Plasmodium Falciparum Multidrug Resistance Protein Research Articles

The spectre of chloroquine—evolutionary conflict at the molecular level in a Plasmodium falciparum multidrug resistance protein

The spectre of chloroquine—evolutionary conflict at the molecular level in a Plasmodium falciparum multidrug resistance protein

The structure of Plasmodium falciparum multidrug resistance protein 1 reveals an N-terminal regulatory domain

Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), an adenosine triphosphate (ATP)-binding cassette (ABC) transporter on the digestive vacuole (DV) membrane of the parasite, is associated with the resistance to antimalarial drugs. To understand the mechanisms of PfMDR1, we determined the cryo-electron microscopy structures of this transporter in different states. The transporter in the apo state shows an inward-facing conformation with a large cavity opening to the cytoplasm. Upon ATP binding and dimerization of the nucleotide-binding domains (NBDs), PfMDR1 displays an outward-facing conformation with a cavity toward the DV lumen. Drug resistance-associated mutations were investigated in both structures for their effects, and Y184F was identified as an allosteric activity-enhancing mutation. The amphiphilic substrate-binding site of PfMDR1 was revealed by the complex structure with the antimalarial drug mefloquine and confirmed by mutagenesis studies. Remarkably, a helical structure was found to hinder NBD dimerization and inhibit PfMDR1 activity. The location of this regulatory domain in the N terminus is different from the well-studied R domain in the internal linker region of other ABC transporter family members. The lack of the phosphorylation site of this domain also suggests a different regulation mechanism.

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.

Drug-resistant gene polymorphisms in Plasmodium falciparum isolated from Bioko Island, Equatorial Guinea in 2018 and 2019

To investigate the genetic polymorphisms of Plasmodium falciparum multidrug resistance protein 1 (PfMDR1), chloroquine resistance transporter (PfCRT) and Kelch 13 (PfK13) genes in Bioko Island, Equatorial Guinea, so as to provide insights into the development of the malaria control strategy in local areas. A total of 85 peripheral blood samples were collected from patients with Plasmodium falciparum infections in Bioko Island, Equatorial Guinea in 2018 and 2019, and genomic DNA was extracted. The PfMDR1, PfCRT and PfK13 genes were amplified using a nested PCR assay. The amplification products were sequenced, and the gene sequences were aligned. There were no mutations associated with artemisinin resistance in PfK13 gene in Bioko Island, Equatorial Guinea, while drug-resistant mutations were detected in PfMDR1 and PfCRT genes, and the proportions of PfMDR1_N86Y, PfMDR1_Y184F and PfCRT_K76T mutations were 35.29% (30/85), 72.94% (62/85) and 24.71% (21/85), respectively. There are mutations in PfMDR1, PfCRT and PfK13 genes in P. falciparum isolates from Bioko Island, Equatorial Guinea.

Expansion of a Specific Plasmodium falciparum PfMDR1 Haplotype in Southeast Asia with Increased Substrate Transport.

Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia.IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.

Implications des pompes membranaires de Plasmodium falciparum dans le transport et la résistance aux antipaludiques

The malaria parasite Plasmodium falciparum has become resistant to most antimalarial drugs and even to the latest marketed ones, like artemisinin-based combinaison therapies (ACT) in Southeast Asia. A possible reason for resistance to antimalarial drugs could be that drugs do not reach their target sites, due to active extrusion out of parasite. More than 100 transporters would be localized at the P. falciparum plasmic membrane and digestive vacuole membrane. Some of these transporters, belonging primarily to the superfamily of DMT (drug/metabolite transporters) like PfCRT (Plasmodium falciparum chloroquine resistance transporter) and to the superfamily of ABC transporters (ATP-binding cassette) like PfMDR1 (Plasmodium falciparum multidrug resistance protein 1), PfMDR2, PfMDR5 and PfMDR6, and PfMRP1 (Plasmodium falciparum multidrug resistance-associated protein 1) and PfMRP2, are involved in antimalarial drug transport and drug resistance. Gene amplification or single nucleotide mutations in these genes can modulate P. falciparum susceptibility level to antimalarial drugs. The use of inhibitors against these transporters is a promising novel therapeutic drug strategy.

