Selection for high-level chloroquine resistance results in deamplification of the pfmdr1 gene and increased sensitivity to mefloquine in Plasmodium falciparum.

A chloroquine resistant cloned isolate of Plasmodium falciparum, FAC8, which carries an amplification in the pfmdr1 gene was selected for high-level chloroquine resistance, resulting in a cell line resistant to a 10-fold higher concentration of chloroquine. These cells were found to have lost the amplification in pfmdr1 and to no longer over-produce the protein product termed P-glycoprotein homologue 1 (Pgh1). The pfmdr1 gene from this highly resistant cell line was not found to encode any amino acid changes that would account for increased resistance. Verapamil, which reverses chloroquine resistance in FAC8, also reversed high-level chloroquine resistance. Furthermore, verapamil caused a biphasic reversal of chloroquine resistance as the high-level resistance was very sensitive to low amounts of verapamil. These data suggest that over-expression of the P-glycoprotein homologue is incompatible with high levels of chloroquine resistance. In order to show that these results were applicable to other chloroquine selected lines, two additional mutants were selected for resistance to high levels of chloroquine. In both cases they were found to deamplify pfmdr1. Interestingly, while the level of chloroquine resistance of these mutants increased, they became more sensitive to mefloquine. This suggests a linkage between the copy number of the pfmdr1 gene and the level of chloroquine and mefloquine resistance.     

pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum

The emergence and spread of multidrug resistant Plasmodium falciparum has severely limited the therapeutic options for the treatment of malaria. With ever-increasing failure rates associated with chloroquine or sulphadoxine-pyrimethamine treatment, attention has turned to the few alternatives, which include quinine and mefloquine. Here, we have investigated the role of pfmdr1 3' coding region point mutations in antimalarial drug susceptibility by allelic exchange in the GC03 and 3BA6 parasite lines. Results with pfmdr1-recombinant clones indicate a significant role for the N1042D mutation in contributing to resistance to quinine and its diastereomer quinidine. The triple mutations S1034C/N1042D/D1246Y, highly prevalent in South America, were also found to enhance parasite susceptibility to mefloquine, halofantrine and artemisinin. pfmdr1 3' mutations showed minimal effect on P. falciparum resistance to chloroquine or its metabolite mono-desethylchloroquine in these parasite lines, in contrast to previously published results obtained with 7G8 parasites. This study supports the hypothesis that pfmdr1 3' point mutations can significantly affect parasite susceptibility to a wide range of antimalarials in a strain-specific manner that depends on the parasite genetic background.     

A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vacuole

Resistance to chloroquine in Plasmodium falciparum bears a striking similarity to the multi-drug resistance (MDR) phenotype of mammalian tumor cells which is mediated by overexpression of P-glycoprotein. We show here that the P. falciparum homologue of the P-glycoprotein (Pgh1) is a 160,000-D protein that is expressed throughout the asexual erythrocytic life cycle of the parasite. Quantitative immunoblotting analysis has shown that the protein is expressed at approximately equal levels in chloroquine resistant and sensitive isolates suggesting that overexpression of Pgh1 is not essential for chloroquine resistance. The chloroquine-resistant cloned line FAC8 however, does express approximately threefold more Pgh1 protein than other isolates which is most likely because of the increased pfmdr1 gene copy number present in this isolate. Immunofluorescence and immunoelectron microscopy has demonstrated that Pgh1 is localized on the membrane of the digestive vacuole of mature parasites. This subcellular localization suggests that Pgh1 may modulate intracellular chloroquine concentrations and has important implications for the normal physiological function of this protein.     

The P-glycoprotein homologues of Plasmodium falciparum: Are they involved in chloroquine resistance?

