Chloroquine transport in Plasmodium falciparum. 1. Influx and efflux kinetics for live trophozoite parasites using a novel fluorescent chloroquine probe.
Several models for how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Distinguishing between these models requires detailed analysis of high-resolution CQ transport data that is unfortunately impossible to obtain with traditional radio-tracer methods. Thus, we have designed and synthesized fluorescent CQ analogues for drug transport studies. One probe places a NBD (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid) group at the tertiary aliphatic N of CQ, via a flexible 6 C amide linker. This probe localizes to the malarial parasite digestive vacuole (DV) during initial perfusion under physiologic conditions and exhibits similar pharmacology relative to CQ, vs both CQ-sensitive (CQS) and CQ-resistant (CQR) parasites. Using live, synchronized intraerythrocytic parasites under continuous perfusion, we define NBD-CQ influx and efflux kinetics for CQS vs CQR parasites. Since this fluorescence approach provides data at much higher kinetic resolution relative to fast-filtration methods using (3)H-CQ, rate constants vs linear initial rates for CQ probe flux can be analyzed in detail. Importantly, we find that CQR parasites have a decreased rate constant for CQ influx into the DV and that this is due to mutation of PfCRT. Analysis of zero trans efflux for CQS and CQR parasites suggests that distinguishing between bound vs free pools of intra-DV drug probe is essential for proper kinetic analysis of efflux. The accompanying paper (DOI 10.1021/bi901035j ) further probes efflux kinetics for proteoliposomes containing purified, reconstituted PfCRT.
Cabrera
Mynthia
M
Department of Chemistry, Georgetown University, NW, Washington, DC 20057, USA.
Natarajan
Jayakumar
J
Paguio
Michelle F
MF
Wolf
Christian
C
Urbach
Jeffrey S
JS
Roepe
Paul D
PD
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D000962:Antimalarials / Q000378:metabolism* / Q000494:pharmacology; D001693:Biological Transport, Active / Q000187:drug effects; D002738:Chloroquine / Q000031:analogs & derivatives* / Q000378:metabolism* / Q000494:pharmacology; D004351:Drug Resistance; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016778:Malaria, Falciparum / Q000378:metabolism / Q000469:parasitology / Q000517:prevention & control; D010963:Plasmodium falciparum / Q000187:drug effects / Q000254:growth & development* / Q000378:metabolism*
D000962:Antimalarials; D005456:Fluorescent Dyes; D002738:Chloroquine; D009327:4-Chloro-7-nitrobenzofurazan