JAC Advance Access originally published online on November 13, 2006
Journal of Antimicrobial Chemotherapy 2007 59(2):197-203; doi:10.1093/jac/dkl461
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Influence of the Plasmodium falciparum P-glycoprotein homologue 1 (pfmdr1 gene product) on the antimalarial action of cyclosporin
1 Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College Dublin 2, Ireland 2 Office of Biostatistics, US Food and Drug Administration/Center for Drug Evaluation and Research, Silver Spring MD 20993-0002, USA
*Corresponding author. Tel: +353-1-896-1414; Fax: +353-1-679-9294; E-mail: abell{at}tcd.ie
Received 2 May 2006; returned 1 September 2006; revised 28 September 2006; accepted 19 October 2006
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Background and objectives: The immunosuppressant cyclosporin A and a number of other cyclosporins have potent and selective antimalarial activity. Their exact mechanism of antimalarial action is unknown but the structure–activity relationships for malarial parasite inhibition and immunosuppression differ markedly. The 3'-keto derivative of cyclosporin D (valspodar) is particularly potent against the human malarial parasite Plasmodium falciparum in culture but causes negligible immunosuppression. Multidrug resistance in mammalian cancer cells, the result of overproduction of the P-glycoprotein, can be reversed by certain cyclosporins, particularly valspodar. We therefore investigated the possibility that the antimalarial target of cyclosporin might be a P-glycoprotein homologue. P. falciparum P-glycoprotein homologue 1 (Pgh1; the pfmdr1 gene product) is located in the digestive vacuole (DV) membrane of the parasite. Its function is unknown but it modulates the susceptibility of parasites to quinolines and related antimalarial drugs, including quinine, mefloquine, halofantrine and chloroquine, and to artemisinin.
Methods and results: Here we demonstrate that (i) sequence polymorphisms in pfmdr1 altered the susceptibility of parasites to cyclosporin A and (ii) pfmdr1-overexpressing strains were slightly less susceptible to the drug. Furthermore, we found synergistic antimalarial interactions between cyclosporin A and quinine, mefloquine or halofantrine and antagonism between cyclosporin A and chloroquine. However, we were unable to detect a direct interaction between cyclosporin and Pgh1.
Conclusions: The amino acid sequence and copy number of Pgh1 may influence cyclosporin susceptibility as a result of a direct interaction between the drug and the protein, or via indirect effects on the physiology of the DV.
Keywords: malaria , cyclosporin A , Pgh1
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Cyclosporin A (CsA) is an immunosuppressive undecapeptide of fungal origin that is used extensively in organ transplantation.1 It also displays marked inhibitory activity against malarial and some other parasites.2 CsA inhibits the growth in the culture of erythrocytic stages of various malarial parasites, including the species most virulent in humans, Plasmodium falciparum, and is effective in vivo against a range of rodent malarial parasites.
CsA has a range of pharmacological activities in humans but its immunosuppressive action appears to be based largely on interference with T-lymphocyte activation.1 The chief molecular basis of this interference is the inhibition of the protein serine/threonine phosphatase, calcineurin, a component of a crucial signalling pathway, by a complex between CsA and its intracellular receptor, cyclophilin.3,4 Several lines of evidence (reviewed in ref. 5) suggest that the antimalarial activity of CsA may be due to a similar mechanism. Erythrocytic P. falciparum parasites produce high levels of at least two cyclophilins, PfCYP19A and PfCYP19B, both of which are cyclosporin-binding proteins.6–8 P. falciparum also possesses a calcineurin homologue whose phosphatase activity is inhibited by CsA–PfCYP19A or CsA–PfCYP19B in vitro.9 Furthermore, five of nine of the CsA-resistant mutant parasites isolated by Kumar et al.9 were found to have lesions in either cyclophilin, causing reduced affinity for CsA, or in calcineurin, causing reduced affinity for CsA–cyclophilin. However, calcineurin is not yet known to play any significant role in the development of intraerythrocytic parasites. Many naturally occurring and semi-synthetic cyclosporins have antimalarial activity in culture but there is no correlation between this property and either immunosuppressive potency or cyclophilin binding.7,8,10 Notably, the cyclosporin that was most antimalarially active, [3'-keto-MeBmt]1-CsD (SDZ PSC 833, valspodar), has negligible immunosuppressive activity and low cyclophilin binding.10 We have therefore explored alternative explanations for the antimalarial activity of cyclosporins.