Chloroquine has been the mainstay of antimalarial chemotherapy but the rapid spread of resistance to this important drug has now compromised its efficacy. The mechanism of chloroquine resistance has not been known but recent evidence from Plasmodium falciparum, the causative agent of the most severe form of human malaria, suggested similarities to the multidrug resistance phenotype (MDR) of mammalian tumour cells which is mediated by a protein molecule termed P-glycoprotein. Two mdr genes (pfmdr1 and pfmdr2) encoding P-glycoprotein homologues have been identified in P. falciparum and one of these (pfmdr1) has several alleles that have been linked to the chloroquine resistance phenotype. In contrast analysis of a genetic cross between chloroquine-resistant and -sensitive P. falciparum has suggested that the genes encoding the known P-glycoprotein homologues are not linked. This review outlines the similarities of the chloroquine resistance phenotype with the MDR phenotype of mammalian tumour cells and explores the possible role of the pfmdr genes.     

Polymorphisms within PfMDR1 alter the substrate specificity for anti-malarial drugs in Plasmodium falciparum

Resistance to several anti-malarial drugs has been associated with polymorphisms within the P-glycoprotein homologue (Pgh-1, PfMDR1) of the human malaria parasite Plasmodium falciparum. Pgh-1, coded for by the gene pfmdr1, is predominately located at the membrane of the parasite's digestive vacuole. How polymorphisms within this transporter mediate alter anti-malarial drug responsiveness has remained obscure. Here we have functionally expressed pfmdr1 in Xenopus laevis oocytes. Our data demonstrate that Pgh-1 transports vinblastine, an established substrate of mammalian MDR1, and the anti-malarial drugs halofantrine, quinine and chloroquine. Importantly, polymorphisms within Pgh-1 alter the substrate specificity for the anti-malarial drugs. Wild-type Pgh-1 transports quinine and chloroquine, but not halofantrine, whereas polymorphic Pgh-1 variants, associated with altered drug responsivenesses, transport halofantrine but not quinine and chloroquine. Our data further suggest that quinine acts as an inhibitor of Pgh-1. Our data are discussed in terms of the model that Pgh-1-mediates, in a variant-specific manner, import of certain drugs into the P. falciparum digestive vacuole, and that this contributes to accumulation of, and susceptibility to, the drug in question.     

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Increased sensitivity to the antimalarials mefloquine and artemisinin is conferred by mutations in the pfmdr1 gene of Plasmodium falciparum

The declining efficacy of chloroquine and pyrimethamine/sulphadoxine in the treatment of human malaria has led to the use of newer antimalarials such as mefloquine and artemisinin. Sequence polymorphisms in the pfmdr1 gene, the gene encoding the plasmodial homologue of mammalian multidrug resistance transporters, have previously been linked to resistance to chloroquine in some, but not all, studies. In this study, we have used a genetic cross between the strains HB3 and 3D7 to study inheritance of sensitivity to the structurally unrelated drugs mefloquine and artemisinin, and to several other antimalarials. We find a complete allelic association between the HB3-like pfmdr1 allele and increased sensitivity to these drugs in the progeny. Different pfmdr1 sequence polymorphisms in other unrelated lines were also associated with increased sensitivity to these drugs. Our results indicate that the pfmdr1 gene is an important determinant of susceptibility to antimalarials, which has major implications for the future development of resistance.     

Evidence for a pfcrt-associated chloroquine efflux system in the human malarial parasite Plasmodium falciparum

Resistance to the antimalarial drug chloroquine has been linked with polymorphisms within a gene termed pfcrt in the human malarial parasite Plasmodium falciparum, yet the mechanism by which this gene confers the reduced drug accumulation phenotype associated with resistance is largely unknown. To investigate the role of pfcrt in mediating chloroquine resistance, we challenged P. falciparum clones differing only in their pfcrt allelic form with the "varying-trans" procedure. In this procedure, movement of labeled substrate across a membrane is measured when unlabeled substrate is present on the trans side of the membrane. If a transporter is mediating the substrate flow, a stimulation of cis-to-trans movement may be observed with increasing concentrations of trans substrate. We present evidence for an association of those pfcrt alleles found in chloroquine-resistant P. falciparum strains with the phenomenon of stimulated chloroquine accumulation under varying-trans conditions. Such an association is not seen with polymorphisms within pfmdr1, which encodes a homologue of the human multidrug resistance efflux pump. Our data are interpreted in terms of a model in which pfcrt is directly or indirectly involved in carrier-mediated chloroquine efflux from resistant cells.     

