Mutations in the pantothenate kinase of the malaria parasite P . falciparum confer resistance or hypersensitivity to diverse pantothenate analogues

1. Research School of Biology, The Australian National University, Canberra, Australia 2. Department of Chemistry, University of Glasgow, Glasgow, United Kingdom 3. Department of Chemistry, McGill University, Montreal, Quebec, Canada 4. Department of Biochemistry, Stellenbosch University, Matieland, South Africa 5. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, Australia 6. Department of Chemistry, Xi’an Jiaotong-Liverpool University, Suzhou, China 7. Medical School, The Australian National University, Canberra, Australia


Introduction
In recent years, the effort to roll back malaria has shown encouraging progress through the increased use of insecticide-treated mosquito nets, improved diagnostics and artemisinin-based combination chemotherapies (ACTs) 1 .Evidence of this includes the decreasing worldwide malaria incidence (266 million cases in 2005 down to 212 million cases in 2015) and mortality (741,000 deaths in 2005 down to 429,000 deaths in 2015) over the past decade 1 .However, there is an alarming trend of ACT-resistant parasites emerging in multiple Asian countries where the disease is endemic 2 .Recently, there have also been multiple reports of patients contracting ACT-resistant Plasmodium falciparum malaria from various African countries 3,4 , which exemplify the clear risk of artemisinin resistance developing in the continent.This threat to the efficacy of ACTs highlights the requirement for a new armoury of antimalarial medicines, with several compounds representing different chemotypes entering the preclinical trial stage.However, the antimalarial drug-discovery pipeline is reliant on just a few known drug targets and the probability of successfully producing a new blood-stage medicine remains low 5 .In order to manage the threat of parasite drug resistance, there needs to be a continued effort to identify new classes of antimalarials.One metabolic pathway that has garnered recent interest for drugdevelopment is the parasite's coenzyme A (CoA) biosynthetic pathway 6,7 .
Early seminal studies have shown that the asexual stage of intra-erythrocytic P. falciparum absolutely requires an exogenous supply of vitamin B 5 (pantothenate; Figure 1) for survival [7][8][9] .Pantothenate is taken up by the parasite 10,11 and converted into CoA, an essential cofactor for many metabolic processes 7 .This conversion is catalysed by a series of five enzymes, the first of which is pantothenate kinase (PfPanK), an enzyme that phosphorylates pantothenate to form 4'phosphopantothenate 11 .By performing this step, the parasite traps pantothenate within its cytosol and commits it to the CoA biosynthetic pathway 10 .The additional four steps are, in turn, catalysed by phosphopantothenoylcysteine synthetase (PfPPCS), phosphopantothenoylcysteine decarboxylase (PfPPCDC), phosphopantetheine adenylyltransferase (PfPPAT) and dephospho-CoA kinase (PfDPCK) 6 .Putative genes coding for each of the enzymes in the pathway (with several enzymes having multiple putative candidates) have been identified in the P. falciparum genome 12,13 and have also been shown to be transcribed during the intraerythrocytic stage of the parasite's lifecycle 14 .In order to capitalise on the pathway as a potential drug-development target, however, it is crucial to ascertain the exact identity of each of these putative genes.This will allow the drug discovery process to become more efficient and targeted.
Investigations aimed at discovering antiplasmodial agents that act by interfering with the parasite's CoA biosynthetic pathway identified several antiplasmodial pantothenate analogues, including pantothenol (PanOH) and CJ-15,801 9,15,16 (see Figure 1 for structures).Subsequent studies identified pantothenamides as antiplasmodial pantothenate analogues with substantially increased potency [17][18][19] .Unfortunately pantothenamides are unstable in vivo because they are degraded by the serum enzyme pantetheinase 17 .Recent reports of structural optimisations of lead pantothenamides have described two compounds, N5-trz-C1-Pan (compound 1e in Howieson et al. 20 ) and N-PE-aMe-PanAm (see Figure 1 for structures), that are potent antiplasmodials (with nanomolar IC 50 values) and also resistant to pantetheinasemediated degradation 20,21 .However, although these compounds have been shown to 4 target CoA biosynthesis or utilisation, their exact mechanism(s) of action has not been elucidated.
In this study, we have used continuous drug-pressuring with PanOH or CJ-15,801 to generate a number of P. falciparum parasite lines that are several-fold resistant to these pantothenate analogues.Whole-genome sequencing revealed mutations in one of the two putative Pfpank genes of all of the clones, Pfpank1.Complementation experiments confirmed that these mutations are responsible for the resistance phenotypes.Characterisation of the effects of the mutations on parasite growth in culture and also PfPanK function, generated data consistent with the mutated gene coding for an active pantothenate kinase in P. falciparum and for the gene to be essential for normal parasite development during the intraerythrocytic stage of their lifecycle.Additional characterisation of the PanOH and CJ-15,801-resistant lines revealed that antiplasmodial pantothenate analogues have at least two distinct mechanisms of action, targeting CoA biosynthesis or utilisation.Both of these mechanisms can be influenced by the Pfpank1 mutations identified here.Furthermore, our study provides genetic evidence validating the importance of the metabolic activation of pantothenate analogues in the antiplasmodial activity of these compounds.

Materials and Methods
Parasite culture and lysate preparation P. falciparum parasites were maintained in RPMI 1640 media supplemented with 11 mM glucose, 200 M hypoxanthine, 24 g/mL gentamicin and 6 g/L Albumax II (referred to as complete medium) as previously described 22 .Synchronisation of ringstage parasite cultures using D-sorbitol 23 and trophozoite-stage parasite isolation from infected erythrocytes via saponin permeabilisation 10 were performed as described previously.Clonal parasite populations were generated through limiting dilution cloning as reported previously 24 , with modifications.Parasite lysates were prepared from saponin-isolated mature trophozoite-stage parasites as described previously 10 , except after trituration (15 times), each lysate was either centrifuged once at 16,100 × g for 30 min or three times at 2,000 × g, with the supernatant transferred to a new centrifuge tube after each centrifugation.Lysates were generally stored at −20 °C before use.

