Synergistic Malaria Parasite Killing by Two Types of Plasmodial Surface Anion Channel Inhibitors

<div><p>Malaria parasites increase their host erythrocyte’s permeability to a broad range of ions and organic solutes. The plasmodial surface anion channel (PSAC) mediates this uptake and is an established drug target. Development of therapies targeting this channel is limited by several problems including interactions between known inhibitors and permeating solutes that lead to incomplete channel block. Here, we designed and executed a high-throughput screen to identify a novel class of PSAC inhibitors that overcome this solute-inhibitor interaction. These new inhibitors differ from existing blockers and have distinct effects on channel-mediated transport, supporting a model of two separate routes for solute permeation though PSAC. Combinations of inhibitors specific for the two routes had strong synergistic action against <i>in vitro</i> parasite propagation, whereas combinations acting on a single route produced only additive effects. The magnitude of synergism depended on external nutrient concentrations, consistent with an essential role of the channel in parasite nutrient acquisition. The identified inhibitors will enable a better understanding of the channel’s structure-function and may be starting points for novel combination therapies that produce synergistic parasite killing.</p></div

Hits act against residual transport and differ from known PSAC inhibitors.

<p>(A) Osmotic lysis kinetics in PhTMA lysis solution without (black trace) or with 200 μM furosemide (green or red traces). Addition of 0.1, 0.3, or 1 μM PRT-1 (top to bottom red traces, respectively) inhibits residual lysis. (B) Dose responses for inhibition of residual permeability (<i>P</i>) of PhTMA⁺ or proline (red and blue circles, respectively; mean ± S.E.M. of up to 10 trials each). (C) Inhibitor concentrations that block uptake by 50% (<i>K</i>_{<i>0</i>.<i>5</i>}). Mean ± S.E.M. for inhibition of residual transport in PhTMA⁺ + 200 μM furosemide or primary transport in sorbitol are shown on a logarithmic scale as black and red bars, respectively. While ISG-21 and TP-52 have higher affinities against primary transport, PRT inhibitors have comparable or greater activity against residual transport. (D) Dose responses for PRT-1 inhibition of residual <i>P</i> in PhTMA⁺ plus either 100 nM ISG-21 or 2 μM TP-52 (blue and green triangles, respectively; mean ± S.E.M. of up to 3 trials each). Solid lines in panels (B) and (D) represent the best fits to the sum of two Langmuir isotherms [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149214#pone.0149214.ref011" target="_blank">11</a>].</p

Effects of external nutrient levels on inhibitor efficacy against parasite growth.

<p>(A, B) Dose responses for growth inhibition by ISG-21 and PRT-1 in standard medium and PGIM (black and red symbols, respectively). While ISG-21 has significantly improved activity in PGIM, the efficacy of PRT-1 is unchanged. Solid lines represent best fits to a logistic decay with a Hill coefficient. (C) Ratio of <i>IC</i>_<i>50</i> values for parasite killing in standard RPMI 1640-based medium to PGIM for indicated inhibitors. Bars represent mean ± S.E.M. of replicates from up to 7 independent trials.</p

<i>In vitro</i> interactions between primary and residual PSAC inhibitors.

<p>Combination growth inhibition experiments, tallied as mean ± S.E.M. sum of FIC values for 50% growth inhibition (∑FIC₅₀). Combinations are grouped under headings to indicate experiments performed with a primary and a residual inhibitor, two primary inhibitors, or two residual inhibitors.</p