Modulation of halofantrine resistance after coadministration of halofantrine with diverse pharmacological agents in a rodent malaria model

The declining efficacy of antimalarial drugs against Plasmodium falciparum strains has been reported from several endemic regions of the world. Strategic evaluation of several pharmacological agents in combination with chloroquine to enhance the sensitivity of the latter against resistant parasites has been documented in several studies. However no attempts have been directed to monitor the efficacy of such biological response modifiers for reversing the resistance to halofantrine. In the present study the comparative efficacy of a total of 22 pharmacological agents representing diverse categories including antihistamines, antidepressants, calcium channel blockers, neuroleptics etc. has been determined in combination with halofantrine against halofantrine resistant Plasmodium yoelii nigeriensis. Results show significant potentiation of the efficacy of halofantrine when administered concurrently with histamine H1 receptor antagonists cyproheptadine and ketotifen. Combination with pheniramine, amitriptyline, verapamil and penfluridol also produced moderate degree of potentiation which was well marked during the early phase of progression of parasitaemia.     

The detection of pfcrt and pfmdr1 point mutations as molecular markers of chloroquine drug resistance, Pahang, Malaysia

ABSTRACT: BACKGROUND: Malaria is still a public health problem in Malaysia with chloroquine (CQ) being the first-line drug in the treatment policy of uncomplicated malaria. There is a scarcity in information about the magnitude of Plasmodium falciparum CQ resistance. This study aims to investigate the presence of single point mutations in the P. falciparum chloroquine-resistance transporter gene (pfcrt) at codons 76, 271, 326, 356 and 371 and in P. falciparum multi-drug resistance-1 gene (pfmdr1) at codons 86 and 1246, as molecular markers of CQ resistance. METHODS: A total of 75 P. falciparum blood samples were collected from different districts of Pahang state, Malaysia. Single nucleotide polymorphisms in pfcrt gene (codons 76, 271, 326, 356 and 371) and pfmdr1 gene (codons 86 and 1246) were analysed by using mutation-specific nested PCR and restriction fragment length polymorphism (PCR-RFLP) methods. RESULTS: Mutations of pfcrt K76T and pfcrt R371I were the most prevalent among pfcrt gene mutations reported by this study; 52% and 77%, respectively. Other codons of the pfcrt gene and the positions 86 and 1246 of the pfmdr1 gene were found mostly of wild type. Significant associations of pfcrt K76T, pfcrt N326S and pfcrt I356T mutations with parasitaemia were also reported. CONCLUSION: The high existence of mutant pfcrt T76 may indicate the low susceptibility of P. falciparum isolates to CQ in Peninsular Malaysia. The findings of this study establish baseline data on the molecular markers of P. falciparum CQ resistance, which may help in the surveillance of drug resistance in Peninsular Malaysia.     

Evidence for a central role for PfCRT in conferring Plasmodium falciparum resistance to diverse antimalarial agents

Chloroquine resistance in Plasmodium falciparum is primarily conferred by mutations in pfcrt. Parasites resistant to chloroquine can display hypersensitivity to other antimalarials; however, the patterns of crossresistance are complex, and the genetic basis has remained elusive. We show that stepwise selection for resistance to amantadine or halofantrine produced previously unknown pfcrt mutations (including S163R), which were associated with a loss of verapamil-reversible chloroquine resistance. This was accompanied by restoration of efficient chloroquine binding to hematin in these selected lines. This S163R mutation provides insight into a mechanism by which PfCRT could gate the transport of protonated chloroquine through the digestive vacuole membrane. Evidence for the presence of this mutation in a Southeast Asian isolate supports the argument for a broad role for PfCRT in determining levels of susceptibility to structurally diverse antimalarials.     

Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events.

The multidrug resistance (MDR) phenotype in mammalian tumor cells can involve amplification of mdr genes that results in overexpression of the protein product termed P-glycoprotein. Chloroquine resistance (CQR) in Plasmodium falciparum has similarities with the MDR phenotype in tumor cells, and some isolates of P. falciparum have amplified levels of the pfmdr1 gene. To investigate the nature and origin of pfmdr1 amplicons, we have cloned large regions of a 110-kb amplicon from the CQR cloned isolate B8 by using the yeast artificial chromosome system. We have identified and sequenced the breakpoints of the amplicon by a novel method employing inverted polymerase chain reaction that is applicable to analysis of any large-scale repeat. We show that the five copies of the amplicon in this isolate are in a head to tail configuration. A string of 30 A's flank the breakpoints on each side of the amplified segment, suggesting a mechanism for the origin of the tandem amplification. Polymerase chain reaction analysis with oligonucleotides that cross the B8 breakpoint has shown in 26 independent CQR isolates, 16 of which contain amplified copies of pfmdr1, that amplification of the pfmdr1 gene in P. falciparum has arisen as multiple independent events. These results suggest that this region of the genome is under strong selective pressure.     

Ex vivo susceptibility of Plasmodium falciparum to antimalarial drugs in Northern Uganda

In Uganda, artemether-lumefantrine was introduced as an artemisinin-based combination therapy (ACT) for malaria in 2006. We have previously reported a moderate decrease in ex vivo efficacy of lumefantrine in Northern Uganda, where we also detected ex vivo artemisinin-resistant Plasmodium falciparum. Therefore, it is necessary to search for candidate partner alternatives for ACT. Here, we investigated ex vivo susceptibility to four ACT partner drugs as well as quinine and chloroquine, in 321 cases between 2013 and 2018. Drug-resistant mutations in pfcrt and pfmdr1 were also determined. Ex vivo susceptibility to amodiaquine, quinine, and chloroquine was well preserved, whereas resistance to mefloquine was found in 45.8%. There were few cases of multi-drug resistance. Reduced sensitivity to mefloquine and lumefantrine was significantly associated with the pfcrt K76 wild-type allele, in contrast to the association between chloroquine resistance and the K76T allele. Pfmdr1 duplication was not detected in any of the cases. Amodiaquine, a widely used partner drug for ACT in African countries, may be the first promising alternative in case lumefantrine resistance emerges. Therapeutic use of mefloquine may not be recommended in this area. This study also emphasizes the need for sustained monitoring of antimalarial susceptibility in Northern Uganda to develop proper treatment strategies.     

Evidence for a substrate specific and inhibitable drug efflux system in chloroquine resistant Plasmodium falciparum strains

The mechanism underpinning chloroquine drug resistance in the human malarial parasite Plasmodium falciparum remains controversial. By investigating the kinetics of chloroquine accumulation under varying-trans conditions, we recently presented evidence for a saturable and energy-dependent chloroquine efflux system present in chloroquine resistant P. falciparum strains. Here, we further characterize the putative chloroquine efflux system by investigating its substrate specificity using a broad range of different antimalarial drugs. Our data show that preloading cells with amodiaquine, primaquine, quinacrine, quinine, and quinidine stimulates labeled chloroquine accumulation under varying-trans conditions, while mefloquine, halofantrine, artemisinin, and pyrimethamine do not induce this effect. In the reverse of the varying-trans procedure, we show that preloaded cold chloroquine can stimulate quinine accumulation. On the basis of these findings, we propose that the putative chloroquine efflux system is capable of transporting, in addition to chloroquine, structurally related quinoline and methoxyacridine antimalarial drugs. Verapamil and the calcium/calmodulin antagonist W7 abrogate stimulated chloroquine accumulation and energy-dependent chloroquine extrusion. Our data are consistent with a substrate specific and inhibitible drug efflux system being present in chloroquine resistant P. falciparum strains.     

