The determinant of verapamil-reversible chloroquine resistance (CQR) in a Plasmodium falciparum genetic cross maps to a 36 kb segment of chromosome 7. This segment harbors a 13-exon gene, pfcrt, having point mutations that associate completely with CQR in parasite lines from Asia, Africa, and South America. These data, transfection results, and selection of a CQR line harboring a novel K761 mutation point to a central role for the PfCRT protein in CQR. This transmembrane protein localizes to the parasite digestive vacuole (DV), the site of CQ action, where increased compartment acidification associates with PfCRT point mutations. Mutations in PfCRT may result in altered chloroquine flux or reduced drug binding to hematin through an effect on DV pH.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with multiple mutations in the digestive vacuole membrane protein PfCRT. The chloroquine-sensitive (CQS) 106/1 line of P. falciparum has six of seven PfCRT mutations consistently found in CQR parasites from Asia and Africa. The missing mutation at position 76 (K76T in reported population surveys) may therefore be critical to CQR. To test this hypothesis, we exposed 106/1 populations (10 9 -10 10 parasites) to a chloroquine (CQ) concentration lethal to CQS parasites. In multiple independent experiments, surviving CQR parasites were detected in the cultures after 28 to 42 days. These parasites showed novel K76N or K76I PfCRT mutations and corresponding CQ IC 50 values that were ϳ8-and 12-fold higher than that of the original 106/1 IC 50 . A distinctive feature of the K76I line relative to 106/1 parasites was their greatly increased sensitivity to quinine (QN) but reduced sensitivity to its enantiomer quinidine (QD), indicative of a unique stereospecific response not observed in other CQR lines. Furthermore, verapamil had the remarkable effect of antagonizing the QN response while potentiating the QD response of K76I parasites. In our single-step drug selection protocol, the probability of the simultaneous selection of two specific mutations required for CQR is extremely small. We conclude that the K76N or K76I change added to the other pre-existing mutations in the 106/1 PfCRT protein was responsible for CQR. The various mutations that have now been documented at PfCRT position 76 (K76T, K76N, K76I) suggest that the loss of lysine is central to the CQR mechanism.

We report on the development of a new SYBR Green I-based plate assay for analyzing the activities of antimalarial drugs against intraerythrocytic Plasmodium falciparum. This assay is considerably faster, less labor-intensive, and less expensive than conventional radiotracer (e.g., [3 H]hypoxanthine and [ 3 H]ethanolamine)-based assays or P. falciparum lactate dehydrogenase activity-based assays. The assay significantly improves the pace at which antimalarial drug discovery efforts may proceed.The continued emergence and spread of multidrug-resistant strains of Plasmodium falciparum and P. vivax are arguably the most pressing problems in the area of infectious diseases today. Also, although the recent deciphering of the P. falciparum genome reveals many promising new drug targets, the financial cost of bringing drugs to the clinic is a major obstacle in the development of new antimalarials (6). A faster, less expensive, high-throughput means of screening the activities of drugs against a variety of malarial parasite strains would greatly assist preclinical drug development.Quantitative assessment of the effects of drugs on parasite growth and development can be achieved by direct (but extremely tedious) microscopic examination of blood smears. An alternative assay is measurement of the effect of drug exposure by determination of the level of incorporation of radiolabeled hypoxanthine. While the latter method can be automated, it requires radioactive materials and is not convenient for detection of parasite stage-specific effects. Another assay measures parasite lactate dehydrogenase activity by methods that do not require radioisotopes. However, this assay requires multiple processing steps and expensive reagents and is not particularly cost-effective for large-scale drug screening efforts.We have thus endeavored to develop more rapid and convenient cell-based assays for quantifying antimalarial drug activities. We have strived to enhance simplicity and reduce cost. In this paper, we report on the development of one such assay that relies on the fluorophore SYBR Green I. MATERIALS AND METHODSCell culture. Asexual culture is routinely performed. Parasite cultures are initiated from stabilates preserved in liquid nitrogen (the level of parasitemia during storage is Ն10%). Following the initiation of a fresh culture, at least two full life cycles (96 h) are completed before parasites are used for assays. In general, cultures are synchronized in the laboratory, and assays are initiated when the parasites are at the ring stage. However, we find that this assay is equally applicable to asynchronous culture and that similar 50% inhibitory concentrations (IC 50 s) are calculated from data with asynchronous and synchronous cultures (data not shown). Prior to assay initiation, the level of parasitemia of an aliquot of a stock culture is measured by light microscopy following Giemsa staining or by fluorescence-activated cell sorter analysis after staining with propidium iodide. In general, stock cultures with 5 to 10% paras...

Data availability: All raw movie frames, micrographs, the particle stack and relevant metadata files have been deposited into EMPIAR, with accession code EMPIAR-10330. The electron density map has been deposited into EMDB, with accession code EMD-20806. The model has been deposited in the PDB, with accession code 6UKJ. All data are available in the manuscript or the supplementary materials.

