Balancing drug resistance and growth rates via compensatory mutations in the Plasmodium falciparum chloroquine resistance transporter

The widespread use of chloroquine to treat Plasmodium falciparum infections has resulted in the selection and dissemination of variant haplotypes of the primary resistance determinant PfCRT. These haplotypes have encountered drug pressure and within‐host competition with wild‐type drug‐sensitive parasites. To examine these selective forces in vitro, we genetically engineered P. falciparum to express geographically diverse PfCRT haplotypes. Variant alleles from the Philippines (PH1 and PH2, which differ solely by the C72S mutation) both conferred a moderate gain of chloroquine resistance and a reduction in growth rates in vitro. Of the two, PH2 showed higher IC₅₀ values, contrasting with reduced growth. Furthermore, a highly mutated pfcrt allele from Cambodia (Cam734) conferred moderate chloroquine resistance and enhanced growth rates, when tested against wild‐type pfcrt in co‐culture competition assays. These three alleles mediated cross‐resistance to amodiaquine, an antimalarial drug widely used in Africa. Each allele, along with the globally prevalent Dd2 and 7G8 alleles, rendered parasites more susceptible to lumefantrine, the partner drug used in the leading first‐line artemisinin‐based combination therapy. These data reveal ongoing region‐specific evolution of PfCRT that impacts drug susceptibility and relative fitness in settings of mixed infections, and raise important considerations about optimal agents to treat chloroquine‐resistant malaria.     

Mechanistic basis for multidrug resistance and collateral drug sensitivity conferred to the malaria parasite by polymorphisms in PfMDR1 and PfCRT

Polymorphisms in the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene and the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene alter the malaria parasite’s susceptibility to most of the current antimalarial drugs. However, the precise mechanisms by which PfMDR1 contributes to multidrug resistance have not yet been fully elucidated, nor is it understood why polymorphisms in pfmdr1 and pfcrt that cause chloroquine resistance simultaneously increase the parasite’s susceptibility to lumefantrine and mefloquine—a phenomenon known as collateral drug sensitivity. Here, we present a robust expression system for PfMDR1 in Xenopus oocytes that enables direct and high-resolution biochemical characterizations of the protein. We show that wild-type PfMDR1 transports diverse pharmacons, including lumefantrine, mefloquine, dihydroartemisinin, piperaquine, amodiaquine, methylene blue, and chloroquine (but not the antiviral drug amantadine). Field-derived mutant isoforms of PfMDR1 differ from the wild-type protein, and each other, in their capacities to transport these drugs, indicating that PfMDR1-induced changes in the distribution of drugs between the parasite’s digestive vacuole (DV) and the cytosol are a key driver of both antimalarial resistance and the variability between multidrug resistance phenotypes. Of note, the PfMDR1 isoforms prevalent in chloroquine-resistant isolates exhibit reduced capacities for chloroquine, lumefantrine, and mefloquine transport. We observe the opposite relationship between chloroquine resistance-conferring mutations in PfCRT and drug transport activity. Using our established assays for characterizing PfCRT in the Xenopus oocyte system and in live parasite assays, we demonstrate that these PfCRT isoforms transport all 3 drugs, whereas wild-type PfCRT does not. We present a mechanistic model for collateral drug sensitivity in which mutant isoforms of PfMDR1 and PfCRT cause chloroquine, lumefantrine, and mefloquine to remain in the cytosol instead of sequestering within the DV. This change in drug distribution increases the access of lumefantrine and mefloquine to their primary targets (thought to be located outside of the DV), while simultaneously decreasing chloroquine’s access to its target within the DV. The mechanistic insights presented here provide a basis for developing approaches that extend the useful life span of antimalarials by exploiting the opposing selection forces they exert upon PfCRT and PfMDR1.     

Metabolic QTL Analysis Links Chloroquine Resistance in Plasmodium falciparum to Impaired Hemoglobin Catabolism

Drug resistant strains of the malaria parasite, Plasmodium falciparum, have rendered chloroquine ineffective throughout much of the world. In parts of Africa and Asia, the coordinated shift from chloroquine to other drugs has resulted in the near disappearance of chloroquine-resistant (CQR) parasites from the population. Currently, there is no molecular explanation for this phenomenon. Herein, we employ metabolic quantitative trait locus mapping (mQTL) to analyze progeny from a genetic cross between chloroquine-susceptible (CQS) and CQR parasites. We identify a family of hemoglobin-derived peptides that are elevated in CQR parasites and show that peptide accumulation, drug resistance, and reduced parasite fitness are all linked in vitro to CQR alleles of the P. falciparum chloroquine resistance transporter (pfcrt). These findings suggest that CQR parasites are less fit because mutations in pfcrt interfere with hemoglobin digestion by the parasite. Moreover, our findings may provide a molecular explanation for the reemergence of CQS parasites in wild populations.     