Assessing the Antimalarial Potentials of Phytochemicals: Virtual Screening, Molecular Dynamics and In-Vitro Investigations

Background: Combined in-silico and in-vitro approaches were adopted to investigate the antiplasmodial activity of Catharanthus roseus and Tylophora indica plant extracts as well as their isolated components (vinblastine, vincristine and tylophorine). </P><P> Methods: We employed molecular docking to prioritize phytochemicals from a library of 26 compounds against Plasmodium falciparum multidrug-resistance protein 1 (PfMDR1). Furthermore, Molecular Dynamics (MD) simulations were performed for a duration of 10 ns to estimate the dynamical structural integrity of ligand-receptor complexes. </P><P> Results: The retrieved bioactive compounds viz. tylophorine, vinblastin and vincristine were found to exhibit significant interacting behaviour; as validated by in-vitro studies on chloroquine sensitive (3D7) as well as chloroquine resistant (RKL9) strain. Moreover, they also displayed stable trajectory (RMSD, RMSF) and molecular properties with consistent interaction profile in molecular dynamics simulations. </P><P> Conclusion: We anticipate that the retrieved phytochemicals can serve as the potential hits and presented findings would be helpful for the designing of malarial therapeutics.

Chloroquine diphosphate bearing dextran nanoparticles augmented drug delivery and overwhelmed drug resistance in Plasmodium falciparum parasites

Chloroquine diphosphate (CHQ) is primarily used for the treatment of Plasmodium falciparum malaria at the dose of 500mg orally or 10mg/kg parenterally. However, point mutations in Plasmodiumfalciparum chloroquine resistance transporter (PfCRT) protein and Plasmodium falciparum multidrug resistance protein 1 (Pfmdr1) localized in digestive vacuole membrane, are responsible for CHQ resistance. Therefore, in present investigation, dextran nanoparticles bearing chloroquine diphosphate (CHQ-DEX-NPs) were formulated by solvent diffusion method of size below 70nm with zeta-potential of −20.1±3.2mV. FT-IR, DSC and PXRD techniques confirmed the successful loading of drug in nanomatrix system with amorphous attributes. In vitro drug release analysis indicated the Higuchi pattern with diffusion controlled drug release. The IC50 of CHQ-DEX-NPs in sensitive (3D7) and resistant (RKL9) Plasmodium falciparum strains was estimated to be 0.031-μg/ml and 0.13-μg/ml significantly lower than 0.059-μg/ml and 0.36-μg/ml of CHQ. The augmented therapeutic efficacy of CHQ-DEX-NPs may be credited to deposition of tailored nanoparticles in food vacuoles of malaria parasites owing to the affinity of parasite towards DEX that consequently lower the drug resistance and improved the therapeutic index. In conclusion, CHQ-DEX-NPs must be evaluated under a set of stringent in vivo parameters to establish its therapeutic efficacy in preclinical model.

Selection of N86F184D1246 haplotype of Pfmrd1 gene by artemether-lumefantrine drug pressure on Plasmodium falciparum populations in Senegal.

Background The use of artemisinin as a monotherapy resulted in the emergence of artemisinin resistance in 2005 in Southeast Asia. Monitoring of artemisinin combination therapy (ACT) is critical in order to detect and prevent the spread of resistance in endemic areas. Ex vivo studies and genotyping of molecular markers of resistance can be used as part of this routine monitoring strategy. One gene that has been associated in some ACT partner drug resistance is the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene. The purpose of this study was to assess the drug susceptibility of P. falciparum populations from Thiès, Senegal by ex vivo assay and typing molecular markers of resistance to drug components of ACT currently used for treatment.Methods The ex vivo susceptibility of 170 P. falciparum isolates to chloroquine, amodiaquine, lumefantrine, artesunate, and artemether was determined using the DAPI ex vivo assay. The high resolution melting technique was used to genotype the pfmdr1 gene at codons 86, 184 and 1246.ResultsA significant decrease in IC50 values was observed between 2012 and 2013: from 13.84 to 6.484 for amodiaquine, 173.4 to 113.2 for lumefantrine, and 39.72 to 18.29 for chloroquine, respectively. Increase of the wild haplotype NYD and the decrease of the mutant haplotype NFD (79 and 62.26 %) was also observed. A correlation was observed between the wild type allele Y184 in pfmdr1 and higher IC50 for all drugs, except amodiaquine.ConclusionThis study has shown an increase in sensitivity over the span of two transmission seasons, marked by an increase in the WT alleles at pfmdr1. Continuous the monitoring of the ACT used for treatment of uncomplicated malaria will be helpful.

