AAC Accepts, published online ahead of print on 28 April 2008

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Antimicrob. Agents Chemother. doi:10.1128/AAC.00057-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Changes in PSAC Reduce Leupeptin Uptake and Can Confer Drug Resistance in P. falciparum-Infected Erythrocytes

Godfrey Lisk, Margaret Pain, Ilya Y. Gluzman, Shivkumar Kambhampati, Tetsuya Furuya, Xin-zhuan Su, Michael P. Fay, Daniel E. Goldberg, and Sanjay A. Desai^*

The Laboratory of Malaria and Vector Research, the Biostatistics Research Branch, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892, and the Howard Hughes Medical Institute, Washington University, Departments of Molecular Microbiology and Medicine, St. Louis, Missouri

^* To whom correspondence should be addressed. Email: sdesai{at}niaid.nih.gov.

Cysteine protease inhibitors kill malaria parasites and are being pursued for antimalarial development. Because they have multiple targets within bloodstream stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. Here, 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. As measured with osmotic fragility, electrophysiology, and a new leupeptin uptake assay, the changes in PSAC activity included selective reductions in organic solute permeability, 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 uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance though selection of PSAC mutants.