Model of the pentamidine binding mode to TbAQP2 and proposed uptake by endocytosis in the flagellar pocket.

<p>(A) Shown are the crystal structure of the prototypical aquaglyceroporin GlpF and a model of TbAQP2. GlpF Arg206 and TbAQP2 Leu264 mark the position of the ar/R selectivity filter. In TbAQP2, the Asp265 sidechain carboxylate binds to an amidine moiety of pentamidine (light blue), whereas in GlpF the space is occupied by the guanidine sidechain of Arg206. The location of the ‘NPA/NPA’ region (white bar) and sequence deviations in TbAQP2 are indicated. (B) Proposed uptake mechanism of pentamidine via high-affinity binding to TbAQP2, endocytosis of the complex, and release of pentamidine in the acidic lysosome due to pH shift or TbAQP2 degradation.</p

Inhibition of TbAQP2 glycerol permeability by pentamidine and derivatives.

<p>(A) Shown are example traces of protoplast light scattering in 300 mM isotonic glycerol gradients in the presence of pentamidine. Red traces indicate uninhibited glycerol influx via TbAQP2 (left) and TbAQP3 (right), effects of pentamidine concentrations from 50 nM to 50 μM are colored light blue, and of 500 μM pentamidine in dark blue. (B) Structure-function evaluation of TbAQP2 inhibition by pentamidine. The chemical structures of the used compounds are shown on the left and respective dose-response curves on the right. (C) Effect of pH titration of TbAQP2 Asp265 and replacement by mutation to alanine on pentamidine inhibition. The dashed lines show the pentamidine inhibition of wild-type TbAQP2 at pH 7.2 (data taken from Fig 2B). The left panel shows dose-response curves of pentamidine pH 3.5 (closed symbols; IC₅₀ 450 nM), and pH 2.5 (open symbols; IC₅₀ 1.1 μM). The effect of the Asp265Ala point mutation (expression confirming Western blot in the inset) on the inhibition by pentamidine is depicted on the right. Data taken at pH 7.2 (IC₅₀ 4.5 μM) are indicated by closed symbols, those at pH 2.5 (IC₅₀ 5.8 μM) by open symbols. Each data point is an average of 5–9 light scattering traces each from at least two independent experiments.</p

Quantitative structure activity relations of PfFNT inhibitors.

<p>Variations of the MMV007839 scaffold (boxed, center) are indicated by red shading in the structures. The efficiency of the compounds on PfFNT in yeast (black IC₅₀ values) and, if available, on the viability of cultured parasites (red values) are shown next to the structures.</p

Discovery of nanomolar PfFNT inhibitors from the <i>malaria box</i>.

<p><b>(A)</b> Energetic flux of <i>Plasmodium</i> parasites. The parasite’s cytoplasm is shielded by three consecutive membranes: the red blood cell membrane (RBCM), the plasmodial vacuolar membrane (PVM), and the plasmodial plasma membrane (PPM). Glucose is taken up via the red blood cell’s (RBC) glucose transporter (GLUT1) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006172#ppat.1006172.ref015" target="_blank">15</a>] and the plasmodial hexose transporter (HT) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006172#ppat.1006172.ref003" target="_blank">3</a>] for anaerobic glycolysis and ATP generation. Lactate dehydrogenase [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006172#ppat.1006172.ref007" target="_blank">7</a>], (LDH) replenishes the pool of NADH + H⁺. l-lactate and protons are released via PfFNT and the erythrocyte monocarboxylate transporter (MCT1) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006172#ppat.1006172.ref010" target="_blank">10</a>]. <b>(B)</b> Screening of the <i>malaria box</i> at 10 μM yields two hits that fully block transport of PfFNT heterologously expressed in yeast: MMV007839 and MMV000972. <b>(C/D)</b> IC₅₀ determinations for PfFNT inhibition in yeast (black) and viability of cultured parasites (red) by the compound hits. <b>(E)</b> Inhibition of the erythrocyte MCT1 by MMV007839. The dashed line indicates efficiency of MMV007839 on PfFNT for comparison. The error bars denote S.E.M (n ≥ 3). <b>(F)</b> Chemical structures of MMV007839 and MMV000972.</p

Selection of resistant 3D7 parasites confirms PfFNT as the MMV007839 target.

<p><b>(A)</b> A single nucleotide exchange in the PfFNT gene of MMV007839 resistant 3D7 parasites. <b>(B)</b> Position of the resulting G107S mutation in the conserved L2 loop of the FNT family (numbering from PfFNT; set with TeXshade [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006172#ppat.1006172.ref021" target="_blank">21</a>]). <b>(C)</b> Model of a PfFNT monomer [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006172#ppat.1006172.ref007" target="_blank">7</a>] with position 107 shown as spheres (glycine in wildtype, left/center; G107S, right) and the L2/L5 loops shaded orange and blue, respectively. <b>(D)</b> Strongly reduced efficiency of MMV007839 on PfFNT G107S in yeast (black) and resistant parasites (red); dashed lines indicate efficiency on wildtype PfNT and parasites for comparison. <b>(E)</b> Confirmation of the MMV007839/FNT interaction by increase in efficiency on the <i>Babesia bovis</i> FNT by mutation of the naturally occurring serine to glycine at the resistance site. Errors denote S.E.M. from ≥ 3 replicates.</p

Circumvention of the PfFNT G107S resistance mutation and binding mode.

<p><b>(A)</b>l-lactate uptake via PfFNT G107 S/A/C/V mutants over time and derived transport rates <b>(B)</b>. <b>(C)</b> Relative selectivity of MMV007839, BH-296, and the pharmacophore for the PfFNT G107S resistance mutation. A ratio of 1 would indicate equal efficiency on wildtype and mutant PfFNT. <b>(D)</b> Efficiency of BH-296 on PfFNT G107S in yeast (black) and resistant parasites (red); dashed lines indicate efficiency on wildtype PfFNT and parasites for comparison. <b>(E)</b> Efficiency of the pharmacophore on PfFNT G107S in yeast compared to PfFNT wildtype (dashed line). The error bars indicate S.E.M. (n ≥ 3). <b>(F)</b> Proposed model of MMV007839 binding to PfFNT and <b>(G)</b> strategy to circumvent the clash with G107S by introducing limited flexibility into the inhibitor scaffold.</p

Binding and dissociation kinetics of MMV007839.

<p><b>(A)</b> Concentration and incubation-time dependent inhibition of PfFNT by MMV007839 in yeast. <b>(B)</b> Full and half-maximally inhibited PfFNT does not re-gain activity within 2 h after removal of the inhibitor from the buffer. <b>(C)</b> Time dependency of the MMV007839 IC₅₀. Prolonged, 24 h treatment of PfFNT expressing yeast with MMV007839 shifts the IC₅₀ from 170 nM (obtained at 20 min treatment; dashed curve) to 15 nM. <b>(D)</b> Determination of the apparent K_i from the inhibition rates, k_obs, observed in (<b>A</b>). The error bars are S.E.M. of at least triplicate values.</p