Complex polymorphisms in the Plasmodium falciparum multidrug resistance protein 2 gene and its contribution to antimalarial response.

Plasmodium falciparum has the capacity to escape the actions of essentially all antimalarial drugs. ATP-binding cassette (ABC) transporter proteins are known to cause multidrug resistance in a large range of organisms, including the Apicomplexa parasites. P. falciparum genome analysis has revealed two genes coding for the multidrug resistance protein (MRP) type of ABC transporters: Pfmrp1, previously associated with decreased parasite drug susceptibility, and the poorly studied Pfmrp2. The role of Pfmrp2 polymorphisms in modulating sensitivity to antimalarial drugs has not been established. We herein report a comprehensive account of the Pfmrp2 genetic variability in 46 isolates from Thailand. A notably high frequency of 2.8 single nucleotide polymorphisms (SNPs)/kb was identified for this gene, including some novel SNPs. Additionally, we found that Pfmrp2 harbors a significant number of microindels, some previously not reported. We also investigated the potential association of the identified Pfmrp2 polymorphisms with altered in vitro susceptibility to several antimalarials used in artemisinin-based combination therapy and with parasite clearance time. Association analysis suggested Pfmrp2 polymorphisms modulate the parasite's in vitro response to quinoline antimalarials, including chloroquine, piperaquine, and mefloquine, and association with in vivo parasite clearance. In conclusion, our study reveals that the Pfmrp2 gene is the most diverse ABC transporter known in P. falciparum with a potential role in antimalarial drug resistance.

Surveillance of artemether-lumefantrine associated Plasmodium falciparum multidrug resistance protein-1 gene polymorphisms in Tanzania.

BackgroundResistance to anti-malarials is a major public health problem worldwide. After deployment of artemisinin-based combination therapy (ACT) there have been reports of reduced sensitivity to ACT by malaria parasites in South-East Asia. In Tanzania, artemether-lumefantrine (ALu) is the recommended first-line drug in treatment of uncomplicated malaria. This study surveyed the distribution of the Plasmodium falciparum multidrug resistance protein-1 single nucleotide polymorphisms (SNPs) associated with increased parasite tolerance to ALu, in Tanzania.MethodsA total of 687 Plasmodium falciparum positive dried blood spots on filter paper and rapid diagnostic test strips collected by finger pricks from patients attending health facilities in six regions of Tanzania mainland between June 2010 and August 2011 were used. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to detect Pfmdr1 SNPs N86Y, Y184F and D1246Y.ResultsThere were variations in the distribution of Pfmdr1 polymorphisms among regions. Tanga region had exceptionally high prevalence of mutant alleles, while Mbeya had the highest prevalence of wild type alleles. The haplotype YFY was exclusively most prevalent in Tanga (29.6%) whereas the NYD haplotype was the most prevalent in all other regions. Excluding Tanga and Mbeya, four, most common Pfmdr1 haplotypes did not vary between the remaining four regions (χ2 = 2.3, p = 0.512). The NFD haplotype was the second most prevalent haplotype in all regions, ranging from 17% - 26%.ConclusionThis is the first country-wide survey on Pfmdr1 mutations associated with ACT resistance. Distribution of individual Pfmdr1 mutations at codons 86, 184 and 1246 varies throughout Tanzanian regions. There is a general homogeneity in distribution of common Pfmdr1 haplotypes reflecting strict implementation of ALu policy in Tanzania with overall prevalence of NFD haplotype ranging from 17 to 26% among other haplotypes. With continuation of ALu as first-line drug this haplotype is expected to keep rising, thus there is need for continued pharmacovigilance studies to monitor any delayed parasite clearance by the drug.

