Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.     

Functional reconstitution of purified chloroquine resistance membrane transporter expressed in yeast

Malaria is one of the major parasitic diseases. Current treatment of malaria is seriously hampered by the emergence of drug resistant cases. A once-effective drug chloroquine (CQ) has been rendered almost useless. The mechanism of CQ resistance is complicated and largely unknown. Recently, a novel transmembrane protein, Plasmodium falciparum chloroquine resistance transporter (PfCRT), has fulfilled all the requirements of being the CQ resistance gene. In order to elucidate the mechanism how PfCRT mediates CQ resistance, we have cloned the cDNA from a CQ sensitive parasite (3D7) and tried to express it in Pichia pastoris (P. pastoris) but with unsuccessful results due to AT-rich sequences in the malaria genome. We have therefore, based on the codon usage in P. pastoris, chemically synthesized a codon-modified pfcrt with an overall 55% AT content. This codon-modified pfcrt has now been successfully expressed in P. pastoris. The expressed PfCRT has been purified with immuno metal affinity chromatography (IMAC) and then reconstituted into proteoliposome. It was found that proteoliposomes have a saturable, concentration and time-dependent CQ transport activity. In addition, we found that proteoliposomes with resistant PfCRT(r) (K76T or K76I) showed an increased CQ transport activity compared to liposomes with lipid alone, or proteoliposomes reconstituted with sensitive PfCRT(s) (K76) protein. This activity could be inhibited by nigericin and decreased with the removal of Cl(-). This work suggests that PfCRT is mediating CQR in P. falciparum by virtue of its changes in CQ transport activity depending on pH gradient and chloride ion in the food vacuole.     

Multiple transporters associated with malaria parasite responses to chloroquine and quinine

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.     

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.     

Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance

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.     

Mutations in transmembrane domains 1, 4 and 9 of the Plasmodium falciparum chloroquine resistance transporter alter susceptibility to chloroquine, quinine and quinidine

Mutations in the Plasmodium falciparum chloroquine (CQ) resistance transporter (PfCRT) can result in verapamil-reversible CQ resistance and altered susceptibility to other antimalarials. PfCRT contains 10 membrane-spanning domains and is found in the digestive vacuole (DV) membrane of intraerythrocytic parasites. The mechanism by which PfCRT mediates CQ resistance is unclear although it is associated with decreased accumulation of drug within the DV. On the permissive background of the P. falciparum 106/1(K76) parasite line, we used single-step drug selection to generate isogenic clones containing unique pfcrt point mutations that resulted in amino acid changes in PfCRT transmembrane domains 1 (C72R, K76N, K76I and K76T) and 9 (Q352K, Q352R). The resulting changes of charge and hydropathy affected quantitative CQ susceptibility and accumulation as well as the stereospecific responses to quinine and quinidine. These results, together with a previously described S163R mutation in transmembrane domain 4, indicate that transmembrane segments 1, 4 and 9 of PfCRT provide important structural components of a substrate recognition and translocation domain. Charge-affecting mutations within these segments may affect the ability of PfCRT to bind different quinoline drugs and determine their net accumulation in the DV.     

A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug-protein interactions involving this mutant residue.     

Dictyostelium discoideum expresses a malaria chloroquine resistance mechanism upon transfection with mutant, but not wild-type, Plasmodium falciparum transporter PfCRT

Chloroquine resistance in Plasmodium falciparum malaria results from mutations in PfCRT, a member of a unique family of transporters present in apicomplexan parasites and Dictyostelium discoideum. Mechanisms that have been proposed to explain chloroquine resistance are difficult to evaluate within malaria parasites. Here we report on the targeted expression of wild-type and mutant forms of PfCRT to acidic vesicles in D. discoideum. We show that wild-type PfCRT has minimal effect on the accumulation of chloroquine by D. discoideum, whereas forms of PfCRT carrying a key charge-loss mutation of lysine 76 (e.g. K76T) enable D. discoideum to expel chloroquine. As in P. falciparum, the chloroquine resistance phenotype conferred on transformed D. discoideum can be reversed by the channel-blocking agent verapamil. Although intravesicular pH levels in D. discoideum show small acidic changes with the expression of different forms of PfCRT, these changes would tend to promote intravesicular trapping of chloroquine (a weak base) and do not account for reduced drug accumulation in transformed D. discoideum. Our results instead support outward-directed chloroquine efflux for the mechanism of chloroquine resistance by mutant PfCRT. This mechanism shows structural specificity as D. discoideum transformants that expel chloroquine do not expel piperaquine, a bisquinoline analog of chloroquine used frequently against chloroquine-resistant parasites in Southeast Asia. PfCRT, nevertheless, may have some ability to act on quinine and quinidine. Transformed D. discoideum will be useful for further studies of the chloroquine resistance mechanism and may assist in the development and evaluation of new antimalarial drugs.     

