Pantothenamides are potent, on-target inhibitors of Plasmodium falciparum growth when serum pantetheinase is inactivated - PubMed (original) (raw)

Pantothenamides are potent, on-target inhibitors of Plasmodium falciparum growth when serum pantetheinase is inactivated

Christina Spry et al. PLoS One. 2013.

Abstract

Growth of the virulent human malaria parasite Plasmodium falciparum is dependent on an extracellular supply of pantothenate (vitamin B(5)) and is susceptible to inhibition by pantothenate analogues that hinder pantothenate utilization. In this study, on the hunt for pantothenate analogues with increased potency relative to those reported previously, we screened a series of pantothenamides (amide analogues of pantothenate) against P. falciparum and show for the first time that analogues of this type possess antiplasmodial activity. Although the active pantothenamides in this series exhibit only modest potency under standard in vitro culture conditions, we show that the potency of pantothenamides is selectively enhanced when the parasite culture medium is pre-incubated at 37°C for a prolonged period. We present evidence that this finding is linked to the presence in Albumax II (a serum-substitute routinely used for in vitro cultivation of P. falciparum) of pantetheinase activity: the activity of an enzyme that hydrolyzes the pantothenate metabolite pantetheine, for which pantothenamides also serve as substrates. Pantetheinase activity, and thereby pantothenamide degradation, is reduced following incubation of Albumax II-containing culture medium for a prolonged period at 37°C, revealing the true, sub-micromolar potency of pantothenamides. Importantly we show that the potent antiplasmodial effect of pantothenamides is attenuated with pantothenate, consistent with the compounds inhibiting parasite proliferation specifically by inhibiting pantothenate and/or CoA utilization. Additionally, we show that the pantothenamides interact with P. falciparum pantothenate kinase, the first enzyme involved in converting pantothenate to coenzyme A. This is the first demonstration of on-target antiplasmodial pantothenate analogues with sub-micromolar potency, and highlights the potential of pantetheinase-resistant pantothenamides as antimalarial agents.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Chemical structures of pantothenate and related compounds.

The hydroxy analogue of pantothenate, pantothenol, inhibits growth of P. falciparum in vitro and in vivo. Amide analogues of pantothenate, pantothenamides (for which the core structure is shown), possess antibacterial activity in vitro. Pantetheine, a naturally occurring pantothenate derivative, is hydrolyzed to pantothenate (and cysteamine) by the enzyme pantetheinase.

Figure 2

Figure 2. Effect of pantothenamides on proliferation of P. falciparum and P. falciparum lysate-catalysed [14C]pantothenate phosphorylation.

The 50% inhibitory concentrations (IC50 values) measured against P. falciparum parasites cultured (for 96 h) in Albumax-complete RPMI containing 1 µM pantothenate, as determined using the SYBR Green I-based growth assay are shown. Assays were performed using Albumax-complete RPMI prepared within 48 h of the assay, stored at 4°C, and incubated at 37°C for a maximum of 1 h (fresh) or Albumax-complete RPMI incubated continuously at 37°C for 40 h immediately after preparation (aged). The IC50 values shown for parasites cultured in fresh Albumax-complete RPMI are averages from between two and eight independent experiments each performed in duplicate or triplicate. Where the IC50 values determined were below 200 µM, they are presented as the mean ± SEM from between three and eight independent experiments. The IC50 values shown for parasites cultures in aged medium are averages from between two and three independent experiments each performed in triplicate. Where the IC50 values determined were below 200 µM, they are presented as the mean ± range/2 or SEM as appropriate. The percentage inhibition of [14C]pantothenate phosphorylation by PanK in P. falciparum lysate caused by pantothenamides (when tested at a concentration of 100 µM) in the presence of 0.2 µM pantothenate are also shown. The percentage inhibition was calculated from the measured amounts of [14C]pantothenate phosphorylated during a 10 min incubation in the presence of pantothenamide and in the presence, instead, of the corresponding concentration of DMSO only. Data are averages ± range/2 from two independent experiments, each performed in duplicate. A value of 100 indicates complete inhibition of [14C]pantothenate phosphorylation was observed in both independent experiments. The amount of [14C]pantothenate phosphorylated by P. falciparum lysate was significantly lower in the presence of all pantothenamides (P<0.0001, ANOVA).