Plasmid preparation and parasite transfection
Several plasmid constructs were generated through the course of this study to be used for different lines of investigations.The Pfpank1-pGlux-1 plasmid consists of the wildtype Pfpank1-coding sequence inserted within multiple cloning site III of pGlux-1, which contains the human dihydrofolate reductase (hdhfr) gene that confers resistance to WR99210 as a positive selectable marker.This places Pfpank1 under the regulation of the Plasmodium falciparum chloroquine resistance transporter (Pfcrt) promoter, and upstream of the GFP-coding sequence.This plasmid construct allows the parasite to express a GFP-tagged PfPanK1 and was used to localise the protein within the parasite.The Pfpank1-stop-pGlux-1 plasmid is identical to the Pfpank1-pGlux-1 construct but contains two stop codons between the Pfpank1-specific sequence and the GFP-coding sequence.This enables the parasite to express wild-type PfPanK1 that is not GFP-tagged and was used to complement the mutant lines with wild-type PfPanK1.The DPfpank1-pCC-1 construct was generated using the pCC-1 plasmid 25 as the backbone (Figure S1).This plasmid contains the hdhfr gene cassette as a positive selectable marker.Two Pfpank1-specific sequences were cloned into sites flanking the hdhfr cassette to allow for homologous integration into the native Pfpank1 locus of the transfected parasite.The plasmid also contains the Saccharomyces cerevisiae cytosine deaminase/phosphoribosyl transferase (Scfcu) gene that confers sensitivity to 5-fluorocytosine (5-FC) as a negative selectable marker.Parasites that integrate the hdhfr gene cassette into Pfpank1 by homologous recombination and no longer carry the DPfpank1-pCC-1 construct as an episome should lose the Scfcu gene and therefore be resistant to 5-FC, while parasites that maintain the episomal DPfpank1-pCC-1 would be killed due to the toxic metabolite generated in the presence of Scfcu.The genomic regions for homologous integration were selected such that if the construct is successfully integrated into the P. falciparum genome by double crossover homologous recombination, the hdhfr gene cassette will replace a 500 base-pair region of the first exon of Pfpank1, and hence will disrupt the gene.
The Pfpank1 sequence used to generate the Pfpank1-pGlux-1 and Pfpank1-stop-pGlux-1 plasmids were amplified from parasite RNA.The RNeasy Mini Kit (QIAGEN) was used to purify total RNA from saponin-isolated P. falciparum parasites (typically 2 × 10 7 cells) according to the manufacturer's protocol for purifying total RNA from animal cells.An optional 15 min DNase I incubation was included in the procedure to eliminate residual genomic DNA (gDNA).Complementary DNA (cDNA) was synthesised from total RNA using SuperScript II Reverse Transcriptase (ThermoFisher), with Oligo(dT) 12-18 primer, and included an optional incubation with RNaseOUT Recombinant Ribonuclease Inhibitor, as per manufacturer's instructions.When required, parasite gDNA was extracted from saponin-isolated trophozoite-stage parasites using DNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's instructions.Pfpank1-specific sequences were subsequently amplified from cDNA (to generate the Pfpank1-pGlux-1 and Pfpank1-stop-pGlux-1 constructs) or gDNA of wildtype 3D7 strain P. falciparum (to generate the DPfpank1-pCC-1 construct) using either KOD Hot Start DNA polymerase (Merck Millipore) or Platinum Pfx DNA polymerase (ThermoFisher), with the oligonucleotide primers listed in Table S1 (Supplementary Information), following each manufacturer's protocol.
To generate the Pfpank1-stop-pGlux-1 and Pfpank1-pGlux-1 constructs, the In-Fusion cloning (Clontech) method was employed to insert the Pfpank1-coding sequence into pGlux-1, with and without the introduction of stop codons prior to the GFP-coding sequence, respectively.Before the cloning step, the pGlux-1 plasmid was linearised by sequential digestions with XhoI (ThermoFisher) and then KpnI (New England Biolabs), according to each manufacturer's recommendation.The In-Fusion reactions were set up with either the In-Fusion Dry-Down PCR Cloning Kit (for Pfpank1-pGlux-1) or the In-Fusion HD EcoDry Cloning Kit (for Pfpank1-stop-pGlux-1) essentially as instructed by the manufacturer's protocol.To generate the DPfpank1-pCC-1 construct, the Pfpank1 flank fragments (amplified from parasite gDNA to contain specific restriction sites; Table S1) were digested with their respective restriction enzymes (SacII and SpeI (both New England Biolabs) for the 5' Pfpank1 flank, and EcoRI (ThermoFisher) and AvrII (New England Biolabs) for the 3' Pfpank1 flank) according to each manufacturer's protocol.Subsequently, the digested 5' Pfpank1 flank was ligated into a linearised pCC-1 plasmid (following SacII and SpeI digestion performed as before) using a Quick T4 DNA ligase (New England Biolabs), following manufacturer's protocol, to generate an intermediate plasmid.This intermediate plasmid was then linearised by EcoRI and AvrII digestion performed as above, and was ligated with the 3' Pfpank1 flank as described above to generate the final DPfpank1-pCC-1 plasmid (Figure S1).
The purity and concentration of all DNA and RNA samples was determined using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies).Sanger sequencing of DNA samples was performed at the Australian Genome Research Facility (Sydney) to confirm the accuracy of DNA sequences.The Pfpank1-pGlux-1, Pfpank1-stop-pGlux-1 and DPfpank1-pCC-1 plasmids were transfected into ring-stage parasites and positive transfectants were selected by introducing WR99210 (10 nM) into the culture as described previously 26 .

SYBR Safe-based parasite proliferation assay
The effect of various compounds on the in vitro proliferation of the different parasite lines were tested using a previously-reported SYBR Safe-based fluorescence assay 17 , with minor modifications.Briefly, for the in vitro inhibition experiments performed in this study, synchronous ring-stage parasite-infected erythrocytes were suspended within wells of a 96-well plate, in a final volume of 200 L complete media (0.5% parasitaemia and 1% haematocrit) in the presence of the test compound dissolved in water or dimethyl sulfoxide (DMSO), with the highest concentration of DMSO not exceeding 0.1% v/v.Parasite-infected erythrocytes incubated in 500 nM chloroquine were used as a no-proliferation control, while those incubated in the absence of inhibitory compounds were used as a 100% parasite proliferation control.The parasite suspensions were then incubated at 37 °C for 96 h under a low-oxygen atmosphere (96% nitrogen, 3% carbon dioxide and 1% oxygen).At the end of the incubation, each 96-well plate was frozen at −20 °C (to lyse the cells) and thawed before being processed as described previously 17 .
In vitro pantothenate requirement experiments were performed in a similar way, except instead of a test compound, ring stage-infected erythrocytes were incubated in pantothenate-free complete RPMI 1640 medium (as described above; Athena Enzyme Systems) supplemented with 2-fold serial dilutions of pantothenate.All solutions were prepared in complete pantothenate-free medium and cells used for these experiments were washed twice in complete pantothenate-free medium (10 mL, 1,500 × g for 5 min) to remove any pantothenate before being placed in the final suspension containing the desired concentration of pantothenate.In these experiments, parasiteinfected erythrocytes incubated in the absence of pantothenate served as a noproliferation control, while those incubated in 1 M pantothenate (the pantothenate concentration normally present in complete medium used for in vitro parasite culture) served as a 100% parasite proliferation control.
Each data set was fitted with sigmoidal curves (typically either  = 0 + () * + * ,) * or  = () * + * ,) * , where  represents percentage parasite proliferation, 0 represents minimum percentage parasite proliferation,  represents the range of percentage parasite proliferation,  represents the Hill coefficient,  represents the IC 50 or SC 50 value and  represents compound concentration) using a nonlinear least squares regression.The IC 50 and SC 50 values for these experiments were then determined from these sigmoidal curves and averaged from several independent experiments.The fold change, relative to the Parent line, in the sensitivity of each mutant clone to a particular compound was calculated as the ratio between the IC 50 value obtained for the mutant clone from a single experiment and the average Parent IC 50 value.The same was done for the corresponding PfPanK1-complemented lines.IC 35 values (obtained in the same method described above) were used to determine the CJ-15,801 fold change for PanOH-B and CJ-A because a full sigmoidal curve, and hence IC 50 values, could not be obtained for all of the experiments performed for these clones.