Investigating the activity of quinine analogues versus chloroquine resistant Plasmodium falciparum

Plasmodium falciparum, the deadliest malarial parasite species, has developed resistance against nearly all man-made antimalarial drugs within the past century. However, quinine (QN), the first antimalarial drug, remains efficacious worldwide. Some chloroquine resistant (CQR) P. falciparum strains or isolates show mild cross resistance to QN, but many do not. Further optimization of QN may provide a well-tolerated therapy with improved activity versus CQR malaria. Thus, using the Heck reaction, we have pursued a structure-activity relationship study, including vinyl group modifications of QN. Certain derivatives show good antiplasmodial activity in QN-resistant and QN-sensitive strains, with lower IC(50) values relative to QN.     

Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance

Recent studies have highlighted the importance of a parasite protein referred to as the chloroquine resistance transporter (PfCRT) in the molecular basis of Plasmodium falciparum resistance to the quinoline antimalarials. PfCRT, an integral membrane protein with 10 predicted transmembrane domains, is a member of the drug/metabolite transporter superfamily and is located on the membrane of the intra-erythrocytic parasite's digestive vacuole. Specific polymorphisms in PfCRT are tightly correlated with chloroquine resistance. Transfection studies have now proven that pfcrt mutations confer verapamil-reversible chloroquine resistance in vitro and reveal their important role in resistance to quinine. Available evidence is consistent with the view that PfCRT functions as a transporter directly mediating the efflux of chloroquine from the digestive vacuole.     

PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX

It is accepted that resistance of Plasmodium falciparum to chloroquine (CQ) is caused primarily by mutations in the pfcrt gene. However, a consensus has not yet been reached on the mechanism by which resistance is achieved. CQ-resistant (CQR) parasite lines accumulate less CQ than do CQ-sensitive (CQS) parasites. The CQR phenotype is complex with a component of reduced energy-dependent CQ uptake and an additional component that resembles energy-dependent CQ efflux. Here we show that the required energy input is in the form of the proton electrochemical gradient across the digestive vacuole (DV) membrane. Collapsing the DV proton gradient (or starving the parasites of glucose) results in similar levels of CQ accumulation in CQS and CQR lines. Under these conditions the accumulation of CQ is stimulated in CQR parasite lines but is reduced in CQS lines. Energy deprivation has no effect on the rate of CQ efflux from CQR lines implying that mutant PfCRT does not function as an efflux pump or active carrier. Using pfcrt-modified parasite lines we show that the entire CQ susceptibility phenotype is switched by the single K76T amino acid change in PfCRT. The efflux of CQ in CQR lines is not directly coupled to the energy supply, consistent with a model in which mutant PfCRT functions as a gated channel or pore, allowing charged CQ species to leak out of the DV.     

Evidence for the early emergence of piperaquine-resistant Plasmodium falciparum malaria and modeling strategies to mitigate resistance

Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter’s central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.     

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.     

Heterologous expression and ATPase activity of mutant versus wild type PfMDR1 protein

