PSAC Inhibitor (Microbiotix)

Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted α-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel's composition and structure, and should guide the development of therapies targeting the PSAC.

The plasmodial surface anion channel (PSAC) increases erythrocyte permeability to many solutes in malaria but has uncertain physiological significance. We used a PSAC inhibitor with different efficacies against channels from two Plasmodium falciparum parasite lines and found concordant effects on transport and in vitro parasite growth when external nutrient concentrations were reduced. Linkage analysis using this growth inhibition phenotype in the Dd2 × HB3 genetic cross mapped the clag3 genomic locus, consistent with a role for two clag3 genes in PSAC-mediated transport. Altered inhibitor efficacy, achieved through allelic exchange or expression switching between the clag3 genes, indicated that the inhibitor kills parasites through direct action on PSAC. In a parasite unable to undergo expression switching, the inhibitor selected for ectopic homologous recombination between the clag3 genes to increase the diversity of available channel isoforms. Broad-spectrum inhibitors, which presumably interact with conserved sites on the channel, also exhibited improved efficacy with nutrient restriction. These findings indicate that PSAC functions in nutrient acquisition for intracellular parasites. Although key questions regarding the channel and its biological role remain, antimalarial drug development targeting PSAC should be pursued.