In mammalian cells, CsA also binds to the P-glycoprotein, a 170 kDa integral-membrane protein of the ATP-binding cassette (ABC) transporter superfamily that is the principal protein responsible for the phenomenon of multiple drug resistance (MDR) in malignant cells.11,12 ‘Classical’ MDR is characterized by the ability of tumours to acquire resistance simultaneously to a number of structurally and mechanistically unrelated chemotherapeutic agents, as a result of efflux by P-glycoprotein, which is overproduced by the resistant cells. A diverse array of compounds, including Ca2+-channel blockers, steroids, calmodulin antagonists, antibiotics and immunosuppressive agents, can reverse MDR in cultured cells.13 CsA is one of the most effective ‘first-generation’ MDR modulators and can restore the effectiveness of various anticancer drugs against many MDR cell lines.11 Cyclosporins are slow substrates of P-glycoprotein and act by inhibiting the efflux of substrates. Valspodar, a second generation P-glycoprotein modulator, is a 
20-fold more potent chemosensitizer of MDR cells than CsA and is less toxic.14
Chloroquine-resistant P. falciparum have a phenotype that is similar in some ways to that of mammalian MDR cells, in that resistant parasites accumulate less drug and resistance can be partially reversed by the P-glycoprotein inhibitor verapamil (reviewed in refs 15 and 16). P. falciparum has several genes with sequence similarity to ABC transporters.17 One of these genes, pfmdr1, encodes a protein of 162 kDa that has been named P-glycoprotein homologue 1 (Pgh1). Pgh1 is located primarily in the membrane of the parasite digestive vacuole (DV), an acidic organelle that is the primary site of breakdown of host haemoglobin, but is also present to a smaller extent in the parasite cytoplasmic membrane.18 Although pfmdr1 (as its name suggests) was originally considered the candidate gene responsible for chloroquine resistance, it was subsequently shown that mutations in another gene pfcrt encoding a DV-membrane protein were both necessary and sufficient for chloroquine resistance in the laboratory and in the field.15 However, overexpression of, or polymorphisms in, pfmdr1 can modulate the level of chloroquine resistance, so the resistance phenotype results from the composite effects of the products of two or possibly more genes.16,19 Moreover, overexpression of pfmdr1 by virtue of gene amplification can be associated with cross-resistance to the structurally related drugs mefloquine, halofantrine and quinine16 and is believed to be significant in many mefloquine-resistant field isolates.20 In addition, allelic-exchange studies conclusively showed that pfmdr1 sequence polymorphisms were associated with changes in susceptibility to mefloquine, halofantrine, quinine, the unrelated endoperoxide drug artemisinin21,22 and the weakly antimalarial verapamil.23 Mefloquine is a known modulator of human P-glycoprotein and it has been suggested that Pgh1 may be its molecular target.17 Taken together, the accumulated evidence indicates that Pgh1 is involved in susceptibility and resistance to several drugs of the quinoline/arylamino alcohol group both in the laboratory and the field, perhaps by directly or indirectly removing the drugs from their sites of action.
We therefore examined the susceptibility to CsA of some P. falciparum strains with different genotypes or expression levels of pfmdr1, with a view to determining whether Pgh1 might be the target for cyclosporins or might otherwise influence their antimalarial action. In some of the strains mentioned above, susceptibility to chloroquine and to mefloquine (and in some cases halofantrine and quinine) are inversely related and some investigators have found antagonism between chloroquine and mefloquine or quinine (reviewed in ref. 24). We therefore tested combinations of CsA with quinolines and related agents for synergism or antagonism. Our data show that Pgh1 influences susceptibility to CsA as a result of either a direct drug–protein interaction or an indirect effect.
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Chemical compounds
All compounds were obtained from Sigma-Aldrich unless otherwise stated. Mefloquine was a gift from Roche AG and halofantrine was from SmithKline Beecham Pharmaceuticals.
