Research ArticleMalaria

Antimalarial pantothenamide metabolites target acetyl–coenzyme A biosynthesis in Plasmodium falciparum

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Science Translational Medicine  18 Sep 2019:
Vol. 11, Issue 510, eaas9917
DOI: 10.1126/scitranslmed.aas9917
  • Fig. 1 Generation of stable pantothenamides.

    (A) Structures of pantothenic acid, the starting point of CoA biosynthesis, and panthetheine, a natural pantothenamide that is the substrate of vanins. (B) The structure of phenethyl-PanAm, the starting point for chemical optimization, and its stabilized analog CXP18.6-006. (C) The antimalarial activities of phenethyl-PanAm and CXP18.6-006 when tested against P. falciparum NF54 asexual blood-stage parasites in vitro. The figure shows mean parasite density relative to control. Error bars indicate SDs determined from four (phenethyl-PanAm) or three (CXP18.6-006) independent replicates. (D) The core structure of pantothenamides described in this paper. Chiral centers are indicated with an asterisk. (E) Structures of lead pantothenamide compounds and their IC50 values against asexual blood stages of P. falciparum. The active compound MMV689258 is in the 2′-R,2-S configuration, whereas the inactive compound MMV884968 is in the 2′-S,2-R configuration (table S1). R1 and R2 indicate positions in the core structure depicted in (D).

  • Fig. 2 In vitro antimalarial activities of pantothenamides.

    (A) The activity of pantothenamides against P. falciparum NF54 asexual blood-stage parasites in vitro. The figure shows mean parasite density relative to vehicle control. (B) The activity of MMV689258 against a panel of drug-resistant P. falciparum strains and, for comparison, against drug-sensitive NF54 parasites. (C) An analysis of the effects of MMV689258 on the development of P. falciparum NF54 parasites in human primary liver cells in vitro. The figure shows mean parasite density relative to vehicle control. (D) The transmission blocking activity of MMV689258. Mature P. falciparum stage V gametocytes from luminescent reporter strain NF54-HGL were exposed to compound for 24 hours before feeding to A. stephensi mosquitoes. Eight days after feeding, the infection status of the mosquitoes was assessed using luminescence analyses to count the number of oocysts. (E) The gametocytocidal activity of MMV689258 against P. falciparum NF54 gametocytes harvested at day 11 postinduction of gametocytogenesis in human red blood cells in vitro. (A), (B), (C), and (E) show average values and SDs from four to six replicates (A and E) or two to four replicates (B and C). (D) shows average values and SE of the mean from two independent mosquito feeding experiments with 15 to 24 mosquitoes per feeding experiment. DHA, dihydroartemisinin.

  • Fig. 3 CoA biosynthesis and pantothenamide metabolism in P. falciparum trophozoites.

    (A) Schematic for the endogenous pantothenate/CoA biosynthesis pathway (left) or pantothenamide metabolism (right). Structures are displayed for the metabolites of interest and colored to denote key functional residues. Enzyme abbreviations are displayed in red between the metabolic routes to denote the utilization of these enzymes in both pathways. PA, pantothenic acid; 4′-P-PA, 4′-phosphopantothenate; 4′-P-PC, 4′-phosphopantothenoyl-l-cysteine; 4′-P-PT, pantetheine-4′-phosphate; deP-CoA, dephosphoCoA; CoA, coenzyme A; PanAm, pantothenamide; 4′-P-PanAm, 4′-phosphopantothenamide analog; dPCoA-PanAm, dephosphoCoA pantothenamide analog; CoA-PanAm, pantothenamide CoA antimetabolite; ETC, electron transport chain; PANK, pantothenate kinase; PPCS, phosphopantothenoylcysteine synthetase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPAT, phosphopantetheine adenylyltransferase; and DPCK, dephospho-CoA kinase. (B) Representative extracted ion chromatograms for pantothenate and pantothenamide metabolism. The top panel displays peaks for endogenous compounds run as pure standards (1 μM); antimetabolite peaks are from cellular extracts. The x axis denotes the retention time of the detected metabolite [colors match schematic in (A)], and the y axis denotes the relative abundance (100% per respective extracted ion chromatogram). All peaks identified were unique to the drug treatment (extracted ion chromatograms from untreated infected human red blood cell extracts not shown for clarity). (C) Endogenous metabolic alterations in trophozoite stage parasites treated with lead pantothenamide compounds at 10× IC50 for 2.5 hours. The y axis denotes the pantothenamide tested, and the x axis is the average peak area fold change (±SE) relative to a paired untreated control. Each sample was collected in technical triplicate for n = 3 biological replicates. ‡ denotes statistical significance at P < 0.01 by one-way ANOVA between all groups; * denotes P < 0.01 using Fisher’s least significant difference post hoc method versus untreated control.

  • Fig. 4 Mutations in ACS and ACS11 lead to pantothenamide-resistant parasites.