Drug pressuring
Two independent drug-pressuring cultures were initiated for each of the pantothenate analogues, PanOH and CJ-15,801.Pressuring was initiated by exposing synchronous ring-stage Parent line parasites (10 mL culture of 2 or 4% parasitaemia and 2% haematocrit) to either analogue at the IC 50 values obtained for the Parent line at the time (PanOH = 400 M and CJ-15,801 = 75 M).Parasites were then exposed to cycles of increasing drug-pressure that lasts about 2 -4 weeks each.During each cycle, each culture was maintained continuously in the presence of a fixed concentration of the pressuring analogue, with a medium replacement every 48 h and fresh erythrocytes (100 L) added at least once a week.Whenever the parasitaemia for each culture reached approximately 5 -10%, a parasite proliferation assay was performed with predominantly ring-stage parasites as described above.If the IC 50 value obtained for the assay was equal to or greater than the pressuring concentration, aliquots of the culture were cryopreserved, and the concentration of the pantothenate analogue was increased.The cycle was then repeated, each time increasing the compound concentration in a step-wise fashion (multiples of 2 from the initial pressuring concentration) until the pressured parasites became approximately 8 × less sensitive than the Parent line to the selecting compounds.This process took 11 -13 weeks.The drug-resistant parasites were subsequently cultured and cloned in the absence of pantothenate analogue pressure for at least 3 months.The clones were then confirmed to have retained resistance to the pantothenate analogue used in the selection process, and were subsequently maintained in a drug-free environment for the remainder of the study.

Competition assay
In order to compare the fitness of the mutant clones with that of the Parent, we set up three competition cultures, each containing a mixture of one mutant line and the Parent line.Equal number of parasites (5 × 10 8 cells in the first experiment and 2.5 × 10 8 cells in the second experiment) from each line were mixed into a single culture.Aliquots (3 to 5 mL) of these cultures were immediately used for a PanOH SYBR Safebased parasite proliferation assay (to generate Week 0 data) as described above.The cultures were then maintained under standard conditions as detailed above for a period of 6 weeks before they were used to perform another PanOH proliferation assay (to generate Week 6 data).

Whole genome sequencing and variant calling
Parasite gDNA for sequencing was extracted (typically from ~10 8 cells) as described above.Next generation whole genome sequencing was performed by the Biomolecular Resource Facility at the Australian Cancer Research Foundation, the Australian National University.Samples were sequenced with the Illumina MiSeq platform with version 2 chemistry (2 × 250 base pairs, paired-end reads) Nextera XT Kit (Illumina).To determine the presence of any Single Nucleotide Polymorphisms (SNPs) in the genome of the drug-pressured clones, the genomic sequencing data were analysed using an integrated variant calling pipeline, PlaTyPus, as previously described 29 , with minor modifications to resolve operating system compatibility.As PlaTyPus does not detect insertions-deletions SNPs ("indels"), the Integrated Genome Viewer (IGV) software (Broad Institute) was used to manually inspect the gene sequences of all putative enzymes in the CoA biosynthetic pathway for indels.

Generation of PfPanK1 model
The structure of PfPanK1 minus its parasite specific inserts was predicted by homology modeling using the AMPPNP and pantothenate-bound human PanK3 structure (PDB ID: 5KPR 30 ) as a template.The model was generated using the one-toone threading module of the Phyre2 webserver (available at http://www.sbg.bio.ic.ac.uk/phyre2) 31 .

Confocal microscopy
Erythrocytes infected with trophozoite-stage 3D7 strain P. falciparum parasites expressing PfPanK1-GFP were observed and imaged either with a Leica TCS-SP2-UV confocal microscope (Leica Microsystems) using a 63 × water immersion lens or a Leica TCS-SP5-UV confocal microscope (Leica Microsystems) using a 63 × oil immersion lens.
To prepare coverslip-bound cells, parasite-infected erythrocytes (5 − 10% parasitaemia) were washed once (centrifuged at 500 × g, 5 min) and resuspended at approximately 2% haematocrit in phosphate buffered saline (PBS), before 1 − 2 mL was added to a polyethylenimine (PEI)-coated coverslip placed within a well of a 6-well plate.Plates were incubated with shaking for 15 min at room temperature, after which unbound cells were washed off the coverslips with PBS (2 mL per well, with a 2 min shaking incubation followed by aspiration).
For imaging of fixed cells, unstained coverslip-bound cells were fixed in 1 mL of PBS containing 4% (w/v) paraformaldehyde (Electron Microscopy Services) and 0.0075% (w/v) gluteraldehyde for 30 min at room temperature without shaking.The fixative was then removed and the coverslips washed in PBS three times as described above, before they were rinsed in water and dried.A drop of SLOWFADE (Invitrogen) containing the nuclear stain 4',6-diamino-2-phenylindole (DAPI) was added to the centre of the coverslip, which was then inverted onto a microscope slide and sealed with nail polish, before being used for imaging.
Live cells were stained with Hoechst 33258 nuclear stain and bound to a coverslip for imaging.Briefly, parasite-infected erythrocytes were prepared in PBS (~4% haematocrit) as described above, pelleted (2,000 × g, 30 s), and resuspended in PBS containing 20 g/mL Hoechst 33258.Cells were incubated in the dye for 20 min at room temperature with shaking, pelleted and washed three times with PBS (2,000 × g, 30 s), before being resuspended in PBS (~2% haematocrit) and bound to PEI-coated coverslips as described above.Shortly prior to imaging, 5 − 10 L of PBS was added to the centre of the coverslip, before it was inverted onto a microscope slide and sealed with nail polish.

