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- PfMDR1: mechanisms of transport modulation by functional polymorphismsPublication . Ferreira, Pedro; Holmgren, Gabrielle; Veiga, Maria Isabel; Uhlen, Per; Kaneko, Akira; Gil, José PedroATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.
- Assessing the cost-benefit effect of a plasmodium falciparum drug resistance mutation on parasite growth In vitroPublication . Froberg, Gabrielle; PE, Ferreira; Martensson, Andreas; Ali, Abdullah; Bjorkman, Anders; Gil, J. P.Plasmodium falciparum mutations associated with antimalarial resistance may be beneficial for parasites under drug pressure, although they may also cause a fitness cost. We herein present an in vitro model showing how this combined effect on parasite growth varies with the drug concentration and suggest a calculated drug-specific cost-benefit index, indicating the possible advantage for mutated parasites. We specifically studied the D-to-Y change at position 1246 encoded by the pfmdr1 gene (pfmdr1 D1246Y) in relation to amodiaquine resistance. Susceptibilities to amodiaquine, desethylamodiaquine, and chloroquine, as well as relative fitness, were determined for two modified isogenic P. falciparum clones differing only in the pfmdr1 1246 position. Data were used to create a new comparative graph of relative growth in relation to the drug concentration and to calculate the ratio between the benefit of resistance and the fitness cost. Results were related to an in vivo allele selection analysis after amodiaquine or artesunate-amodiaquine treatment. pfmdr1 1246Y was associated with decreased susceptibility to amodiaquine and desethylamodiaquine but at a growth fitness cost of 11%. Mutated parasites grew less in low drug concentrations due to a predominating fitness cost, but beyond a breakpoint concentration they grew more due to a predominating benefit of increased resistance. The cost-benefit indexes indicated that pfmdr1 1246Y was most advantageous for amodiaquine-exposed parasites. In vivo, a first drug selection of mutant parasites followed by a fitness selection of wild-type parasites supported the in vitro data. This cost-benefit model may predict the risk for selection of drug resistance mutations in different malaria transmission settings.
- Plasmodium falciparum multidrug resistance protein 1 and artemisinin-based combination therapy in AfricaPublication . Dahlstrom, Sabina; Ferreira, Pedro; Veiga, Maria Isabel; Sedighi, Nazli; Wiklund, Lisa; Martensson, Andreas; Farnert, Anna; Sisowath, Christin; Osorio, Lyda; Darban, Hamid; Andersson, Bjoern; Kaneko, Akira; Conseil, Gwanaelle; Bjorkman, Anders; Gil, José PedroPlasmodium falciparum response mechanisms to the major artemisinin-based combination therapies (ACTs) are largely unknown. Multidrug-resistance protein (MRP)-like adenosine triphosphate (ATP)-binding cassette transporters are known to be related to multidrug resistance in many organisms. Therefore, we hypothesized that sequence variation in pfmrp1 can contribute to decreased parasite sensitivity to ACT. Through sequencing of the pfmrp1 open reading frame for 103 geographically diverse P. falciparum infections, we identified 27 single-nucleotide polymorphisms (SNPs), of which 21 were nonsynonymous and 6 synonymous. Analyses of clinical efficacy trials with artesunate-amodiaquine and artemether-lumefantrine detected a specific selection of the globally prevalent I876V SNP in recurrent infections after artemether-lumefantrine treatment. Additional in silico studies suggested an influence of variation in amino acid 876 on the ATP hydrolysis cycle of pfMRP1 with potential impact on protein functionality. Our data suggest for the first time, to our knowledge, the involvement of pfMRP1 in P. falciparum in vivo response to ACT.