Chloroquine susceptibility and reversibility in a Plasmodium falciparum genetic cross

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) are major determinants of verapamil (VP)‐reversible CQ resistance (CQR). In the presence of mutant PfCRT, additional genes contribute to the wide range of CQ susceptibilities observed. It is not known if these genes influence mechanisms of chemosensitization by CQR reversal agents. Using quantitative trait locus (QTL) mapping of progeny clones from the HB3 × Dd2 cross, we show that the P. falciparum multidrug resistance gene 1 (pfmdr1) interacts with the South‐East Asia‐derived mutant pfcrt haplotype to modulate CQR levels. A novel chromosome 7 locus is predicted to contribute with the pfcrt and pfmdr1 loci to influence CQR levels. Chemoreversal via a wide range of chemical structures operates through a direct pfcrt‐based mechanism. Direct inhibition of parasite growth by these reversal agents is influenced by pfcrt mutations and additional loci. Direct labelling of purified recombinant PfMDR1 protein with a highly specific photoaffinity CQ analogue, and lack of competition for photolabelling by VP, supports our QTL predictions. We find no evidence that pfmdr1 copy number affects CQ response in the progeny; however, inheritance patterns indicate that an allele‐specific interaction between pfmdr1 and pfcrt is part of the complex genetic background of CQR.     

Molecular interaction of selected phytochemicals under the charged environment of Plasmodium falciparum chloroquine resistance transporter (PfCRT) model

Phytochemicals of Catharanthus roseus Linn. and Tylophora indica have been known for their inhibition of malarial parasite, Plasmodium falciparum in cell culture. Resistance to chloroquine (CQ), a widely used antimalarial drug, is due to the CQ resistance transporter (CRT) system. The present study deals with computational modeling of Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein and development of charged environment to mimic a condition of resistance. The model of PfCRT was developed using Protein homology/analogy engine (PHYRE ver 0.2) and was validated based on the results obtained using PSI-PRED. Subsequently, molecular interactions of selected phytochemicals extracted from C. roseus Linn. and T. indica were studied using multiple-iterated genetic algorithm-based docking protocol in order to investigate the translocation of these legends across the PfCRT protein. Further, molecular dynamics studies exhibiting interaction energy estimates of these compounds within the active site of the protein showed that compounds are more selective toward PfCRT. Clusters of conformations with the free energy of binding were estimated which clearly demonstrated the potential channel and by this means the translocation across the PfCRT is anticipated.     

A Process Similar to Autophagy Is Associated with Cytocidal Chloroquine Resistance in Plasmodium falciparum

Resistance to the cytostatic activity of the antimalarial drug chloroquine (CQ) is becoming well understood, however, resistance to cytocidal effects of CQ is largely unexplored. We find that PfCRT mutations that almost fully recapitulate P. falciparum cytostatic CQ resistance (CQR^CS) as quantified by CQ IC₅₀ shift, account for only 10–20% of cytocidal CQR (CQR^CC) as quantified by CQ LD₅₀ shift. Quantitative trait loci (QTL) analysis of the progeny of a chloroquine sensitive (CQS; strain HB3)×chloroquine resistant (CQR; strain Dd2) genetic cross identifies distinct genetic architectures for CQR^CS vs CQR^CC phenotypes, including identification of novel interacting chromosomal loci that influence CQ LD₅₀. Candidate genes in these loci are consistent with a role for autophagy in CQR^CC, leading us to directly examine the autophagy pathway in intraerythrocytic CQR parasites. Indirect immunofluorescence of RBC infected with synchronized CQS vs CQR trophozoite stage parasites reveals differences in the distribution of the autophagy marker protein PfATG8 coinciding with CQR^CC. Taken together, the data show that an unusual autophagy – like process is either activated or inhibited for intraerythrocytic trophozoite parasites at LD₅₀ doses (but not IC₅₀ doses) of CQ, that the pathway is altered in CQR P. falciparum, and that it may contribute along with mutations in PfCRT to confer the CQR^CC phenotype.     

Multiple Drugs Compete for Transport via the Plasmodium falciparum Chloroquine Resistance Transporter at Distinct but Interdependent Sites

Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.     