Attempted disruption of Pfpank1
The PfPanK1 disruption plasmid, ∆Pfpank1-pCC-1, was transfected into wild-type 3D7 strain P. falciparum, and positive transfectants were selected as described earlier.P. falciparum parasites have previously been shown to survive equally well in a pantothenate-free complete RPMI 1640 medium supplemented with ≥100 M CoA as compared to standard complete medium, consistent with them having the capacity to take up exogenous CoA, hence bypassing the need for any PfPanK activity 7 .Therefore, to support the growth of any Pfpank1 gene-disrupted parasites generated with the ∆Pfpank1-pCC-1 construct, parasites were continuously maintained in complete medium supplemented with 100 M CoA following transfection.WR99210resistant ∆Pfpank1-pCC-1-transfectant parasites were subsequently maintained in the absence of WR99210 for three weeks to promote the loss of episomal copies of the construct.To select for parasites that had integrated the hdhfr positive selection cassette into the genome, WR99210 (10 nM) was re-introduced to predominantly ringstage ΔPfpank1-pCC-1-transfectant cultures.Complete medium and WR99210 were replaced daily after the re-introduction of WR99210, and fresh erythrocytes added when appropriate.Parasites from this culture (1 cycle off and on WR99210) were then either subjected to negative selection or maintained in the absence of WR99210 for a second three-week period to promote further loss of episomal copies of the ΔPfpank1-pCC-1 construct.The second WR99210 cycle was carried out exactly as the first.
Negative selection was performed to isolate parasites in which the hdhfr selection cassette of the ΔPfpank1-pCC-1 construct had been integrated into the genome by double crossover homologous recombination.In these parasites, the Scfcu gene that confers sensitivity to 5-FC should have been lost.Negative selection was initiated with parasites that had been through either 1 or 2 rounds of WR99210 cycling.5-FC (232 nM) was introduced to cultures of predominantly trophozoite-stage parasites, while selection with WR99210 (10 nM) was maintained and the medium was replaced daily.When parasites were no longer observable in Giemsa-stained culture smears, the medium (along with WR99210 and 5-FC) was replaced every second day and fresh erythrocytes (100 L) were added once a week.Once WR99210-and 5-FC-resistant parasites were observed in culture, the medium (containing WR99210 and 5-FC) was replaced daily, with fresh erythrocytes added as appropriate.

Southern blot analysis
To identify transfectants in which the hdhfr selection cassette of the ΔPfpank1-pCC-1 construct had been successfully integrated into the Pfpank1 gene locus, gDNA samples were extracted from saponin-isolated, trophozoite-stage WR99210-and 5-FC-resistant parasites as described above.These gDNA samples (~2 g) were digested with the restriction enzyme AflII (New England Biolabs), before being analysed by southern blotting using the digoxigenin (DIG) system (Roche) according to the Roche DIG Applications Manual for Filter Hybridisation.

[ 14 C]Pantothenate phosphorylation by parasite lysate
The phosphorylation of [ 14 C]pantothenate by parasite lysates prepared from the Parent and mutant clonal lines was measured using Somogyi reagent (which precipitates phosphorylated compounds from solution) as outlined previously 32 , with some modifications.In general, each reaction contained a final concentration of 50 mM Tris-HCl (pH 7.4), 5 mM ATP, 5 mM MgCl 2 and 2 M (0.1 Ci/mL) [ 14 C]pantothenate (American Radiolabeled Chemicals).The phosphorylation reactions were initiated by the addition of parasite lysate (at a concentration corresponding to ~1.0 × 10 7 cells/mL) and were maintained at 37 °C throughout the experiment.For each condition, a reaction with identical components to the corresponding test reaction but lacking parasite lysate was prepared and this served as the negative (no phosphorylation) control.Reactions were terminated by transferring 50 L aliquots (in duplicate) into wells of a 96-well white polystyrene microplate with either 0.45 m polypropylene filter bottom wells and short drip directors (Whatman), or 0.2 m hydrophilic PVDF membrane filter bottom wells (Corning), which had been pre-loaded with 50 L 150 mM barium hydroxide.To determine total radioactivity, 50 L aliquots of each phosphorylation reaction were transferred, in duplicate, and mixed thoroughly with 150 L Microscint-40 by pipetting the mixture at least 50 times in the wells of an OptiPlate-96 microplate (Perkin-Elmer).All of these samples were then processed, measured and analysed as described previously 32 .
Activity profiles of the PfPanK enzymes from the different mutant clones and the Parent line were determined across the following ranges of pantothenate concentrations, with the range of [ 14 C]pantothenate concentrations in brackets: 0.1 -2 M (0.005 -0.1 Ci/mL) for the Parent, 3.125 -200 M (0.1 Ci/mL) for PanOH-A and (0.156 -0.313 Ci/mL) PanOH-B, and 6.25 -800 M (0.3 -0.313 Ci/mL) for CJ-A.The pantothenate phosphorylation reactions in these kinetic experiments were set up as described previously 32 , with the following minor modifications.Each mL of the reactions contained lysates prepared from ~1.0 × 10 8 cells.All of the reactions were designed such that the amount of pantothenate phosphorylated by the lysate increased linearly with time during the experiment (15 -40 min for Parent, 13 -90 min for PanOH-A, 0.5 -4.2 h for PanOH-B and 1 -28 h for CJ-A).The initial rate of each reaction was determined from the gradient of its individual linear regression.These rates were then plotted as a function of pantothenate concentration, and the Michaelis-Menten equation " = ()

3,)
" was fitted to the data, where  represents V 0 ,  represents V max ,  represents K m and  represents substrate concentration.The Michaelis constant (K m ) and maximal velocity (V max ) parameters of the PfPanK variants were then determined from each fitted curve.The specificity constant of each PfPanK variant was calculated by dividing its V max value by the corresponding K m value.The relative specificity constant was in turn calculated by dividing the specificity constant value obtained for each variant in each independent repeat to the average Parent specificity constant value.
Pantothenate phosphorylation experiments were also performed in the presence of antiplasmodial pantothenate analogues to evaluate what effects the different PfPanK1 mutations have on the inhibitory activities of these analogues.The reactions (each containing lysates equivalent to 5 × 10 7 cells/mL and 0.1 Ci/mL of [ 14 C]pantothenate) were set up essentially as described previously 32 and were incubated at 37 °C for 10 min (PanOH-A), 40 min (Parent), 2 h (PanOH-B) or 5 h (CJ-A), during which the amount of pantothenate phosphorylated increased linearly with time in the absence of analogue.The pantothenate analogues were dissolved in water or DMSO, with the highest concentration of DMSO not exceeding 0.1% v/v.The amount of pantothenate phosphorylated in each reaction was expressed as a percentage of total pantothenate phosphorylation (wells without any test analogue).The data was then plotted as a function of analogue concentration and a suitable sigmoidal curve (either  = 0 +

