, 1982) is insufficient to accommodate the needs of the parasite. Permeabilization to amino acids may be crucial for yet another reason. The finding that most of the amino acids produced by host cell digestion egress from the infected cell (Zarchin et al., 1986) suggests that such digestion may be needed primarily to provide living space for the parasite, rather than to supply substrates for protein synthesis. Rapid egress of amino acids through the new pathways is therefore required to avoid feedback inhibition of proteolysis or an intracellular accumulation of osmotically active solute which could lead to swelling and eventual lysis. (The digestion of one mole of hemoglobin monomer results in 143 moles of amino acids). Finally, the membrane of normal erythrocytes is virtually impermeable to myoinositol, yet this compound is needed for the synthesis of phosphatidylinositol by the parasite (Vial et al., 1982). The new pathways accommodate this substrate (Elford et al., 1985; Ginsburg et al., 1985) and there is a close correlation between the appearance of the new pathways and the rate of metabolic incorporation of rnyo-inositol. POSSIBLE M E C H A N I S M S OF P E R M E A B I L I Z A T I O N
The plasma membrane of infected cells differs in many aspects from that of the normal erythrocyte. Its fluidity is substantially higher (Allred et al., 1983; Howard and Sawyer, 1980; Sherman and Greenan, 1984), the decrease in viscosity being probably due to the depletion of cholesterol from the host cell membrane (Seed and Kreier, 1972; Holz, 1977). Fluidization could account for the observed increased transport through the native systems (see Yeagle, 1985, for a recent review) but it cannot explain the appearance of the new pathways, since uninfected cells--either in the infected animal or in culture--show similar alterations of their membrane lipids, but do not possess the new pathways. The protein composition of the membrane of infected cells is also dramatically modulated, notably by the insertion of new polypeptides of parasite origin (Howard, 1982; Braun-Breton et al., 1986) which confer upon it new antigenic properties. One or
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:more of these new polypeptides may have the specific function of conferring upon the membrane the novel permeability properties. It is quite possible, however, that the new polypeptides have quite another function but are not perfectely well adapted to the ]lipid constitution of the native red cell membrane and hence fail to maintain proper sealing of the membrane permeability barrier. Thus, a consequence of protein insertion could be the permeabilization of the membrane. The selectivity properties of this modified membrane obviously depend on the chemical nature of the proteins and their interaction with the lipids. A definite elucidation of the true mechanism will be obtained only through the reconstitution of purified proteins in phospholipid membranes. It has been recently suggested that the parasite could exert an oxidative stress on its host with resulting damage to the membrane (Sherman, 1985). Auto-oxidation of hemoglobin is a normal process in red cells and is probably enhanced in infected cells (Etkin and Eaton, 1975; Friedman et al., 1979). As a result, superoxide and hydroxyl radicals and also peroxides are produced which could affect membrane lipids and proteins, leading eventually to cell lysis (Flynn et al., 1983). Induction of oxidative stress in normal erythrocytes indeed increases the permeability of their membranes (Deuticke, 1986), but the selectivity pattern resulting from this effect (Deuticke et al., 1983) is essentially different from that observed in malaria-infected cells (Ginsburg and Stein, 1987) and thus is very unlikely to account for the appearance of the new pathways. BIOPHYSICAL C H A R A C T E R I Z A T I O N O F T H E N E W PATHWAYS The permeability of human erythrocytes infected with P. falciparum to some 30 different carbohydrates and amino acids has been recently determined (Ginsburg et al., 1[985), as indicated in Table 1. These data were subsequently analyzed rigorously by Ginsburg and Stein (1987) in order to identify the mechanism of transport. Using the Table 1. Solutes which are accommodatedby the new permeabilitypathways Carbohydrates
Amino acids
Anions
Others
Glycerol Erythritol Arabitol Xylitol Sorbitol Mannitol Dulcitol Ribose 2-Deoxyglucose Rhamnose Arabinose D-glucose L-glucose Sedoheptulose myo-inositol
Glycine Alanine Valine Leucine Isoleucine Cysteine Serine Threonine Aspartate Glutamate Asparagine Glutamine Histidine
Chloride NBD-taurine Distilbene sulfonate Gluconate Dipicolinate*
Thiourea NBD-alanine Phlorizin
* Many other anions have been shown by Professor Z. I. Cabantchik of our Department, to penetrate through the new pathways. A detailed report will be published shortly.