Mutation of the P. falciparum chloroquine resistance transporter (PfCRT) causes resistance to chloroquine (CQ) and other antimalarial drugs. Mutation and/or overexpression of one of the multidrug resistance protein homologues found in this malarial parasite (PfMDR1) may further modify or tailor the degree of multidrug resistance. However, considerable controversy surrounds the precise contribution of PfMDR1, in part because no direct biochemical studies of PfMDR1 have yet been possible. Using codon optimization and other principles, we have designed and constructed a yeast optimized version of the wild type pfmdr1 gene and have successfully overexpressed PfMDR1 protein in P. pastoris yeast. The protein is well expressed in either full length form or as two separate half transporters, is well localized to the yeast plasma membrane and is fully functional as evidenced by ATPase activity measurements. We have also expressed mutants that have previously been hypothesized to influence drug resistance in parasites. Using purified plasma membrane fractions, we have analyzed antimalarial drug effects on ATPase activity for wild type versus mutant proteins. Relative to other ABCB transporters involved in drug resistance, PfMDR1 is unusual. It has similar pH, [ATP], and Mg++ dependencies for ATP hydrolysis, yet relatively high Km and Vmax values for ATP hydrolysis, and ATPase activity is only mildly stimulated by antimalarial drugs. The largest measured drug effect is for CQ (to which PfMDR1 is not believed to confer resistance), and it is strongly inhibitory for WT PfMDR1. Drug resistance associated PfMDR1 mutants show either elevated (Dd2 allele encoded) or reduced (7G8 allele) basal ATPase activity and different patterns of drug stimulation or inhibition, relative to WT PfMDR1. The Dd2 PfMDR1 isoform also shows a slightly more alkaline pH optimum. Surprisingly, verapamil alone (1-300 microM) does not significantly affect WT ATPase activity but inhibits the Dd2 isoform at 1 microM. These data should assist ongoing analysis of the contribution of PfMDR1 to antimalarial drug resistance.     

Analysis of chloroquine resistance transporter (CRT) isoforms and orthologues in S. cerevisiae yeast

Previous work from our laboratory optimized MeOH-inducible expression of the P. falciparum malarial parasite transporter PfCRT in P. pastoris yeast. These strains are useful for many experiments but do not allow for inducible protein expression under ambient growth conditions. We have therefore optimized galactose-inducible expression of PfCRT in S. cerevisiae yeast. We find that expression of PfCRT confers CQ hypersensitivity to growing yeast and that this is due to plasma membrane localization of the transporter. We use quantitative analyses of growth rates to compare hypersensitivity for yeast expressing various PfCRT isoforms. We also report successful high level inducible expression of the P. vivax orthologue, PvCRT, and compare CQ hypersensitivity for PvCRT vs PfCRT expressing yeast. We test the hypothesis that hypersensitivity is due to increased transport of CQ into yeast expressing the transporters via direct (3)H-CQ transport experiments and analyze the effect that membrane potential has on transport. The data suggest important new tools for rapid functional screening of PfCRT and PvCRT isoforms and provide further evidence for a model wherein membrane potential promotes charged CQ transport by PfCRT. Data also support our previous conclusion that wild type PfCRT is capable of CQ transport and provide a basis for understanding the lack of correspondence between PvCRT mutations and resistance to CQ in the important malarial parasite P. vivax.     

Is PfCRT a channel or a carrier? Two competing models explaining chloroquine resistance in Plasmodium falciparum

Chloroquine (CQ), an antimalarial drug with a long history, now frequently fails in the field owing to the rapid spread of resistant Plasmodium falciparum strains. CQ resistance is linked to a K76T mutation in PfCRT, a membrane-located food vacuolar protein and member of the drug-metabolite transporter superfamily, but there is as yet no agreed mechanism of how mutated PfCRT brings about CQ resistance. Current models suggest that mutated PfCRT acts either as a channel or a transporter of CQ, enabling CQ to leave the digestive food vacuole of the parasite, in which the CQ accumulates. Here, we review the pros and cons of the carrier and transporter models in light of recent developments in the field.     

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.     

On the mechanism of chloroquine resistance in Plasmodium falciparum

Resistance to chloroquine of malaria strains is known to be associated with a parasite protein named PfCRT, the mutated form of which is able to reduce chloroquine accumulation in the digestive vacuole of the pathogen. Whether the protein mediates extrusion of the drug acting as a channel or as a carrier and which is the protonation state of its chloroquine substrate is the subject of a scientific debate. We present here an analytical approach that explores which combination of hypotheses on the mechanism of transport and the protonation state of chloroquine are consistent with available equilibrium experimental data. We show that the available experimental data are not, by themselves, sufficient to conclude whether the protein acts as a channel or as a transporter, which explains the origin of their different interpretation by different authors. Interestingly, though, each of the two models is only consistent with a subset of hypotheses on the protonation state of the transported molecule. The combination of these results with a sequence and structure analysis of PfCRT, which strongly suggests that the molecule is a carrier, indicates that the transported species is either or both the mono and di-protonated forms of chloroquine. We believe that our results, besides shedding light on the mechanism of chloroquine resistance in P. falciparum, have implications for the development of novel therapies against resistant malaria strains and demonstrate the usefulness of an approach combining systems biology strategies with structural bioinformatics and experimental data.     

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.