基因逆转肿瘤多药耐药的研究进展

化疗在恶性肿瘤的综合治疗中占有非常重要的地位,而耐药性是严重影响肿瘤病人化疗效果及生存的主要原因之一,其中多药耐药(multi-drug resistance,MDR)最具临床意义。多药耐药是指肿瘤细胞对某一化疗药物产生耐药性后,对其他化学结构及机理不同的化疗药物也产生交叉耐药性。研究表明MDR是一个多阶段发展、多因素参与的复杂事件。逆转肿瘤多药耐药是目前肿瘤化疗的研究热点之一。近年随着基础科学研究的不断深入,基因逆转肿瘤多药耐药的研究已从分子水平上,定点、多位点阻断多药耐药基因的表达,已取得一些显著的进展。本文对肿瘤多药耐药机制以及逆转肿瘤多药耐药性的相关基因做一简要综述。     

The ATP-binding cassette (ABC) gene family of Plasmodium falciparum

Multidrug resistance (MDR) in mammalian tumour cells is mediated by P-glycoproteins. The apparent similarities between MDR and the chloroquine-resistance phenotype (CQR) in Plasmodium falciparum suggests that homologous proteins may be involved. In mammals, P-glycoproteins are encoded by mdr genes that are a subset of a super-family characterized by ATP-binding cassettes (ABC). Three genes, pfmdr1, pfmdr2 and pfef3-rl, have been identified in P. falciparum that have homology to the ABC transporter gene family. Each protein encoded by these genes has a distinct structure, suggesting functional differences between the three. Justin Rubio and Alan Cowman here discuss the structure and possible function of the ABC proteins from P. falciparum and evidence that the protein encoded by the pfmdr1 gene can influence quinoline-containing antimalarial drug-resistance phenotypes.     

Spread and evolution of Plasmodium falciparum drug resistance

Worldwide spread of Plasmodium falciparum drug resistance to conventional antimalarials, chloroquine and sulfadoxine/pyrimethamine, has been imposing a serious public health problem in many endemic regions. Recent discovery of drug resistance-associated genes, pfcrt, pfmdr1, dhfr, and dhps, and applications of microsatellite markers flanking the genes have revealed the evolution of parasite resistance to these antimalarials and the geographical spread of drug resistance. Here, we review our recent knowledge of the evolution and spread of parasite resistance to chloroquine and sulfadoxine/pyrimethamine. In both antimalarials, resistance appears to be largely explained by the invasion of limited resistant lineages to many endemic regions. However, multiple, indigenous evolutionary origins of resistant lineages have also been demonstrated. Further molecular evolutionary and population genetic approaches will greatly facilitate our understanding of the evolution and spread of parasite drug resistance, and will contribute to developing strategies for better control of malaria.     

Plasmodium falciparum resistant to chloroquine and to pyrimethamine in Comoros

We report the outcome of chloroquine treatment and the prevalence of mutations at codon 86 of the pfmdr1 gene, at codon 76 of the pfcrt gene, and at codon 108 of the pfdhfr gene in clinical isolates of Plasmodium falciparum collected from 30 children under 10 years of age living in the Comoros Union. This in vivo study was carried out in February and March 2001 in Moroni. Chloroquine treatment failed in 23 children (76.6%; 95% confidence interval: 57.7 to 90.1%). Subsequent genotyping showed that all P. falciparum isolates (100%) harboured a tyrosine residue at position 86 in pfMDR1. 83.3% (25/30) of these isolates harboured a mutation at position 76 in pfCRT and half (15/30) of these isolates also harboured a mutation at position 108 in pfDHFR. Chloroquine resistance is a real concern in the Comoros Union. The prevalence of pfDHFR mutant parasites is alarming. The alternative drugs proposed as a replacement for chloroquine as first-line treatment in Comoros, and the strategy to monitor the drug susceptibility of Plasmodium sp in this part of the Indian Ocean sub-region are discussed.