A 2-amino quinoline, 5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethylcarbamyl-4,6-dithiaoctanoic acid, interacts with PfMDR1 and inhibits its drug transport in Plasmodium falciparum

Malaria is a major disease in the tropics where chemotherapy remains the main mode of treatment and as such the rise and spread of drug-resistant malaria can lead to human tragedy. Two membrane transport proteins, PfMDR1 (Plasmodium falciparum multidrug resistance protein 1) and PfCRT (P. falciparum chloroquine resistance transporter), have been shown to cause resistance to several antimalarials. Both PfMDR1 and PfCRT are localized to the digestive vacuolar membrane and appear to regulate the transport of drugs and physiological metabolites. In this study we have used MK571, a 2-amino quinoline, to explore its interaction with PfMDR1 and PfCRT in chloroquine-sensitive and -resistant strains of P. falciparum. Our results show that chloroquine-resistant strains (e.g., K1, Dd2, and 7G8) are consistently more sensitive to MK571 than chloroquine-sensitive strains (e.g., 3D7, 106/1 and D10). This association, however, was not maintained with the chloroquine-resistant strain FCB which IC50 value was similar to chloroquine-sensitive strains. Moreover, the susceptibility of chloroquine-sensitive and -resistant strains to MK571 does not correlate with mutated PfCRT, nor is it reversible with verapamil; but correlates with mutations in PfMDR1. Furthermore, MK571 appears to target the parasite's digestive vacuole (DV), as demonstrated by the ability of MK571 to: (1) block the accumulation of the fluorescent dye Fluo-4 AM, a PfMDR1 substrate, into the digestive vacuole; (2) reduce the transvacuolar pH gradient; and (3) inhibit the formation of β-hematin in vitro. Moreover, the presence of non-toxic concentrations of MK571 sensitized both chloroquine-sensitive and -resistant parasites to mefloquine and halofantrine, likely by competing against PfMDR1-mediated sequestering of the drugs into the DV compartment and away from the drugs’ cytosolic targets. Our data, nevertheless, found only a minimal decrease in MK571 IC50 value in FCB parasite which second pfmdr1 copy was inactivated via gene disruption. Taken together, the findings of this study suggest that MK571 interacts with native and mutant PfMDR1 and modulates the import of drugs or solutes into the parasite's DV and, as such, MK571 may be a useful tool in the characterization of PfMDR1 drug interactions and substrate specificity.

Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar.

Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) has recently emerged as an important determinant of drug resistance and mutations in the gene have been associated with several drugs. The aim of this study was to understand the level of genetic diversity in pfmrp1 and to determine the association of different mutations with altered drug susceptibilities of P. falciparum. We analysed 193 sequences of pfmrp1 from South-East Asia, west Asia, Africa, Oceania and South America. We measured the level of genetic diversity and determined signatures of selection on the gene. In vitro susceptibilities of 28 P. falciparum isolates from north-east Myanmar to a panel of seven commonly used antimalarials were determined. Statistical analysis was performed to determine the association of different mutations with in vitro drug susceptibilities. A total of 28 single nucleotide polymorphisms were identified in 193 sequences, of which 22 were non-synonymous. Whereas mutations in the pfmrp1 gene were conserved among different countries within a continent, they were different between continents. Seven non-synonymous mutations were identified in the north-east Myanmar isolates; all were relatively frequent in this region as well as in other neighbouring countries. Molecular evolutionary analysis detected signatures of positive selection on the gene. Moreover, some mutations in this gene were found to be associated with reduced susceptibilities to chloroquine, mefloquine, pyronaridine and lumefantrine. Evidence of the positive selection of pmfrp1 and its association with the susceptibilities of parasites to multiple drugs signifies its potential as an important candidate for monitoring drug resistance.