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Renkin equation (Renkin, 1953), they concluded that the data did not fit a model of an aqueous pore. Since the data indicated almost no dependence of the permeability on the molecular volume of the permeant, they also rejected a "non-Stokesian" diffusion mechanism (Lieb and Stein, 1986) through membrane lipid domains or through waterfilled spaces which could be produced by the aggregation of membrane proteins. The best fit was obtained when the permeability data were plotted against the number of hydrogen bonds that the permeating molecules can form (calculated according to Stein, 1967). This analysis indicated that for every additional OH group (equivalent to 2 H-bonds), the permeability decreased by a factor of 3 to 4. This suggested that the hydrogen-bonding capacity of the permeant could be a crucial factor determining its permeability. (The inclusion of the data for the amino acids in this analysis required one to allow for the effect of the zwitterionic nature of these permeants. It was found by trial and error that adding eleven H-bonds to these compounds allowed all of the available data to be put on one straight line in a plot of permeability against the putative number of H-bonds). This description was interpreted as showing that permeation through the membrane of the malaria-infected erythrocyte is via a route that is very loose, nonstructured, but very weakly hydrophobic. The fact that each H-bond reduces the permeability by a factor of 2, can be compared with the effect of an OH group on the partitioning of polar solutes into iso-butanol (Collander, 1950). One might therefore infer that the polarity of the rate-determining membrane barrier for solute permeation is similar to iso-butanol. Such polarity could exist in the region of the phospholipids' headgroup and glycerol backbone. The pathways' preference for anions over cations (Kutner et al., 1983; Ginsburg et al., 1985) is understandable if one simply recalls that in phospholipid membranes the free energy of ion translocation contributed by the dipoles of the phospholipid headgroups establishes a 6-8 kcal/mole preference for anions over cations (see Honig et al., 1986 for a recent review). Both the anion selectivity and the iso-butanol-like polarity of the partitioning region are compatible with a permeability barrier at or near the glycerol backbone of the headgroups of the phospholipids. We have speculated (Ginsburg and Stein, 1987) that the parasite-produced new proteins inserted into the host cell membrane do not form tight seals with the hydrocarbon chains of the phospholipids. The barrier for transmembrane diffusion then remains at the interface between the phospholipid headgroups and the hydrophilic domains of these proteins, lipids and proteins being held together by electrostatic forces. A misadjustment between phospholipids and proteins has been shown to increase the leak permeability in reconstituted membranes (Van Hoogevest et al., 1984) and a similar effect could constitute the basis for the altered permeability of malaria-infected erythrocytes. It should be underscored that transport pathways of similar selectivity properties have not been described hitherto in any other type of cell. THE POTENTIAL USE OF THE NEW PATHWAYS FOR TARGETING OF DRUGS The unique biophysical properties of the transport pathways induced by the parasite in the membrane of the host red cell, opens a cornucopia of new potential
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avenues for the chemotherapy of malaria. One has at hand a transport agency which is unique to malaria-infected cells and which could therefore be used to target compounds specifically into those cells. One can perhaps tailor-make drugs so as to fit them to the selectivity properties of the new pathways. Thus for example, different antimetabolites which could affect both the invader and the host and which usually gain access into cells by virtue of their hydrophobicity, could be altered so as to make them tess hydrophobic and thus, preferentially permeable into the parasitepermeabilized erythrocytes. Such physico-chemical modulation could improve the antimalarial therapeutic index of these antimetabolites. Thus, different antimetabolites, even those having an effect on the host by virtue of their accessibility to the cells, can now be considered as potential antimalarials, provided their alteration to meet the selectivity properties of the new pathways will not compromise their action on their target processes. Furthermore, in the course of development of new drugs, many compounds of proven antimalarial activity are discarded because of their relatively high toxicity to tlhe host. Such toxicity usually can be diminished by the reduction of the availability of tlhe drug to the somatic cells of the host. However, in order to act on the parasite, one must ascertain that the modified compound should be able to cross two other membranes, namely, the parasitophorous and the parasite's cell membrane. The permeability properties of these membranes have not yet been investigated although we already know that they must be highly permeable to amino acids (Zarchin et al., 1986) and that they display a selective permeability to cations (Ginsburg et al., 1986a). Nevertheless, even if the permeability of the parasite's membranes would be ratelimiting, the new pathways could yet be utilized