Paramagnetic metal centers [such as Fe(III) found within ferriprotoporphyrin IX heme (FPIX)] exert through space effects on the relaxation rate of nearby proton spins that depend critically on the metal-proton distance. We have measured these effects for all protons of several antimalarial drugs that bind to FPIX by systematically varying the drug:heme molar ratio in high field NMR experiments. These measurements allow us to determine precise FPIX Fe-drug H distances for the solution structures of noncovalent complexes formed between FPIX mu-oxo dimers and the antimalarial drugs chloroquine (CQ), quinine (QN), and quinidine (QD). Using these distances, we then performed distance restraint calculations to determine the lowest-energy solution structures of these complexes. Structures were solved for neutral, monoprotic (+1), and diprotic (+2) forms of the drugs. Analysis of these structures allows us to visualize for the first time the stereospecific differences between QN and QD binding to FPIX and the differences in populations of QN and QD solution structures upon changes in digestive vacuolar pH for drug resistant malarial parasites [Dzekunov, S. M., et al. (2000) Mol. Biochem. Parasitol. 110, 107-124]. The data indicate a previously unrecognized key role for the CQ aliphatic chain in stabilizing FPIX-CQ complexes, and suggest how lengthening or shortening the chain might perturb stability. We also define FPIX:drug stoichiometries of 2:1 for the complexes formed at physiological FPIX concentrations, in contrast to the 4:1 and 5:1 stoichiometries previously determined at higher FPIX concentrations [Dorn, A., et al. (1998) Biochem. Pharmacol. 55, 727-736]. These atomic resolution antimalarial drug-heme structures should help elucidate how these drugs inhibit formation of hemozoin during metabolism of heme within the malarial parasite Plasmodium falciparum and assist ongoing development of strategies for circumventing antimalarial drug resistance.

The mechanistic basis for chloroquine resistance (CQR) in Plasmodium falciparum recently has been linked to the polymorphic gene pfcrt. Alleles associated with CQR in natural parasite isolates harbor threonine (T), as opposed to lysine (K) at amino acid 76. P. falciparum CQR strains of African and Southeast Asian origin carry pfcrt alleles encoding an amino acid haplotype of CVIET (residues 72-76), whereas most South American CQR strains studied carry an allele encoding an SVMNT haplotype; chloroquine-sensitive strains from malarious regions around the world carry a CVMNK haplotype. Upon investigating the origin of pfcrt alleles in Papua New Guinean (PNG) P. falciparum we found either the chloroquine-sensitive-associated CVMNK or CQR-associated SVMNT haplotypes previously seen in Brazilian isolates. Remarkably we did not find the CVIET haplotype observed in CQR strains from Southeast Asian regions more proximal to PNG. Further we found a previously undescribed CQR phenotype to be associated with the SVMNT haplotype from PNG and South America. This CQR phenotype is significantly less responsive to verapamil chemosensitization compared with the effect associated with the CVIET haplotype. Consistent with this, we observed that verapamil treatment of P. falciparum isolates carrying pfcrt SVMNT is associated with an attenuated increase in digestive vacuole pH relative to CVIET pfcrt-carrying isolates. These data suggest a key role for pH-dependent changes in hematin receptor concentration in the P. falciparum CQR mechanism. Our findings also suggest that P. falciparum CQR has arisen through multiple evolutionary pathways associated with pfcrt K76T.

Recently [Roepe, P.D. (1992) Biochemistry 31, 12555-12564], increased steady-state levels of chemotherapeutic drug efflux from multidrug-resistant (MDR) myeloma cells were correlated with intracellular alkalinization. To better understand elevated pHi in MDR cells, Na(+)- and Cl-dependent recovery of pHi upon intracellular acid or alkaline shock has been examined for this same series of MDR cell lines. In agreement with another recent report [Boscoboinik, D., Gupta, R.S., & Epand, R.M. (1990) Br. J. Cancer 61, 568-572], we find that the rate of Na(+)-induced alkalinization after an intracellular acid shock is increased in the MDR cells, relative to the drug-sensitive parent. Interestingly, we also now find that mRNA encoding the human Na+/H+ exchanger (NHE) is overexpressed in these MDR cells, but the level of overexpression does not correlate with the relative drug resistance or steady-state pHi. It is also found that the efficiency of Cl(-)dependent reacidification of pHi, after an intracellular alkaline shock is reduced in the MDR cells. This effect appears to correlate with the relative expression of MDR protein, but not the relative expression of Cl-/HCO3- exchanger (AE), which we now find is also altered in the series of cells. Since elevated pHi will increase delta pH across the plasma membrane, we have also measured the electrical potential for these cells using three different methods. Most interestingly, the magnitude of the plasma membrane electrical potential (delta psi) decreases concomitant with increased expression of the MDR protein. Energy provided by increased delta pH compensates for the lowered delta psi, such that the total electrochemical membrane potential (delta mu H+) remains similar among the cells in this series (delta mu H+ = delta psi - Z delta pH). These data, along with other recent experiments that associated an increased Cl- conductance with the expression of MDR protein [Valverde, M., Diaz, M., Sepúlveda, F.V., Gill, D.R., Hyde, S.C., & Higgins, C.F. (1992) Nature 355, 830-833], are consistent with a model for MDR protein-mediated multidrug resistance that does not entail direct active transport of lipophilic drugs by the MDR protein.

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