title: Changes in the plasmodial surface anion channel reduce leupeptin uptake and can confer drug resistance in Plasmodium falciparum-infected erythrocytes.
abstract: Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.
authors.affiliation: Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892, USA.
authors.forename: Godfrey
authors.initials: G
authors.lastname: Lisk
authors.forename: Margaret
authors.initials: M
authors.lastname: Pain
authors.forename: Ilya Y
authors.initials: IY
authors.lastname: Gluzman
authors.forename: Shivkumar
authors.initials: S
authors.lastname: Kambhampati
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Furuya
authors.forename: Xin-Zhuan
authors.initials: XZ
authors.lastname: Su
authors.forename: Michael P
authors.initials: MP
authors.lastname: Fay
authors.forename: Daniel E
authors.initials: DE
authors.lastname: Goldberg
authors.forename: Sanjay A
authors.initials: SA
authors.lastname: Desai
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000493:pharmacokinetics; D001693:Biological Transport, Active; D002463:Cell Membrane Permeability; D015853:Cysteine Proteinase Inhibitors / Q000493:pharmacokinetics; D004351:Drug Resistance / Q000502:physiology*; D004912:Erythrocytes / Q000469:parasitology*; D017125:Genes, Protozoan; D006801:Humans; D066298:In Vitro Techniques; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D007976:Leupeptins / Q000493:pharmacokinetics*; D016778:Malaria, Falciparum / Q000188:drug therapy; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D015853:Cysteine Proteinase Inhibitors; D007473:Ion Channels; D007976:Leupeptins; D015800:Protozoan Proteins; C505335:surface anion channel protein, Plasmodium falciparum; C032854:leupeptin