for the metabolic alteration of the infected erythrocyte. It is well established that the intracellular environment of the infected cell plays a crucial role in the ability of the parasite to successfully complete its cell cycle (Sherman, 1979). This is probably best examptified by the development, in malarious areas, of red cell variants such as sickle cells, thalassemia and glucose-6phosphate-dehydrogenase deficient cells (see Friedman, 1981 for a recent review). The mere fact that the parasite has not evolved to meet the altered environment presented by these erythrocytes to the invading organism, suggests that the co-habitation of the parasite and the variant erythrocytes is virtually impossible. The variant erythrocytes are characterized by multiple alterations which involve their house-keeping metabolism as well as their membranes' structural and functional integrity. It is very likely that the biochemical stress exerted by the parasite on its otherwise fragile variant host cell, could either cause the host cell to lyse or render it sufficientlyabnormal to be recognized by the host's reticuloendothetial system as a damaged cell and be removed from the circulation. In both cases the host cell is destroyed prematurely, that is, before the parasite has the chance to complete its cell cycle and produce mature merozoites to penetrate into new cells. Thus, in principle, any agent which can be introduced into the infected cell through the new pathways and can cause a chain of events that would rnimick the situation in the variant erythrocytes (or in any other type of hemolytic anemia for that matter), could be considered for the chemotherapy of malaria. Examples which come immediately into mind are the various affectors of the complex antioxidant defence mechanism of erythrocytes, inhibitors of host energy metabolism, inhibitors of the host cell membranes' ion pumps, etc. The limit seems to be set by the horizons of the imagination of the ingenious pharmacologist.
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DEMONSTRATION OF THE PHARMACOLIGICAL USE OF THE NEW PATHWAYS The applicability of drug-targeting in the inhibition of parasite growth has been demonstrated in in vitro cultures of P. falciparum. Two different examples will be briefly discussed here. The efficiency of heavy-metal chelators as antimalarials has been demonstrated (Scheibel and Adler, 1982). Many of the compounds which were tested are sufficiently hydrophobic so as to cross cell membranes and are therefore expected to affect any type of cell, i.e. to be toxic to the malaria-infected host. In order to demonstrate how a chelator can be targeted specifically into infected cells, we used the polar zinc chelator dipicolinic acid (DPA, pyridine di carboxylic acid), a divalent anion which demonstrably does not penetrate into the normal red blood cell. The rationale of using DPA is obvious: both the host cell and the parasite depend on the activity of zinccontaining or zinc-dependent enzymes, and in the presence of DPA, those enzymes which are accessible to the chelator would evidently be inhibited. As expected, it was found that the chelator entered readily into infected cells and inhibited parasite growth (Ginsburg et al., 1986b). The effective inhibitory concentration of DPA was that known to be required to withdraw zinc from catalase, a key enzyme in the protection of red cells against oxidative radicals, as well as from other enzymes such as those involved in nucleic acid synthesis. Although the precise site of action of DPA has not been established, these findings serve a clear demonstration of the potential pharmacological use of the new pathways. Another example is phlorizin, which has been used in the past as an antimalarial drug (The Merck Index, 8th Edition, 1968). It is known, like its aglycon phloretin, to inhibit the anion and hexose transport systems of the red blood cell (Le Fevre, 1948; Bowyer, 1957), acting at the cytoplasmic side of the membrane (Lepke and Passow, 1973). Phlorizin inhibits the growth of P. falciparum in culture with an IDso of 17 + 6/~M (Cabantchik et al., 1983), displaying its greatest efficacity against the mature stages of the parasite, when the permeability of the parasite-induced pathways reaches its maximum. In the same concentration range both phlorizin and phloretin block efficiently the pathway to the permeation of other solutes (Kutner et al., 1986). Detailed studies have shown that phlorizin exerts its inhibitory activity at the cytoplasmic aspect of the new pathway. These results bear obvious consequences for the pharmacological use of the new pathways: the hydrophobie phloretin acts on transport systems both in normal and in infected cells. Its hydrophilic glycoside does not penetrate into normal red cells, yet enters readily into infected cells through the new pathways and subsequently blocks them to its own passage as well as to that of other solutes, thus causing starvation and/or intoxication of the parasite. Hence, rendering phloretin more hydrophilic by glycosylation targets it specifically to malaria-infected cells. Synthesis of new compounds with similar targeting properties or modification of existing inhibitors to meet the selectivity properties of the new pathways, could pave new avenues in the chemotherapy of malaria, avenues which are so badly and so urgently needed.
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