Parasite strains and culture
P. falciparum line 3D7 was obtained from M. Grainger, National Institute for Medical Research, Mill Hill, London, UK. Transgenic pfmdr1 allelic-exchange lines (7G8-mdrD103' and D10-mdr7G8/1), the corresponding parent lines transfected with their own genes (7G8-mdr7G8 and D10-mdrD10), drug-selected pfmdr1-overexpressing lines (K1Mef and K1Mef2) and their parent strain K1 (see Results for further details) were kindly provided by A. F. Cowman and J. K. Thompson, Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia. Parasites were maintained in continuous culture in human O+ erythrocytes and RPMI 1640 medium supplemented with 25 mM HEPES, 0.05% gentamicin, 50 µg/mL hypoxanthine and 0.18% sodium bicarbonate in candle jars as described previously.25 Albumax (0.5%; Life Technologies) replaced serum in the culture medium for all strains except FCH5.C2,26 which is adapted to grow in horse serum. Drug susceptibilities of asynchronous, asexual blood-stage parasites were assessed using the spectrophotometric parasite lactate dehydrogenase (pLDH) assay as described previously.27,28 Dose–response curves were used to determine the 50% inhibitory concentration (IC50) values. Results were expressed as the geometric means of the IC50 from between three and seven separate experiments and results for different strains were compared using the Student's t-test and/or analysis of variance (ANOVA) with Duncan's multiple range test, assuming log–normal distribution.
Drug interactions
Parasite growth in the presence of drug combinations was measured as for single drugs (see above). Each inhibitor was tested in a series of eight 2-fold dilutions, alone and in combination with another inhibitor at each of eight 2-fold dilutions. Dose–response curves were constructed and IC50 determined as described above. Isobolograms at the IC50s were constructed to assess drug interactions graphically.24 Since the isobolograms indicate the nature and degree of the interaction but offer no proof of statistical significance24 we analysed the raw data using the response-surface model developed previously by Machado.29,30 In this analysis, the value of 
(eta) indicates the degree of deviation from additivity such that = 1 for an additive combination, < 1 indicates a positive interaction (synergism) and > 1 a negative (antagonistic) interaction. The stronger the deviation from = 1, the stronger the interaction. The statistical significance of the deviation from = 1 can be assessed from the confidence interval (CI, often 95%) for , and observing if the value 1 lies outside the interval. An additional test is the likelihood ratio test, which compares the fit of the response-surface model that allows to vary and the fit of the model that constrains to be 1; the resulting test statistic has approximately a 
2 distribution with 1 degree of freedom (see refs 29 and 30 for a fuller explanation).
Drug–protein interactions
SDS–PAGE, silver staining, western immunoblotting and isolation of cyclosporin-binding proteins were performed as described previously.8 Isolation of DV was done by the method of Saliba et al.31 Antiserum to Pgh1 was a gift from A. F. Cowman.
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Effects of pfmdr1 polymorphisms on susceptibility to CsA
Chloroquine-resistant P. falciparum 7G8 parasites have a pfmdr1 allele with four amino acid substitutions relative to the chloroquine-susceptible D10 allele: Y184F, S1034C, N1042D and D1246Y.21 Replacement of the 7G8 mutations with D10 sequence by allelic exchange leads to the construction of 7G8-mdrD103' parasites that encode D10 amino acids at positions 1034, 1042 and 1246. Reed et al.21 showed that in comparison with the 7G8 parent or the transgenic 7G8-mdr7G8 control, 7G8-mdrD103' parasites are less susceptible to melfoquine, halofantrine and artemisinin but more susceptible to chloroquine and quinine. We investigated whether these polymorphisms in pfmdr1 were associated with changes in susceptibility to CsA (Table 1). The phenotypes of the transgenic parasites were confirmed by testing their susceptibilities to mefloquine, halofantrine, quinine and chloroquine. There were quantitative differences between our data and those of Reed et al. that can be ascribed to the different susceptibility tests employed but the results were on the whole qualitatively similar. We observed that the pfmdr1 effect on CsA susceptibility in the 7G8 background mimicked the results seen with mefloquine, halofantrine and artemisinin, i.e. 7G8-mdrD103' was more than 2-fold less susceptible to CsA than 7G8-mdr7G8. Reed et al.21 also showed that introducing the single Pgh1 amino acid substitution at position 1246 into D10 parasites to create the transgenic line D10-mdr7G8/1 increases susceptibility to mefloquine, halofantrine, and artemisinin and decreases susceptibility to quinine but has no effect on susceptibility to chloroquine (which requires the appropriate mutation in pfcrt). In our studies, D10-mdr7G8/1 parasites grew slightly better than D10-mdrD10 at supra-IC50 concentrations of CsA (data not shown) but there was no significant difference in the IC50 between the two lines (Table 1). Taken together, these results suggest that polymorphisms in pfmdr1 affect susceptibility to CsA.
