Fig 1.
Discovery of nanomolar PfFNT inhibitors from the malaria box.
(A) Energetic flux of Plasmodium 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) [15] and the plasmodial hexose transporter (HT) [3] for anaerobic glycolysis and ATP generation. Lactate dehydrogenase [7], (LDH) replenishes the pool of NADH + H+. l-lactate and protons are released via PfFNT and the erythrocyte monocarboxylate transporter (MCT1) [10]. (B) Screening of the malaria box at 10 μM yields two hits that fully block transport of PfFNT heterologously expressed in yeast: MMV007839 and MMV000972. (C/D) IC50 determinations for PfFNT inhibition in yeast (black) and viability of cultured parasites (red) by the compound hits. (E) 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). (F) Chemical structures of MMV007839 and MMV000972.
Fig 2.
Binding and dissociation kinetics of MMV007839.
(A) Concentration and incubation-time dependent inhibition of PfFNT by MMV007839 in yeast. (B) Full and half-maximally inhibited PfFNT does not re-gain activity within 2 h after removal of the inhibitor from the buffer. (C) 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. (D) Determination of the apparent Ki from the inhibition rates, kobs, observed in (A). The error bars are S.E.M. of at least triplicate values.
Fig 3.
Quantitative structure activity relations of PfFNT inhibitors.
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.
Fig 4.
Reversible hemiketal prodrug principle and identification of the pharmacophore.
(A) 1H / 13C HSQC NMR reveals two structural species of MMV007839 in CDCl3, a cyclic hemiketal form (blue, 65%) and a vinylogous acid form (red, 35%). The carbons and respective NMR signals are labeled with lowercase roman numerals. The acidic proton is shown inverse. (B) Structures of two MMV007839 derivatives, i.e. BH-296 (black), lacking the phenolic hydroxyl, and an ethyl ester prodrug of BH-296 (blue). The efficiency of BH-296 on PfFNT inhibition in yeast (black) and on the viability of cultured parasites (red; prodrug in blue) are shown in the graph. (C) Identification of the minimal structural requirements of a PfFNT inhibitor compound, the “pharmacophore”. Error bars indicate S.E.M. from at least three replicates.
Fig 5.
Selection of resistant 3D7 parasites confirms PfFNT as the MMV007839 target.
(A) A single nucleotide exchange in the PfFNT gene of MMV007839 resistant 3D7 parasites. (B) Position of the resulting G107S mutation in the conserved L2 loop of the FNT family (numbering from PfFNT; set with TeXshade [21]). (C) Model of a PfFNT monomer [7] with position 107 shown as spheres (glycine in wildtype, left/center; G107S, right) and the L2/L5 loops shaded orange and blue, respectively. (D) 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. (E) Confirmation of the MMV007839/FNT interaction by increase in efficiency on the Babesia bovis FNT by mutation of the naturally occurring serine to glycine at the resistance site. Errors denote S.E.M. from ≥ 3 replicates.
Fig 6.
Circumvention of the PfFNT G107S resistance mutation and binding mode.
(A) l-lactate uptake via PfFNT G107 S/A/C/V mutants over time and derived transport rates (B). (C) 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. (D) 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. (E) Efficiency of the pharmacophore on PfFNT G107S in yeast compared to PfFNT wildtype (dashed line). The error bars indicate S.E.M. (n ≥ 3). (F) Proposed model of MMV007839 binding to PfFNT and (G) strategy to circumvent the clash with G107S by introducing limited flexibility into the inhibitor scaffold.