Efflux of a range of antimalarial drugs and ‘chloroquine resistance reversers’ from the digestive vacuole in malaria parasites with mutant PfCRT

Chloroquine‐resistant malaria parasites (Plasmodium falciparum) show an increased leak of H⁺ ions from their internal digestive vacuole in the presence of chloroquine. This phenomenon has been attributed to the transport of chloroquine, together with H⁺, out of the digestive vacuole (and hence away from its site of action) via a mutant form of the parasite's chloroquine resistance transporter (PfCRT). Here, using transfectant parasite lines, we show that a range of other antimalarial drugs, as well as various ‘chloroquine resistance reversers’ induce an increased leak of H⁺ from the digestive vacuole of parasites expressing mutant PfCRT, consistent with these compounds being substrates for mutant forms, but not the wild‐type form, of PfCRT. For some compounds there were significant differences observed between parasites having the African/Asian Dd2 form of PfCRT and those with the South American 7G8 form of PfCRT, consistent with there being differences in the transport properties of the two mutant proteins. The finding that chloroquine resistance reversers are substrates for mutant PfCRT has implications for the mechanism of action of this class of compound.     

No Seasonal Accumulation of Resistant P. falciparum when High-Dose Chloroquine Is Used

Background

Potentially chloroquine resistant P. falciparum, identified by the 76T haplotype in the chloroquine resistance transporter (pfcrt 76T), are highly prevalent throughout Africa. In Guinea-Bissau, normal and double dose chloroquine have respective efficacies of 34% and 78% against P.falciparum with pfcrt 76T and approximately three times the normal dose of chloroquine is routinely taken. Proportions of pfcrt 76T generally increase during high transmission seasons, as P.falciparum with pfcrt 76T commonly survive treatment with normal dose chloroquine. In Guinea-Bissau, there should be no seasonal increase of pfcrt 76T if the high doses of CQ commonly used are effective.

Methods and Findings

P. falciparum parasite density, age, sex, the proportion of chloroquine resistance associated haplotypes pfcrt 76T and P. falciparum multidrug resistance gene 1 86Y were assessed in 988 samples collected from children between 2002 and 2007. There was no seasonal accumulation of any allele. During the high and low transmission periods the pfcrt 76T proportions were 24% (95% CI, 21–27%) and 26% (95% CI, 20–33%). There was no significant change of pfcrt 76T (OR 1.05, 95% CI; 0.94–1.16 p = 0.39) or pfmdr1 86Y (OR 0.92, 95%CI; 0.83–1.01 p = 0.08) proportions between 2003 and 2007. Lower median parasite density (P.falciparum/µl) was associated with pfcrt 76T (15254 [95% CI, 12737–17772]; n = 164) compared to pfcrt 76K (18664 [95% CI, 16676–20653]; p = 0.003; n = 591). Similarly, pfmdr1 86Y was associated with a lower median parasite density (16320 [95% CI, 13696–18944]; n = 224) compared to pfmdr1 86N, (18880 [95% CI, 16701–21059]; P = 0.018; n = 445).

Conclusions

In contrast to the rest of Africa, P. falciparum parasites resistant to normal dose chloroquine do not have a selective advantage great enough to become the dominant P.falciparum type in Guinea-Bissau. This is most likely due to the efficacy of high-dose chloroquine as used in Guinea-Bissau, combined with a loss of fitness associated with pfcrt 76T.     

Double-drug development against antioxidant enzymes from Plasmodium falciparum

Abstract

New drugs against malaria are urgently and continuously needed. Plasmodium parasites are exposed to higher fluxes of reactive oxygen species and need high activities of intracellular antioxidant systems. A most important antioxidative system consists of (di)thiols which are recycled by disulfide reductases (DR), namely both glutathione reductases (GR) of the malarial parasite Plasmodium falciparum and man, and the thioredoxin reductase (TrxR) of P. falciparum. The aim of our interdisciplinary research is to substantiate DR inhibitors as antimalarial agents. Such compounds are active per se but, in addition, they can reverse thiol-based resistance against other drugs in parasites. Reversal of drug resistance by DR inhibitors is currently investigated for the commonly used antimalarial drug chloroquine (CQ). Our recent strategy is based on the synthesis of inhibitors of the glutathione reductases from parasite and host erythrocyte. With the expectation of a synergistic or additive effect, double-headed prodrugs were designed to be directed against two different and essential functions of the malarial parasite P. falciparum, namely glutathione regeneration and heme detoxification. The prodrugs were prepared by linking bioreversibly a GR inhibitor to a 4-aminoquinoline moiety which is known to concentrate in the acidic food vacuole of parasites. Drug-enzyme interaction was correlated with antiparasitic action in vitro on strains resistant towards CQ and in vivo in Plasmodium berghei-infected mice as well as absence of cytotoxicity towards human cells. Because TrxR of P. falciparum was recently shown to be responsible for the residual glutathione disulfide-reducing capacity observed after GR inhibition in P. falciparum, future development of antimalarial drug-candidates that act by perturbing the redox equilibrium of parasites is based on the design of new double-drugs based on TrxR inhibitors as potential antimalarial drug candidates.     