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Susceptibilities of pfmdr1-overexpressing strains to CsA
Amplification of the pfmdr1 gene and overproduction of Pgh1 are associated with resistance to mefloquine (and sometimes halofantrine and quinine) in both field isolates and laboratory-selected lines.17 If Pgh1 were the target of CsA, we would expect overproduction of this protein to increase the amount of CsA required to inhibit growth. We therefore tested the susceptibility of isolates K1, K1Mef and K1Mef2 (ref. 32) to CsA. Parasite lines K1Mef and K1Mef2 were derived from cloned, chloroquine-resistant K1/Thailand by continued exposure to mefloquine: they have pfmdr1 copy numbers of 1.9 and 2.4, respectively, compared with 1.0 for K1 and they overproduce Pgh1 by 2- and 2.6-fold, respectively.32 As can be seen from Table 2, K1Mef and K1Mef2 lines were significantly less susceptible to CsA. Resistance of K1Mef and K1Mef2 to mefloquine and halofantrine was confirmed but under the conditions used the expected slight decrease in resistance to chloroquine was not apparent. In summary, the results are consistent with a role for Pgh1 in the action of CsA but are complicated by the fact that the three lines used are not known to have identical genetic backgrounds.
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Effects of combinations of CsA with mefloquine, halofantrine, quinine or chloroquine
In view of the existing data on antimalarial interactions between quinoline and related drugs24 we predicted that if Pgh1 were the target of CsA, then CsA might act synergistically with mefloquine, halofantrine and possibly quinine but antagonistically with chloroquine. These predictions were borne out by the analysis of drug interactions using both 3D7 and FCH5.C2 lines (Figure 1 and data not shown). The statistical analyses for the FCH5.C2 line (Table 3) indicated that the CsA + mefloquine and CsA + halofantrine combinations were strongly synergistic, CsA + quinine was a weakly, but significantly, synergistic combination, while CsA + chloroquine was slightly antagonistic but the effect was on the border of statistical significance. The analysis was therefore repeated for 3D7 (Table 4), in which there was also significant antagonism between CsA and chloroquine. In summary, CsA was synergistic with mefloquine, halofantrine or quinine and antagonistic with chloroquine.
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Investigation of possible CsA–Pgh1 interaction
The function of Pgh1 is unknown and there is no validated assay that could be used to demonstrate a direct effect of CsA on this protein. We therefore attempted to demonstrate interactions between CsA and Pgh1 in parasites or parasite extracts using photoaffinity labelling, co-immunoprecipitation or affinity chromatography. As reported previously, the two major cyclosporin-binding proteins in parasites are cyclophilins PfCYP19A and PfCYP19B (ref. 8) and the detection of cyclosporin–Pgh1 interaction may be compromised by the sequestration of cyclosporin by large quantities of cyclophilins. In no case were we able to detect binding of cyclosporin to Pgh1 in whole cells (C. S. Gavigan and A. Bell, unpublished data).8 In an attempt to increase the ratio of Pgh1 to cyclophilins and in recognition that the DV was the major site of accumulation of labelled dihydrocyclosporin,33 we repeated the experiments of Gavigan et al.8 using extracts of isolated DV in place of whole-parasite extracts. No detectable Pgh1 bound to the cyclosporin column in these experiments (data not shown). Western immunoblotting with antiserum to Pgh1 confirmed that the protein was present in DV extracts but was highly labile, appearing as multiple lower-molecular weight bands (data not shown).