Metabolism of N5-trz-C1-Pan
Cultures of predominantly trophozoite-stage P. falciparum-infected erythrocytes (Parent line) were concentrated to > 95% parasitaemia using magnet-activated cell sorting as described elsewhere 33 .Following a 1 h-recovery at 37 °C in complete medium and an atmosphere of 96% nitrogen, 3% carbon dioxide and 1% oxygen, N5trz-C1-Pan (1 M) or an equivalent volume of the solvent control (0.01% v/v DMSO) was added in triplicate or quadruplicate to suspensions of the P. falciparum-infected erythrocytes in complete medium.After a 4 h exposure to N5-trz-C1-Pan and/or DMSO at 37 °C (again under an atmosphere of 96% nitrogen, 3% carbon dioxide and 1% oxygen), 3 × 10 7 cells from each sample were pelleted by centrifugation at 650 × g for 3 min.The supernatant was discarded and the cells washed in ice-cold culture medium (1 mL, 650 × g, 3 min, 4 °C) followed by ice-cold PBS (500 L, 650 × g, 3 min, 4 °C).To extract metabolites, the washed cells were resuspended in ice-cold methanol containing internal standards (1 M Tris and 1 M CHAPS) and incubated on a vortex mixer for 1 h at 4 °C.The samples were clarified by centrifugation (at 17,000 × g, 10 min, 4 °C) before being processed for liquid chromatography-mass spectrometry (LC-MS) analysis.
Metabolite samples were analysed by LC-MS, using a Dionex RSLC U3000 LC system (ThermoFisher) coupled with a high-resolution, Q-Exactive MS (ThermoFisher), as described previously 34 .Briefly, samples (10 L) were separated over a ZIC-pHILIC column (5 m, 4.6 × 150 mm; Merck) using 20 mM ammonium carbonate and acetonitrile as the mobile phases.The Q-Exactive MS operated at a mass resolution of 35,000 from m/z 85 to 1,050 and was fitted with a heated electrospray source that switched between positive and negative ionisation modes.Pooled technical replicates were analysed using data-dependent tandem mass spectrometry (MS/MS) to confirm the identity of unique features in downstream analysis.LC-MS data were analysed in a non-targeted fashion using the IDEOM workflow, as described elsewhere 35 .Unique features identified in N5-trz-C1-Pantreated samples were manually assessed by visualising high resolution accurate mass LC-MS data with Xcalibur Quanbrowser (ThermoFisher) software.

Statistical analysis
Statistical analysis of means was carried out with unpaired, two-tailed, Student's t test using GraphPad 6 (GraphPad Software, Inc) from which the 95% confidence interval of the difference between the means (95% CI) was obtained.All regression analysis was done using SigmaPlot version 11.0 for Windows (Systat Software, Inc).

Pfpank1 mutations mediate parasite resistance to PanOH and CJ-15,801
The 3D7 P. falciparum strain was cloned through limiting dilution, and a single parasite line (henceforth referred to as the Parent line) was used to generate all of the subsequent lines tested in this study (unless otherwise specified).This was done to ensure that all of the parasite lines generated during the course of this study would share a common genetic background.Using the Parent line, three independent drugpressuring cultures were set up (two with PanOH and one with CJ-15,801).When these parasites had attained approximately 8-fold decrease in sensitivity (~11 -13 weeks of continuous pressuring), they were subsequently cloned by limiting dilution and maintained in the absence of drug pressure.In this manner, three parasite clones were generated: PanOH-A and PanOH-B were generated from the two independent PanOH-pressured cultures while CJ-A was generated from the CJ-15,801-pressured culture.The clones are significantly resistant (95% confidence interval (CI) exclude 0) to the pantothenate analogues they were pressured with.The 50% inhibitory concentration (IC 50 ) values of PanOH against PanOH-A and PanOH-B, and the IC 50 value of CJ-15,801 against CJ-A are approximately 7 -8-fold higher than those measured against the Parent line (Figure 2 a & b and Table 1).Significant crossresistance towards the other pressuring analogue was observed for these clones, as compared to the Parent line (95% CI exclude 0).The PanOH-A and PanOH-B lines were found to be 4 -6-fold less sensitive to CJ-15,801 while CJ-A was 13-fold less sensitive to PanOH (Figure 2 a & b and Table 1).To ensure that the clones did not develop a general drug-resistance phenotype during the selection process, we tested them against chloroquine, an antiplasmodial with a mechanism of action that is unrelated to the parasite's CoA biosynthetic pathway 36 .We found that all of the drugpressured lines have chloroquine IC 50 values that are indistinguishable from that of the Parent line (Table 1).
The PanOH and CJ-15,801 resistance phenotypes observed in the clones was stable for several months of continuous culture in the absence of the pressuring analogue (³ 3 months), consistent with a genetic alteration in these parasites.To determine the mutation(s) responsible for these phenotypes, gDNA was extracted from each clone and subjected to whole genome sequencing.All of the drug-resistant clones were found to harbour a unique mutation in the putative pantothenate kinase gene, Pfpank1 (PF3D7_1420600), as shown in Table 2. Other non-synonymous mutations were detected for each clone (Table S2) but we did not find another gene that was mutated in all three clones.The mutation found in the Pfpank1 of PanOH-A results in the substitution of Asp507 for Asn.The other two drug-resistant clones have a mutation at position 95 of the protein: the Pfpank1 of PanOH-B harbours a deletion of the entire codon leading to a loss of the residue in the coded PfPanK1 protein, while the PfPanK1 of CJ-A has a Gly to Ala substitution.Since the structure of PfPanK1 has not yet been resolved, we generated a three-dimensional model in order to map the mutations within the enzyme.Figure 3 shows a PfPanK1 model structure (pink) based on the solved structure of human PanK3 in complex with adenylyl-imidodiphosphate (AMPPNP) and pantothenate (PDB ID: 5KPR), overlaid on this structure (blue).The spheres shown in the model indicate the relative positions of the mutated residues, while the bound AMPPNP and pantothenate indicate the active site of the enzymes.Although the mutations are far apart in the primary amino acid sequence of PfPanK1, they are positioned in closer proximity to each other in the folded protein and are situated adjacent to the active site.
To confirm that the resistance phenotypes observed for the clones are directly caused by the mutations in Pfpank1, each clone was transfected with an episomal plasmid (Pfpank1-stop-pGlux-1) that enables the parasites to express the wild-type copy of Pfpank1 (in addition to the endogenous mutated copy).These complemented lines are indicated with a superscripted "+WTPfPanK1".From Figure 4 (and Table S3), it can be observed that the complemented mutant clones (grey bars) are significantly less resistant to PanOH (Figure 4 S3).These data are consistent with the mutations observed in Pfpank1 being responsible for the resistance phenotype observed in the mutant clones.

Pfpank1 mutations impair parasite proliferation
To determine whether the Pfpank1 mutations impart a fitness cost to the parasite clones, we set up competition cultures, each by mixing an equal number of parasites from the Parent line and one of the mutant clonal lines, and maintained them under standard conditions for a period of 6 weeks (Figure 5 a).The sensitivity of each competition culture to PanOH was tested on the day the lines were mixed (Week 0) and again at the end of the 6-week period (Week 6).As expected, each Week 0 (dashed line) PanOH dose-response curve is between those obtained for the Parent and the respective mutant clone (dotted lines).A shift of the dose-response curve obtained at Week 6 (solid line) towards the dose-response curve of the Parent line would indicate that the mutant PfPanK1 imparts a fitness cost on the clone.As shown in Figure 5 b, the Week 6 curve for the PanOH-A competition culture only exhibited a marginal leftward shift from Week 0, whereas those for PanOH-B (Figure 5 c) and CJ-A (Figure 5 d) exhibited a more substantial shift, almost reaching the dose-response curve of the Parent line (dotted line, white circles).These results are consistent with the mutations at position 95 in the PfPanK1 of PanOH-B and CJ-A having a negative impact on the in vitro growth of the parasites.We also investigated the importance of PfPanK1 expression for parasite growth by attempting to disrupt the Pfpank1 locus in wild-type 3D7 parasites through homologous recombination (Figure S1 a).However, using southern blots, we failed to detect the presence of transfectants with the expected gene-knockout integration event (Figure S1 b), consistent with this gene being essential during the parasite's intraerythrocytic stage.