    (A) Listed in the table are mutations found in pantothenamide-resistant parasite lines that result in protein coding amino acid changes. Resistance against CXP18.6-052 was induced in NF54 parasites in two independent experiments. The mutations were identified from whole-genome sequences by comparison to the 3D7 reference genome and wild-type NF54 parental line. Multiple clones from each independently derived parasite line were sequenced. (B) Drug sensitivity assays for P. falciparum NF54 drug-induced resistant parasites (F49C11 and F50D5) and genetically engineered parasites. ACS and ACS11 point mutations were either introduced in NF54 parasites or reverted in drug-induced resistant parasites using the CRISPR-Cas9 system. The graphs show average values for mean parasite density relative to controls for asexual blood-stage replication assays. Error bars indicate SEM determined from three independent replicates, with two to three technical replicates per experiment. The data were analyzed using nonlinear regression on GraphPad Prism. The amino acid changes in the different parasite lines are indicated in the key. Square, no mutation; triangle, single mutation; and circle, two mutations. rev, revertant; SM, single mutant; and DM, double mutant. (C) Schematic of protein domains and amino acid mutations. National Center for Biotechnology Information protein sequences were used to map protein domains annotated by the Conserved Domain Database. Black vertical bars denote conserved amino acids within a protein feature. Arrows demonstrate the mutation position within the protein feature; mutations that occurred in conserved amino acids are denoted in red.

  • Fig. 5 In vivo efficacy and pharmacokinetics of MMV689258.

    Female NODscidIL2Rγnull mice were engrafted with human red blood cells and infected with P. falciparum at day 0 at 1% and 2% parasitemia for the experiments depicted in (A) and (C), respectively. At day 3 after infection, mice were dosed with the compounds by oral gavage at doses indicated once daily (QD). (A) Parasitemia after a single dose of each compound (black arrow). (B) Blood concentrations of MMV689258 as a function of time after the first administration of compound at the doses indicated in the key. (C) Parasitemia after daily dosing with each compound on four consecutive days (black arrows). The graphs show average values and standard deviations from two mice per drug/dose combination.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/510/eaas9917/DC1

    Materials and Methods

    Fig. S1. Pantothenamide stability.

    Fig. S2. Parasite life cycle.

    Fig. S3. Pantothenate competition assays.

    Fig. S4. PANK activity.

    Fig. S5. Expression of PfPANK1.

    Fig. S6. Cellular pantothenamide metabolism and targeted metabolomics.

    Fig. S7. Pantothenate and pantothenamide metabolism in saponin-isolated parasites versus uninfected red blood cells.

    Fig. S8. Erythrocytes preexposed to MMV689258 are less susceptible to malaria infection.

    Fig. S9. Drug-resistant parasite (ACS-T627A and ACS11-E660K) transmission to mosquitoes.

    Fig. S10. Pantothenamide-resistant parasites (ACS-T627A and ACS11-E660K) have reduced fitness.

    Fig. S11. Sequence verification of CRISPR-Cas9–engineered mutations.

    Fig. S12. Metabolomics of wild-type versus drug-resistant parasites.

    Fig. S13. Pharmacokinetics of MMV689258 in rodents.

    Fig. S14. Dose-normalized plasma exposure of MMV689258 in NODscidIL2Rγnull mice.

    Fig. S15. Red blood cell counts in PfSCID mice treated with MMV689258.

    Fig. S16. Cell-type specificity and primary human hepatocyte metabolomics.

    Table S1. Selection of compounds to illustrate structure-activity relationship.

    Table S2. IC50 values of compounds shown in Fig. 1.

    Table S3. Description of strains used in resistance panel.

    Table S4. Targeted metabolomics values for select compounds of interest.

    Table S5. Pharmacokinetic parameters derived from the data shown in fig. S13.

    Table S6. Renal excretion of MMV689258 in rats.

    Table S7. Biliary excretion of MMV689258 in rats.

    Table S8. In vitro ADME parameters of MMV689258.

    Table S9. Primers used for genetic studies.

    Table S10. Source data for Figs. 1C, 2 (A to E), 3C, 4B, and 5 (A to C).

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Pantothenamide stability.
    • Fig. S2. Parasite life cycle.
    • Fig. S3. Pantothenate competition assays.
    • Fig. S4. PANK activity.
    • Fig. S5. Expression of PfPANK1.
    • Fig. S6. Cellular pantothenamide metabolism and targeted metabolomics.
    • Fig. S7. Pantothenate and pantothenamide metabolism in saponin-isolated parasites versus uninfected red blood cells.
    • Fig. S8. Erythrocytes preexposed to MMV689258 are less susceptible to malaria infection.
    • Fig. S9. Drug-resistant parasite (ACS-T627A and ACS11-E660K) transmission to mosquitoes.
    • Fig. S10. Pantothenamide-resistant parasites (ACS-T627A and ACS11-E660K) have reduced fitness.
    • Fig. S11. Sequence verification of CRISPR-Cas9–engineered mutations.
    • Fig. S12. Metabolomics of wild-type versus drug-resistant parasites.
    • Fig. S13. Pharmacokinetics of MMV689258 in rodents.
    • Fig. S14. Dose-normalized plasma exposure of MMV689258 in NODscidIL2Rγnull mice.
    • Fig. S15. Red blood cell counts in PfSCID mice treated with MMV689258.
    • Fig. S16. Cell-type specificity and primary human hepatocyte metabolomics.
    • Table S1. Selection of compounds to illustrate structure-activity relationship.
    • Table S2. IC50 values of compounds shown in Fig. 1.
    • Table S3. Description of strains used in resistance panel.
    • Table S4. Targeted metabolomics values for select compounds of interest.
    • Table S5. Pharmacokinetic parameters derived from the data shown in fig. S13.
    • Table S6. Renal excretion of MMV689258 in rats.
    • Table S7. Biliary excretion of MMV689258 in rats.
    • Table S8. In vitro ADME parameters of MMV689258.
    • Table S9. Primers used for genetic studies.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S10 (Microsoft Excel format). Source data for Figs. 1C, 2 (A to E), 3C, 4B, and 5 (A to C).

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