A Genome Wide Association Study of Plasmodium falciparum Susceptibility to 22 Antimalarial Drugs in Kenya

Background

Drug resistance remains a chief concern for malaria control. In order to determine the genetic markers of drug resistant parasites, we tested the genome-wide associations (GWA) of sequence-based genotypes from 35 Kenyan P. falciparum parasites with the activities of 22 antimalarial drugs.

Methods and Principal Findings

Parasites isolated from children with acute febrile malaria were adapted to culture, and sensitivity was determined by in vitro growth in the presence of anti-malarial drugs. Parasites were genotyped using whole genome sequencing techniques. Associations between 6250 single nucleotide polymorphisms (SNPs) and resistance to individual anti-malarial agents were determined, with false discovery rate adjustment for multiple hypothesis testing. We identified expected associations in the pfcrt region with chloroquine (CQ) activity, and other novel loci associated with amodiaquine, quinazoline, and quinine activities. Signals for CQ and primaquine (PQ) overlap in and around pfcrt, and interestingly the phenotypes are inversely related for these two drugs. We catalog the variation in dhfr, dhps, mdr1, nhe, and crt, including novel SNPs, and confirm the presence of a dhfr-164L quadruple mutant in coastal Kenya. Mutations implicated in sulfadoxine-pyrimethamine resistance are at or near fixation in this sample set.

Conclusions/Significance

Sequence-based GWA studies are powerful tools for phenotypic association tests. Using this approach on falciparum parasites from coastal Kenya we identified known and previously unreported genes associated with phenotypic resistance to anti-malarial drugs, and observe in high-resolution haplotype visualizations a possible signature of an inverse selective relationship between CQ and PQ.     

The antimalarial drug resistance protein Plasmodium falciparum chloroquine resistance transporter binds chloroquine

Recently, mutations in the novel polytopic integral membrane protein PfCRT were shown to cause chloroquine resistance (CQR) in the malarial parasite Plasmodium falciparum. PfCRT is not a member of the well-known family of ABC proteins that have previously been associated with other drug resistance phenomena. Thus, the mechanism(s) whereby mutant PfCRT molecules confer antimalarial drug resistance is (are) unknown. Previously, we succeeded in overexpressing PfCRT to high levels in Pichia pastoris yeast by synthesizing a codon-optimized version of the pfcrt gene. Using purified membranes and inside-out plasma membrane vesicles (ISOV) isolated from strains harboring either wild-type or CQR-associated mutant PfCRT, we now show that under deenergized conditions the PfCRT protein specifically binds the antimalarial drug chloroquine (CQ) with a K(D) near 400 nM but does not measurably bind the related drug quinine (QN) at physiologically relevant concentrations. Transport studies using ISOV show that QN is passively accumulated as expected on the basis of previous measurement of the ISOV DeltapH for the different strains. However, passive accumulation of CQ is lower than expected for ISOV harboring mutant PfCRT, despite higher DeltapH for these ISOV.     

Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine

Background

Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance. Expressed in yeast, PfCRT resembles an anion channel. Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin. The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel. It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity. Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own.

Methods

Changes in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined.

Results and conclusions

The results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole. Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76. This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT.     

Characterization of the Chloroquine Resistance Transporter Homologue in Toxoplasma gondii

Mutations in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein confer resistance to the antimalarial drug chloroquine. PfCRT localizes to the parasite digestive vacuole, the site of chloroquine action, where it mediates resistance by transporting chloroquine out of the digestive vacuole. PfCRT belongs to a family of transporter proteins called the chloroquine resistance transporter family. CRT family proteins are found throughout the Apicomplexa, in some protists, and in plants. Despite the importance of PfCRT in drug resistance, little is known about the evolution or native function of CRT proteins. The apicomplexan parasite Toxoplasma gondii contains one CRT family protein. We demonstrate that T. gondii CRT (TgCRT) colocalizes with markers for the vacuolar (VAC) compartment in these parasites. The TgCRT-containing VAC is a highly dynamic organelle, changing its morphology and protein composition between intracellular and extracellular forms of the parasite. Regulated knockdown of TgCRT expression resulted in modest reduction in parasite fitness and swelling of the VAC, indicating that TgCRT contributes to parasite growth and VAC physiology. Together, our findings provide new information on the role of CRT family proteins in apicomplexan parasites.     