PfPanK1 is a functional pantothenate kinase located within the parasite cytosol
The phosphorylation of radiolabelled pantothenate by lysates prepared from each of the mutant clones and the Parent line was measured to determine if the mutations in the putative Pfpank1 gene affect PanK activity, thereby demonstrating that the gene codes for a functional PanK.As shown in Figure 6, at the end of the 75 min timecourse, the lysate prepared from PanOH-A phosphorylated approximately 3 × more [ 14 C]pantothenate than the lysate prepared from the Parent line, while the lysate of PanOH-B generated about 3 × less phosphorylated [ 14 C]pantothenate compared to the Parent line.By comparison, the lysate prepared from CJ-A only produced a small amount of phosphorylated [ 14 C]pantothenate in the same time period.The inset in Figure 6 more readily demonstrates that PanK activity can be detected in CJ-A lysates when the experiment is carried out in the presence of a 100-fold higher pantothenate concentration (200 M) and for an extended time (420 min).These observations provide strong evidence that the Pfpank1 gene codes for a functional PanK.To characterise further PfPanK1, we generated from the wild-type 3D7 strain a transgenic parasite line that episomally expresses a GFP-tagged copy of PfPanK1 in order to localise the protein within the parasite.We found that PfPanK1 is largely localised throughout the cytosol of trophozoite-stage parasites, and is not excluded from the nucleus (Figure 7).
To investigate further the effects of the PfPanK1 mutations present in the PanOH and CJ-15,801 resistant clones, we analysed the PanK activity profiles using lysates prepared from each mutant and the Parent, and determined their kinetic parameters from the Michaelis-Menten equation (Figure 8).The maximal velocity (V max ) of pantothenate phosphorylation by lysates prepared from PanOH-A, PanOH-B and CJ-A are significantly higher (95% CI exclude 0) than that of the Parent.The apparent pantothenate K m values of the mutant clones are 26 -609-fold higher (95% CI exclude 0) than that of the Parent line.Assuming that the enzyme concentration was identical across the different parasite lines when the lysates were generated, we calculated the PfPanK relative specificity constant for each parasite line.The relative specificity constant values indicate the catalytic efficiency of each variant of PfPanK relative to that of the Parent line.The relative specificity constant obtained for PanOH-A (0.74 ± 0.04, mean ± SEM) is not significantly different (95% CI include 0) from that of the Parent.However, those of PanOH-B (0.058 ± 0.004, mean ± SEM) and CJ-A (0.019 ± 0.005, mean ± SEM) are significantly lower (95% CI exclude 0).These data are consistent with all three PfPanK1 mutations observed reducing the enzyme's affinity for pantothenate, although the associated increase in the enzyme V max compensates for the reduced affinity: fully in the PanOH-A clone, but to a much lesser extent in PanOH-B and CJ-A (resulting in a 17-fold and 52-fold reduction in the enzyme's catalytic efficiency, respectively).

CJ-A requires a higher extracellular pantothenate concentration
An extracellular supply of pantothenate is essential for the in vitro proliferation of the intraerythrocytic stage of P. falciparum 8 .Given the impact that the PfPanK1 mutations have on PanK activity (Figures 8 and 9), we set out to determine whether a higher extracellular concentration of pantothenate is required to support the proliferation of the different mutant clones relative to that required by the Parent line.As observed in Figure 9 a, the proliferation of the Parent line (white circles) increased as the extracellular pantothenate concentration was increased, reaching the 100% control level (parasites maintained in the presence of 1 M pantothenate, the concentration of pantothenate in the RPMI medium used to maintain all of the parasite cultures) at approximately 100 nM.In order to compare the extracellular pantothenate requirement between the different lines, we determined the SC 50 (50% stimulatory concentration; i.e. the concentration of pantothenate required to support parasite proliferation to a level equivalent to 50% of the control level) values for the mutants (with and without complementation) and Parent.From Figure 9 b, it can be seen that the SC 50 values of PanOH-A and PanOH-B are not different from that of the Parent line (95% CI include 0).Conversely, as illustrated by the rightward shift in its dose-response curve (black diamonds, Figure 9 a), the pantothenate SC 50 of CJ-A is approximately 3-fold higher than that of the Parent (Figure 9 b; 95% CI = 2.7 to 30.9).Furthermore, consistent with the data from the complementation experiments (Figure 4), the SC 50 value of CJ-A +WTPfPanK1 is comparable to that of the Parent line and also the control line, Parent +WTPfPanK1 , indicating that the episomal expression of wild-type PfPanK1 is sufficient to reverse the phenotypic effects of the mutation.Previous work has shown that, in bacteria, pantothenamides are metabolised by the CoA biosynthetic pathway to form CoA antimetabolites 37 , consistent with PanK activity being important for metabolic activation of pantothenamides.Additionally, it has been reported recently that pantothenamides are also phosphorylated by the PanK in P. falciparum 38 , in line with their metabolic activation in bacteria.We therefore set out to determine whether the modified, pantetheinase-resistant, pantothenamides are metabolised and to what extent.In order to do so we exposed intact P. falciparuminfected erythrocytes to N5-trz-C1-Pan (at ~10 × the IC 50 for 4 h) and subjected lysates from the treated (and control) samples to untargeted LC-MS.Among the metabolites extracted from parasite-infected erythrocytes treated with N5-trz-C1-Pan (but not untreated control samples) were molecules with masses corresponding to  3, right side.All of the analogues tested are significantly less effective (95% CI exclude 0) at inhibiting pantothenate phosphorylation by lysates generated from the mutant clones compared to their ability to inhibit pantothenate phosphorylation by lysates prepared from the Parent line.The exception being N-PE-αMe-PanAm, which did not reach statistical significance when tested against lysate prepared from PanOH-A.Additionally, the effectiveness of the analogues at inhibiting of pantothenate phosphorylation by lysates prepared from the mutant lines all observe the following order: PanOH-A > PanOH-B > CJ-A.

