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

Synergistic killing by combinations of primary and residual inhibitors, but not by combinations from one class only.

<p>(A) Isobologram showing effect of fixed ratio combinations of the primary component inhibitor cpd <b>1</b> and the residual transport inhibitor PRT1-20. Each symbol represented the <i>IC</i>_<i>50</i> for a fixed ratio of the two inhibitors, determined from a full dose response experiment with replicates. Error bars, shown for single compound <i>IC</i>_<i>50</i> values (symbols on the <i>x</i> and <i>y</i> axes), represent S.E.M. values. Solid line connecting these intercept values is the expected profile for additive parasite killing. Strong synergistic killing was found for this drug combination as the symbols are markedly below the additive line. (B) Isobologram for two primary component inhibitors, showing additive interaction. (C) Isobologram for two residual component inhibitors, showing additive interaction.</p

A PRT-1 derivative with improved potency and specificity.

<p>(A) Structure of PRT1-20. A longer alkoxy side chain distinguishes this compound from PRT-1 (red highlight). (B) Mean ± S.E.M. inhibitor <i>K</i>_{<i>0</i>.<i>5</i>} values in PhTMA⁺ + 200 μM furosemide (red bars) and sorbitol (black). When compared to PRT-1, PRT1-20 has improved greater potency against residual transport and reduced activity against the primary mechanism.</p

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

Effect of external nutrient levels on synergistic killing.

<p>(A) Isobologram for ISG-21 and PRT-1 interactions in PGIM. Note that synergistic action is lost in this medium. (B) Isobologram for an identical growth experiments after increasing the culture medium isoleucine concentration to 34 μM, showing restored synergy. The ISG-21 <i>IC</i>_<i>50</i> values in (A) and (B)—17.3 ± 1 nM and 1.25 ± 0.1 μM, <i>x</i> axis intercepts—differ markedly because extracellular nutrient levels affect killing by primary component blockers.</p

A cell-based high-throughput screen identifies novel residual transport inhibitors.

<p>(A) Kinetics of osmotic lysis in indicated solutes with 0, 200, or 2000 μM furosemide (black, red, and green traces where present in each panel, respectively). Notice that 200 μM furosemide abolishes lysis due to sorbitol or alanine uptake, but yields residual uptake of PhTMA⁺ and proline (indicated with arrow and “R”). (B) All points histogram of compound activities against osmotic lysis in PhTMA⁺ + 200 μM furosemide, as measured at 3 h and normalized to 0% block in DMSO-only wells and to 100% block in positive controls (HBS+1600 μM furosemide). Ordinate shows the number of compounds at each block level on a logarithmic scale, revealing that most compounds fell within 3*SD of negative control wells (grey bars). A small number of hits inhibited or accentuated lysis (red and blue bars, respectively). Two of the three compounds producing ≥ 50% block were unavailable for downstream experiments; the third did not reproduce in secondary studies. (C) Histogram comparing compound activities against primary and residual transport (<i>x</i> and <i>y</i> axes reflecting inhibition in parallel sorbitol and PhTMA⁺ + furosemide screens, respectively). Notice that mean ± SEM % activity against residual PhTMA⁺ transport gradually increases with activity in the sorbitol screen (grey histogram bars). Key hits reported previously in the sorbitol screen (compounds <b>1–8</b> from ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149214#pone.0149214.ref010" target="_blank">10</a>]) exhibited weak activity in the PhTMA⁺ screen (black circles). Novel hits from the present screen are superimposed as red circles. (D) Structures and names of key hits from the PhTMA⁺ screen, with the prefix PRT referring to action against the <u>P</u>SAC <u>r</u>esidual <u>t</u>ransport.</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