Figure 3

Figure 3. Antiplasmodial effect of pantothenamides and pantothenol in fresh and aged Albumax-complete RPMI.

The concentration-response curves show the effect of increasing concentrations of compound 3 (A), compound 12 (B), compound 19 (C), and pantothenol (D) on proliferation of P. falciparum parasites cultured (for 96 h) in Albumax-complete RPMI containing 1 µM pantothenate, as measured using the SYBR Green I-based growth assay. Assays were performed using Albumax-complete RPMI stored for a maximum of 48 h at 4°C, and incubated at 37°C for a maximum of 1 h (fresh; open circles) or Albumax-complete RPMI incubated continuously at 37°C for 40 h immediately after preparation (aged; closed circles). The data obtained with parasites cultured in fresh Albumax-complete RPMI are from between three and eight independent experiments performed in duplicate or triplicate and error bars represent SEM. The data obtained with parasites cultured in aged Albumax-complete RPMI are from between two and three independent experiments performed in duplicate or triplicate and error bars represent range/2 or SEM. For clarity, in D, the concentration-response curves represented by the closed circles are shown with negative error bars only, and the concentration-response curves represented by the open circles are shown with positive error bars only. Where not shown, error bars are smaller than the symbol.

Figure 4

Figure 4. Effect of pantothenate supplementation on pantothenamide and pantothenol potency in fresh and aged Albumax-complete RPMI.

The bars represent the fold-increases in the IC50 values of compound 12, compound 19 and pantothenol in Albumax-complete RPMI following supplementation with 100 µM pantothenate. The fold-increases in IC50 values were calculated by dividing each IC50 value measured against P. falciparum cultured in the presence of 100 µM pantothenate by the corresponding IC50 value measured against P. falciparum cultured in the presence of 1 µM pantothenate. The fold-increases in IC50 values were determined in assays performed using Albumax-complete RPMI stored for a maximum of 48 h at 4°C, and incubated at 37°C for a maximum of 1 h (fresh; open bar) or Albumax-complete RPMI stored for a minimum of one week at 4°C and incubated intermittently at 37°C, or, soon after preparation, incubated continuously at 37°C for up to 40 h (aged; closed bar). The fold-increases in IC50 values are averaged from between two and four independent experiments in which test compounds were tested in Albumax-complete RPMI containing 1 µM pantothenate and Albumax-complete RPMI supplemented with 100 µM pantothenate in parallel. Each experiment was performed in duplicate or triplicate, and error bars represent SEM or range/2. Pantothenol bars are shown with a broken edge to indicate that only a lower limit on the fold-increase in IC50 could be determined. This is because less than 50% inhibition of growth was observed at the highest pantothenol concentration tested (2 mM).

Figure 5

Figure 5. Effect of Albumax II supplementation on pantothenamide and pantothenol potency in aged Albumax-complete RPMI.

The concentration-response curves show the effect of increasing concentrations of compound 12 (A) and pantothenol (B) on proliferation of P. falciparum parasites cultured (for 96 h) in Albumax-complete RPMI as measured using the SYBR Green I-based growth assay. Assays were performed using (i) Albumax-complete RPMI incubated immediately after preparation at 37°C for 40 h (aged; closed circles); (ii) aged Albumax-complete RPMI supplemented with additional Albumax II (0.6%, w/v) immediately prior to the assay (aged+(fresh) Albumax; open circles); (iii) aged Albumax-complete RPMI supplemented with additional Albumax II (0.6%, w/v) and heated at 37°C for 40 h immediately prior to the assay (aged+(fresh) Albumax+heat; grey circles); and (iv) aged Albumax-complete RPMI heated at 37°C for 40 h immediately prior to the assay (aged+heat; closed squares). The data presented in A are averaged from three independent experiments, each performed in triplicate, and error bars represent SEM. The data presented in B are averaged from two independent experiments, performed in duplicate or triplicate, and error bars represent range/2. For clarity, in A, the concentration-response curves represented by the closed circles are shown with negative error bars only, and the concentration-response curves represented by the grey circles are shown with positive error bars only. In B, the concentration-response curves represented by the closed circles are shown with positive error bars only, and the concentration-response curves represented by the open circles are shown with negative error bars only. Where not shown, error bars are smaller than the symbol.