Discussion
The putative Pfpank1 gene codes for a functional pantothenate kinase When PfPanK1 is compared to type II PanKs from other organisms in a multiple protein sequence alignment, the three nucleotide-binding motifs characteristic of this superfamily can be seen to be conserved in PfPanK1, consistent with it being a functional PanK 7 .However, biochemical confirmation of its putative function as a PanK has not been demonstrated.In this study, we show that mutations in the putative PfPanK1 lead to substantial changes in pantothenate kinase activity (Figure 8) providing, for the first time, biochemical evidence that the cytosolic PfPanK1 (Figure 7) is a functional pantothenate kinase.Furthermore, the fact that multiple independent experiments aimed at generating parasites resistant to PanOH and CJ-15,801 always selected for mutations in PfPanK1 (Table 2 and Table S2) is consistent with the kinase being the primary PanK involved in the metabolic activation of pantothenate analogues, at least during the intraerythrocytic stage.
Although it is clear that the PfPanK1 residues at position 95 and 507 are required for normal PfPanK1 activity (Figure 8), and the PfPanK1 model structure (Figure 3) shows that both residues are situated adjacent to the enzyme active site, their exact role(s) in modifying the activity of the protein is less obvious.The Gly residue at position 95 is conserved in eukaryotic PanKs and is the residue at the cap of the α2helix 7 in the inactive conformation of the protein (Figure S3 a).One possibility is that the change to Ala at this position could affect the structure of the helix and consequently the overall stability of the protein (at least when the protein is in the inactive state), as Gly has been shown to be much better than Ala at conferring structural stability when located at the caps of helices 39 .Alternatively, Gly residues have been shown to be present at a higher frequency in the active sites of some enzymes where they likely confer the flexibility to alternate between open and closed conformations 40 .Although the Gly95 residue is not part of the PfPanK1 active site per se, it is within close proximity to the site (Figure 3), and the α2-helix certainly undergoes a conformational change when PanK switches from the inactive conformation to the active one 30 .This conformational change is demonstrated in Figure S3 a, which shows an overlay of the human PanK3 crystal structures in the active 30 and inactive 41 state (Gly95 in PfPanK1 is equivalent to Gly117 in the human enzyme).It is worth noting that acetyl-CoA (an inhibitor of the enzyme) can only be accommodated in the binding site when the enzyme is in the inactive state, as when the enzyme is in the active state, the α2-helix encroaches on the space occupied by acetyl-CoA (Figure S3 a).This lends further support to the importance of this helix for the enzyme to transition from the inactive to the active state (and vice versa) and, therefore, to the role that Gly95 could play in this process.Either way, these suggestions may explain why a mutation at this position has a greater impact on PfPanK1 function than the mutation at position 507.The Asp residue at position 507 is replaced by a different amino acid (Glu) in most other eukaryotic PanKs, although they are both negatively-charged 7 .A substitution to the uncharged Asn could disrupt any important salt-bridges or hydrogen bonds with the residue at this position.In human PanK3, the amino acid equivalent to Asp507 is Glu354.In the enzyme's inactive state, Glu354 is within ionic bonding distance of Arg325, which in turn interacts with the 3'phosphate of acetyl-CoA (Figure S3 b) 41 and may therefore stabilise the inactive state of the enzyme.Conversely, in the enzyme's active state, Glu354 and Arg325 are not within ionic bonding distance (Figure S3 b).If Asp507 in PfPanK1 plays a similar role to that proposed for Glu354 in human PanK3, the change at position 507 to an Asn (abolishing the negative charge) could prevent stabilisation of the inactive state, providing an explanation for the increased activity observed in PanOH-A (Figure 6).Determining the crystal structure of PfPanK1 bound with pantothenate may provide a better understanding of the roles these residues play in PanK function.

PfPanK1 is essential for normal intraerythrocytic proliferation of P. falciparum
It has been established that almost all of the 4'-phosphopantothenate found in P. falciparum-infected erythrocytes is generated within the parasite by PfPanK as part of its metabolism into CoA 10 , which is in line with PfPanK activity being essential for the parasite's survival.In the present study, our inability to knock out PfPanK1 (Figure S1) is consistent with the protein being essential for the intraerythrocytic stage of P. falciparum, although we cannot exclude the unlikely possibility that the regions we targeted for the required double-crossover recombination event are genetically intractable.Our observation that clones PanOH-B and CJ-A, which harbour mutations at position 95 of PfPanK1, can be outcompeted by the Parent parasites in competition assays over approximately 20 intraerythrocytic cycles (Figure 5) is consistent with the mutations incurring a fitness cost.This, in turn, indicates that PfPanK1 is essential for normal parasite development, at least during the blood stage of its lifecycle.It was also found here that clone CJ-A requires an approximately 3-fold higher extracellular pantothenate concentration in order to survive (Figure 9), coinciding with this clone having the PfPanK with the highest K m .This is not surprising given the reported importance for the substrate concentration to exceed the enzyme K m for optimal enzyme efficiency 42,43 .More importantly, the requirement by this clone for a higher concentration of extracellular pantothenate is also congruent with PfPanK1 being essential for the normal development of P. falciparum during its asexual blood stage.
Our observation that PfPanK1 is essential for normal parasite growth during the blood stage is at odds with recent reports that show both PanK1 and PanK2 from Plasmodium yoelli and Plasmodium berghei are expendable during the blood stage of those parasites 44,45 .However, both of these murine malaria parasite species preferentially infect reticulocytes 46,47 .Unlike the mature erythrocytes preferred by P. falciparum, reticulocytes have been shown to provide a rich pool of nutrients for the parasite, allowing the murine parasites to survive metabolic or genetic changes that would have been deleterious in P. falciparum 48 .It is therefore conceivable that unlike the condition faced by P. falciparum, the reticulocyte-residing parasites are able to salvage sufficient CoA or CoA intermediates from the host cell for their survival, rendering the two PanK proteins dispensable during their intraerythrocytic stage, a possibility acknowledged by the authors of the P. berghei study 45 .

PanOH and CJ-15,801 share a common mechanism of action
We have presented data consistent with the observed PfPanK1 mutations being the genetic basis for the PanOH and CJ-15,801 resistance phenotypes observed in all of the drug-pressured clones we generated (Figure 4).As shown by the data presented in Figure 10, both PanOH and CJ-15,801 inhibited pantothenate phosphorylation by the mutated PfPanK1 proteins less effectively than their inhibition of pantothenate phosphorylation by the wild-type PfPanK1 (the order of their IC 50 values is Parent < PanOH-A < PanOH-B < CJ-A).Importantly, this order is also reflected in the level of resistance of the mutant clones to these two analogues, although the magnitude is not preserved (Figure 2 and Table 1).These data are consistent with PfPanK1 being involved in the antiplasmodial activity of these analogues either as a target or a metabolic activator.Previous studies have demonstrated that the antiplasmodial activity of both PanOH and CJ-15,801 involves the inhibition of pantothenate phosphorylation by PfPanK 9,15 .More specifically, PanOH has been shown to inhibit PfPanK-mediated pantothenate phosphorylation by serving as its substrate 49 .CJ-15,801 is also likely to be a substrate of PfPanK, especially since it has been shown to be phosphorylated by the S. aureus PanK 50 , another type II PanK 51 .In addition, the same study showed that the second enzyme in the CoA biosynthetic pathway, PPCS, subsequently accepts phosphorylated CJ-15,801 as a substrate, and performs the first step of the PPCS reaction (cytidylylation) on it.However, unlike what happens with 4'phosphopantothenate, this cytidylylated phospho-CJ-15,801 acts as a tight-binding, dead-end inhibitor of the enzyme 50 .A separate study also concluded that PanOH targets the PPCS enzyme in Escherichia coli and Mycobacterium tuberculosis 52 .Based on the data generated in this study and the published reports that PanOH and CJ-15,801 both inhibit PPCS in other systems, we propose a similar mechanism of action for these compounds in P. falciparum, whereby they are phosphorylated by PfPanK1 and subsequently block PfPPCS as dead-end inhibitors (Figure 11).The overlapping pattern of cross-resistance between the two compounds (Figure 2) is also in line with them having a similar mechanism of action.We propose that the observed resistance to PanOH and CJ-15,801 is due to the mutated PfPanK1 having a reduced capacity to phosphorylate these analogues relative to pantothenate.This would have the effect of reducing the amount of phosphorylated PanOH or CJ-15,801 generated relative to 4'-phosphopantothenate, thereby allowing the parasites to survive at higher concentrations of the drugs.Furthermore, our observation that the mutant clones have comparable levels of PanOH and CJ-15,801 resistance (Figure 2 and Table 1), despite having PfPanK1 proteins of vastly different efficiency (Figure 8), is likely due to the pathway flux control at the PfPPCS-mediated step in the CoA biosynthetic pathway of P. falciparum, as shown in a previous study 33 .