Characterization of the Commercially-Available Fluorescent Chloroquine-BODIPY Conjugate,LynxTag-CQGREEN,as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQ_GREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQ_GREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQ_GREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC₅₀s were determined. These data were compared with LynxTag-CQ_GREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC₅₀ of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.     

Surveillance of Travellers: An Additional Tool for Tracking Antimalarial Drug Resistance in Endemic Countries

Introduction

There are growing concerns about the emergence of resistance to artemisinin-based combination therapies (ACTs). Since the widespread adoption of ACTs, there has been a decrease in the systematic surveillance of antimalarial drug resistance in many malaria-endemic countries. The aim of this work was to test whether data on travellers returning from Africa with malaria could serve as an additional surveillance system of local information sources for the emergence of drug resistance in endemic-countries.

Methodology

Data were collected from travellers with symptomatic Plasmodium falciparum malaria returning from Senegal (n = 1,993), Mali (n = 2,372), Cote d’Ivoire (n = 4,778) or Cameroon (n = 3,272) and recorded in the French Malaria Reference Centre during the period 1996–2011. Temporal trends of the proportion of parasite isolates that carried the mutant genotype, pfcrt 76T, a marker of resistance to chloroquine (CQ) and pfdhfr 108N, a marker of resistance to pyrimethamine, were compared for travellers and within-country surveys that were identified through a literature review in PubMed. The in vitro response to CQ was also compared between these two groups for parasites from Senegal.

Results

The trends in the proportion of parasites that carried pfcrt 76T, and pfdhfr 108N, were compared for parasites from travellers and patients within-country using the slopes of the curves over time; no significant differences in the trends were found for any of the 4 countries. These results were supported by in vitro analysis of parasites from the field in Senegal and travellers returning to France, where the trends were also not significantly different.

Conclusion

The results have not shown different trends in resistance between parasites derived from travellers or from parasites within-country. This work highlights the value of an international database of drug responses in travellers as an additional tool to assess the emergence of drug resistance in endemic areas where information is limited.     

Association of molecular markers in Plasmodium falciparum crt and mdr1 with in vitro chloroquine resistance: A Philippine study

Specific mutations in the pfcrt and pfmdr1 genes have been reported to be associated with chloroquine-resistant falciparum malaria parasites worldwide. These genetic markers are considered to be useful tools for the elucidation of several aspects of the epidemiology of drug resistant malaria. In this study, Plasmodium falciparum isolates from three distinct areas of the Philippines were analyzed for drug-resistance-associated genetic mutations, and their association with the in vitro chloroquine (CQ) response. Two novel pfcrt 72–76 allelic types, CVMDT and SVMDT, were detected. The frequency of the pfcrt K76T mutation in the isolates that were successfully tested for in vitro CQ susceptibility was found to be 100% in Kalinga, 80% in Palawan, and 87% in Mindanao. The frequency of the pfmdr1 N86Y mutation was 39% in Kalinga, 35% in Palawan, and 93% in Mindanao isolates. No mutations were found at positions 1042 and 1246 of pfmdr1. However, there were no significant associations found between polymorphisms in these genes and in vitro CQ susceptibility. The results of this study indicate that mutations in pfcrt and pfmdr1 are not predictive of in vitro CQ resistance in Philippine isolates and may therefore not be suitable as molecular markers for surveillance.     

Antiretroviral protease inhibitors potentiate chloroquine antimalarial activity in malaria parasites by regulating intracellular glutathione metabolism

Antiretroviral protease inhibitors significantly potentiated the sensitivity of chloroquine-resistant malaria parasites to the antimalarial drug in vitro and in vivo. Ritonavir was found to be potent in potentiating CQ antimalarial activities in both -resistant and -sensitive lines. The mechanism by which the APIs modulate the CQ resistance in malaria parasites was further investigated. CQ-resistant parasites showed increased intracellular glutathione levels in comparison with the CQ-sensitive parasites. Treatment with APIs significantly reduced the levels of GSH and glutathione S-transferase activities in CQ-resistant parasites. Ritonavir also decreased glutathione reductase activities and glutathione peroxidase activities in CQ-resistant parasite line. Taken together, these results demonstrate that parasite GSH and GST may play an important role in CQ resistance and APIs are able to enhance the sensitivity of CQ-resistant malaria parasite to the drug by influencing the levels of GSH and the activities of the related enzymes.