PfMDR1 Transport Rates Assessed in Intact Isolated Plasmodium falciparum Digestive Vacuoles Reflect Functional Drug Resistance Relationship with pfmdr1 Mutations

Abstract

Drug resistance often emerges from mutations in solute transporters. Single amino acid exchanges may alter functionality of transporters with ‘de novo’ ability to transport drugs away from their site of action. The PfMDR1 transporter (or P-glycoprotein 1) is located in the membrane of the digestive vacuole (DV), functions as an ATP-dependent pump, and transports substrates into the DV. In this study, four strains of Plasmodium falciparum, carrying various pfmdr1 gene mutations, were analysed for their transport characteristics of Fluo-4 in isolated DVs of parasites. To obtain quantitative estimates for PfMDR1 DV surface expression, PfMDR1 protein amounts on each strain’s DV membrane were evaluated by quantitative ELISA. Fluo-4, acting as a substrate for PfMDR1, was applied in DV uptake assays (‘reverse Ca2+ imaging’). Viable DVs were isolated from trophozoite stages with preserved PfMDR1 activity. This newly developed assay enabled us to measure the number of Fluo-4 molecules actively transported into isolated DVs per PfMDR1 molecule. The drug-resistant strain Dd2 presented the highest transport rates, followed by K1 and the drug-sensitive strain 3D7, compatible with their copy numbers. With this assay, an evaluation of the probability of resistance formation for newly developed drugs can be implemented in early stages of drug development.

Access to Artemisinin–Triazole Antimalarials via Organo-Click Reaction: High In Vitro/In Vivo Activity against Multi-Drug-Resistant Malaria Parasites

“…The compounds 1 – 18 were tested in vitro against Plasmodium falciparum, and the IC 50 values against parasite growth (SYBR green assay) are given in Table . To gain a broader overview of antimalarial activity, three strains with different resistance profiles were tested: the chloroquine-sensitive 3D7 strain and the two multi-drug-resistant strains Dd2 and K1. , All new 13 artemisinin–triazole hybrids exhibit high antimalarial activity with IC 50 values down to 2.3 nM (for 7 / 12 ) against 3D7 as well as down to 2.1 nM (for 4 ) and 1.8 nM (for 2 ) for the multi-drug-resistant Dd2 and K1 strains, mostly outperforming the activity of established drugs artemisinin and chloroquine. All five previously reported antiviral benzimidazole-containing artemisinin–triazole hybrids 14 – 18 were also active against P.…”

Antiplasmodial Cyclodecapeptides from Tyrothricin Share a Target with Chloroquine

“…The role of Pf MDR1 in CQ resistance has been shown to involve diminished uptake of the drug into food vacuole and active transport out due to mutations in this transporter [ 64 , 75 ]. There is evidence that Pf MDR1 functions in transport of small peptides which arise from incomplete catabolism of hemoglobin from the food vacuole to the cytoplasm [ 76 , 77 ]. The presence of an analogous MDR1 in mammalian cells led to cross-resistance to a synthetic tripeptide (N-acetyl-leucyl-leucyl-norleucinal) [ 78 ], gramicidin D (15 residue linear peptide from tyrothricin) [ 79 , 80 ] and valinomycin (a cyclic dodecadepsipeptide) [ 79 ].…”

Critical interdependencies between Plasmodium nutrient flux and drugs

“…While not the focus of this opinion article, it is worth noting that perturbing the flux of drugs internally within the parasite can mediate resistance. The best-known example is the reduction in chloroquine present in the DV of resistant parasites which is underpinned by altered activity of Pf CRT, Pf AAT1, and P. falciparum multidrug-resistance protein 1 [ 46 , 48 , 69 , 70 ].…”