Figure 6

Figure 6. Antiplasmodial effect of a pantothenamide and pantothenol in fresh and aged human serum-complete RPMI.

The concentration-response curves show the effect of increasing concentrations of compound 12 (A) and pantothenol (B) on the proliferation of P. falciparum parasites cultured (for 96 h) in human serum-complete RPMI as measured using the SYBR Green I-based growth assay. Assays were performed using human serum-complete RPMI prepared immediately prior to experimentation (fresh; open circles) or human serum-complete RPMI heated at 37°C for 40 h immediately following preparation (aged; closed circles). The data obtained with parasites cultured in fresh human serum-complete RPMI are from three independent experiments, each performed in duplicate or triplicate, and error bars represent SEM. The data obtained with parasites cultured in aged human serum-complete RPMI are from two independent experiments, each performed in duplicate or triplicate, and error bars represent range/2. For clarity, in B, concentration-response curves represented by the closed circles are shown with positive error bars only, and the concentration-response curves represented by the open circles are shown with negative error bars only. Where not shown, error bars are smaller than the symbol.

Figure 7

Figure 7. Hydrolysis of a pantothenamide in the presence of recombinant human pantetheinase and Albumax II.

The time-courses show the concentration of isobutylamine (a product of compound 12 hydrolysis) detected during incubation of compound 12 with recombinant human pantetheinase (100 ng/mL; closed circles), Albumax II (0.6%, w/v; grey circles), or an equivalent volume of water (open circles), and during incubation of Albumax II (0.6%, w/v) in the absence of compound 12 (grey squares). At each time-point, the amount of primary amine was measured using a fluorescamine-based fluorescence assay. Fluorescence measurements were converted to isobutylamine concentrations using a standard curve generated using isobutylamine samples of known concentration that had been processed in the same manner as the test samples. The data are from three or four independent experiments, each performed in duplicate, and error bars represent SEM. For clarity, the time-courses represented by the open circles are shown with positive error bars only, and the time-courses represented by the grey squares are shown with negative error bars only. Where not shown, error bars are smaller than the symbol.

Figure 8

Figure 8. Effect of pantetheinase supplementation on the potency of a pantothenamide in aged Albumax-complete RPMI.

The concentration-response curves show the effect of increasing concentrations of compound 12 on proliferation of P. falciparum parasites cultured (for 96 h) in Albumax-complete RPMI as measured using the SYBR Green I-based growth assay. Assays were performed using (i) Albumax-complete RPMI incubated immediately after preparation at 37°C for 40 h (aged; closed circles); (ii) aged Albumax-complete RPMI supplemented with recombinant human pantetheinase (100 ng/mL) immediately prior to the assay (aged+pantetheinase; open circles); (iii) aged Albumax-complete RPMI supplemented with recombinant human pantetheinase (100 ng/mL) and heated at 37°C for 40 h immediately prior to the assay (aged+pantetheinase+heat; grey circles); and (iv) aged Albumax-complete RPMI heated at 37°C for 40 h before being supplemented with recombinant human pantetheinase (100 ng/mL) immediately prior to the assay (aged+heat+pantetheinase; grey squares). The data are from three independent experiments, each performed in triplicate, and error bars represent SEM. For clarity, the time-courses represented by the open circles are shown with negative error bars only, and the time-courses represented by the grey squares are shown with positive error bars only. Where not shown, error bars are smaller than the symbol.

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Grants and funding

Aspects of this work were supported by grants from the South African Malaria Initiative (SAMI) to ES and KJS and the American Lebanese Syrian Associated Charities (ALSAC), St. Jude Children’s Research Hospital, to REL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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