UV-triggered Affinity Capture Identifies Interactions between the Plasmodium falciparum Multidrug Resistance Protein 1 (PfMDR1) and Antimalarial Agents in Live Parasitized Cells

A representative of a new class of potent antimalarials with an unknown mode of action was recently described. To identify the molecular target of this class of antimalarials, we employed a photo-reactive affinity capture method to find parasite proteins specifically interacting with the capture compound in living parasitized cells. The capture reagent retained the antimalarial properties of the parent molecule (ACT-213615) and accumulated within parasites. We identified several proteins interacting with the capture compound and established a functional interaction between ACT-213615 and PfMDR1. We surmise that PfMDR1 may play a role in the antimalarial activity of the piperazine-containing compound ACT-213615.

Lack of multiple copies of pfmdr1 gene in Papua New Guinea

We describe here the results of an analysis of Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene copy number from 440 field isolates from Papua New Guinea. No multiple copies of the gene were found, which corresponds to the lack of usage of mefloquine. These data extend regional knowledge about the distribution of multidrug-resistant P. falciparum.

Inhibition of Efflux of Quinolines as New Therapeutic Strategy in Malaria

Plasmodium falciparum is one of the most lethal parasite responsible for human malaria. Until now, the only one solution to counter malaria is the use of antimalarial drugs. Unfortunately, the extensively use of drugs, such as quinolines (i.e. chloroquine, quinine or mefloquine), have led to the emergence of drug resistance. Chloroquine and probably other quinolines act in interfering in the detoxification of hematin in the digestive vacuole. Quinolines are accumulated in P. falciparum digestive vacuole and the accumulation varies from a susceptible strain to a resistant one. Nevertheless, the mechanisms of quinoline resistance are still investigating. Genetic polymorphisms in some strains have been linked to drug resistance. The modifications observed are mutations on genes that encode transport proteins localized in the membrane of digestive vacuole. Three transporters were involved in quinoline resistance: PfCRT (Plasmodium falciparum chloroquine resistance transporter), Pgh1 (P-glycoprotein homologue 1) and PfMRP (Plasmodium falciparum multidrug resistance protein). They could be involved in accumulation or efflux mechanisms of drugs. In order to understand their role in resistance, localization, encoding gene structure, protein structure and endogenous function of these three transporters are reported. Some molecules that have no intrinsic antimalarial effect have been shown to reverse drug resistance when they are combined to chloroquine, quinine or mefloquine. These molecules are a solution to counter resistance but also they are precious tools to elucidate the resistance mechanisms. The molecules that have already shown a capacity to reverse chloroquine, quinine or mefloquine resistances were reported. Some of them could act on one of the three transporters involved in drug resistance, by confirming their role in quinoline resistance. Here we summarize the main elements of quinoline resistance and reversion of quinoline resistance related to malaria.

Contribution of the pfmdr1 gene to antimalarial drug-resistance

The emergence of drug-resistance poses a major obstacle to the control of malaria. A homolog of the major multidrug-transporter in mammalian cells was identified, Plasmodium falciparum multidrug resistance protein-1, pfmdr1, also known as the P-glycoprotein homolog 1, Pgh-1. Several studies have demonstrated strong, although incomplete, associations between resistance to the widely used antimalarial drug chloroquine and mutation of the pfmdr1 gene in both laboratory and field isolates. Genetic studies have confirmed a link between mutation of the pfmdr1 gene and chloroquine-resistance. Although not essential for chloroquine-resistance, pfmdr1 plays a role in modulating levels of resistance. At the same time it appears to be a significant component in resistance to the structurally related drug quinine. A strong association has been observed between possession of the wildtype form of pfmdr1, amplification of pfmdr1 and resistance to hydrophobic drugs such as the arylaminoalcohol mefloquine and the endoperoxide artemisinin derivatives in field isolates. This is supported by genetic studies. The arylaminoalcohol and endoperoxide drugs are structurally unrelated drugs and this resistance resembles true multidrug resistance. Polymorphism in pfmdr1 and gene amplification has been observed throughout the world and their usefulness in predicting resistance levels is influenced by the history of drug selection of each population.