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In both laboratory and field isolates of P. falciparum, it is common to find a broadly reciprocal relationship between susceptibility to chloroquine and susceptibility to the arylamino alcohols mefloquine and halofantrine.16 This observation holds true for the allelic-exchange lines constructed by Cowman and co-workers21 in which the D10-type pfmdr1 is associated with decreased susceptibility to mefloquine and halofantrine but increased susceptibility to chloroquine, and the 7G8 sequence vice versa. In a similar vein, overproduction of Pgh1 as a result of amplification of pfmdr1 in parasites derived from chloroquine-resistant lines is associated with resistance to mefloquine, halofantrine and sometimes quinine but enhanced susceptibility to chloroquine.16 In this study, sequence polymorphisms and altered expression levels of pfmdr1 affected susceptibility to CsA in a similar but not identical way to their effect on susceptibility to mefloquine and halofantrine. CsA can therefore be added to the list of drugs whose actions are influenced by this protein. In some P. falciparum strains there is antagonism between chloroquine and either mefloquine or quinine.24 Again, CsA followed the mefloquine pattern in being antagonistic with chloroquine. There are, therefore, certain similarities between parasite susceptibilities to CsA and mefloquine/halofantrine that have not to our knowledge been noted before.
Our data could support the idea that Pgh1 is the molecular target of CsA and that binding of CsA to the transporter inhibits its function, perhaps by affecting chemical gradients across the DV membrane or by reducing the intracellular transport of crucial metabolites. This would be consistent with the known cyclosporin-binding properties of other P-glycoproteins34,35 and the superior antimalarial activity of valspodar.10 It may be relevant that besides cyclosporins and mefloquine a number of known modulators of P-glycoprotein-mediated MDR, including quinine, quinidine, verapamil, FK506, azole antifungal agents and HIV-protease inhibitors,13 have antimalarial activity. However, we were unable to provide evidence for a direct interaction between CsA and Pgh1. While this paper was under review, Rohrbach et al.36 reported that subcellular distribution of Fluo-4 acetoxymethyl ester could be used as a surrogate assay for Pgh1 function in live cells. The accumulation of Fluo-4 in the DV was inhibited by CsA, suggesting a possible direct effect of the drug on Pgh1, but the concentration of CsA used was very high (10 µM). Another possibility is that CsA is transported by Pgh1. The orientation of its nucleotide-binding sites suggests that Pgh1 is likely to transport substrates into rather than out of the DV.37 This might tally with the observation by Scheibel et al.33 that labelled dihydrocyclosporin accumulates in the DV and to a lesser extent in the cytosol. According to this idea, our finding that overproduction of Pgh1 appears to confer a small degree of resistance to CsA might imply that Pgh1 transports the drug away from its site of action (in the cytosol or DV membrane). However, the cytosol contains abundant CsA-binding cyclophilins8 and the bound drug may not be a substrate for any such transporter. Also, CsA is transported only slowly by mammalian P-glycoprotein and valspodar probably not at all.11 Another possibility is that the effects of pfmdr1 polymorphisms and overexpression are indirect, perhaps acting via alterations in some aspect of DV physiology such as luminal pH regulation. Adjudication between these different hypotheses will probably depend on a better understanding of the function of Pgh1. Chloroquine is widely believed to interfere with the process of biomineralization of haem in the DV (reviewed in ref. 38). While there is evidence for effects of the structurally related arylamino alcohols and quinine on this process, their mechanisms of antimalarial action, like that of CsA, have not been clarified.
In summary, the susceptibility of P. falciparum to CsA was influenced by polymorphisms in and expression levels of pfmdr1 but it is not clear whether this is the result of a direct CsA–Pgh1 interaction or an indirect effect. In view of the potent antimalarial activity and low host toxicity of the cyclosporin derivative valspodar, elucidation of the mechanism of antimalarial action of cyclosporins would seem to be a very promising line of inquiry in the search for new and better antimalarial drugs.
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None to declare.
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Present address. Abbott Ireland Diagnostic Division, Finisklin Business Park, Sligo, Ireland. 
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We thank Professor Alan Cowman and Jenny Thompson for providing parasite lines and antisera to Pgh1. This work was supported by grant RP13/2001 from the Health Research Board of Ireland to A. B. The views expressed in this article represent the opinions of the authors, and not necessarily those of the US Food and Drug Administration.
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