N5-trz-C1-Pan is converted into an antiplasmodial CoA analogue -N-PE-αMe-PanAm likely shares the same mechanism of action
N5-trz-C1-Pan and N-PE-αMe-PanAm are pantothenamide-mimics that harbour modifications to prevent them from being substrates of pantetheinase, thereby preventing their degradation: N-PE-αMe-PanAm is methylated at the α-carbon 21 while N5-trz-C1-Pan harbours a triazole instead of the labile amide 20 .Our LC-MS data clearly show that N5-trz-C1-Pan is converted into a CoA antimetabolite (Figure S2), and it is therefore likely to go on to inhibit CoA-utilising enzymes, killing the parasite.Such a mechanism has previously been put forward to explain the antibiotic activity of two prototypical pantothenamides (N5-Pan and N7-Pan) whereby the compounds are phosphorylated by PanK and subsequently metabolised by PPAT and DPCK to generate analogues of CoA (ethyldethia-CoA and butyldethia-CoA) 37,53,54 .These CoA analogues then mediate their antibacterial effect/s primarily by inhibiting CoA-requiring enzymes and acyl carrier proteins 37,53,54 .Similarly, phospho-N5-trz-C1-Pan is not expected to interact with PfPPCS because it lacks the carboxyl group (Figure 1) required for the nucleotide activation by nucleotide transfer 50 .It is therefore expected to bypass the PfPPCS and PfPPCDC steps of the CoA biosynthetic pathway on its way to being converted into the CoA antimetabolite version of N5-trz-C1-Pan (Figure 11).Furthermore, the antiplasmodial activity rank order of N5-trz-C1-Pan against the various mutant clones is very similar to that of N-PE-αMe-PanAm -with PanOH-A being hypersensitive to both compounds, CJ-A resistant to both and PanOH-B, by comparison, exhibiting only small changes in sensitivity to the two compounds (Figure 10  The order of antiplasmodial activity of N5-trz-C1-Pan and N-PE-αMe-PanAm against the mutant clones can be explained on the basis of (i) the difference in the rate of PfPanK1 activity in the various clones at the concentrations of pantothenate and N5trz-C1-Pan / N-PE-αMe-PanAm used (Figure 6), (ii) the fact that the PfPPCSmediated step imposes pathway flux control 33 and (iii) the fact that this pathway flux control is bypassed by N5-trz-C1-Pan (and almost certainly also by N-PE-αMe-PanAm) en route to its conversion into a CoA antimetabolite.As seen in Figure 6, in the presence of 2 M pantothenate (a similar concentration to the 1 M present in the antiplasmodial assay), the pantothenate phosphorylation rate of the different clones has the following rank order: PanOH-A > Parent > PanOH-B > CJ-A, approximately the inverse of the antiplasmodial IC 50 values of N5-trz-C1-Pan and N-PE-αMe-PanAm (described above).Therefore, PanOH-A, for example, would be expected to generate more 4'-phosphopantothenate and phosphorylated N5-trz-C1-Pan (or N-PE-αMe-PanAm), based on the assumption that the mutation also leads to increased phosphorylation activity towards the pantothenate analogues (point i).Whilst the increased levels of 4'-phosphopantothenate would not be expected to result in a concomitant increase in CoA (due to the pathway flux control at PfPPCS; point ii), the increased production of phospho-N5-trz-C1-Pan would result in increased levels of the N5-trz-C1-Pan CoA antimetabolite as the flux control step is bypassed (point iii).This would immediately explain the increased level of sensitivity observed by PanOH-A to both N5-trz-C1-Pan and N-PE-αMe-PanAm and also the sensitivity rank order of the other parasite lines.
In conclusion, our study confirms for the first time that PfPanK1 functions as the active pantothenate kinase in the asexual blood stage of P. falciparum.Our data show that the sites of mutation in PfPanK1 reported here are important residues for normal PfPanK function and are essential for normal intraerythrocytic parasite growth, although further structural and functional studies are required to elucidate their exact role(s).Furthermore, we propose that following phosphorylation by PfPanK1, PanOH and CJ-15,801 compete with 4'-phosphopantothenate and serve as dead-end inhibitors of PfPPCS (depriving the parasite of CoA).In contrast, N-PE-αMe-PanAm and N5-trz-C1-Pan are further metabolised (by PfPPAT and PfDPCK) into CoA analogues that kill the parasite by inhibiting CoA-utilising metabolic processes.Finally, we provide the first genetic evidence consistent with pantothenate analogue activation being a critical step in their antiplasmodial activity.
) * or  = () * + * ,) * , where  represents percentage pantothenate phosphorylation, 0 represents minimum percentage pantothenate phosphorylation,  represents the range of percentage pantothenate phosphorylation,  represents the Hill coefficient,  represents the IC 50 value and  represents analogue concentration) fitted to determine the IC 50 of each analogue.
a) and CJ-15,801 (Figure 4 b) compared to the noncomplemented mutant clones (black bars; 95% CI exclude 0).As expected, the relative sensitivity of the mutant clones to chloroquine is unchanged by the presence of the PfPanK1-encoding plasmid (Figure 4 c).Transfection of the PfPanK1-encoding plasmid into the Parent line did not alter its sensitivity to PanOH, CJ-15,801 or chloroquine (Table N5-trz-C1-Pan and N-PE-αMe-PanAm have a different mechanism of action to PanOH and CJ-15,801 To determine whether the resistance of the clones to PanOH and CJ-15,801 extends to other pantothenate analogues, we tested the mutant clones against the two recently-described, modified pantothenamides with potent antiplasmodial activities, namely N5-trz-C1-Pan 20 and N-PE-αMe-PanAm 21 .We found that PanOH-A is 3-fold more sensitive to N5-trz-C1-Pan, PanOH-B is 2-fold more resistant and CJ-A is 9-fold more resistant when compared to the Parent line (95% CI exclude 0; Figure 10 a and Table 3, left side).Similarly, we found that relative to the Parent line, PanOH-A was more sensitive to N-PE-αMe-PanAm (~2-fold), while CJ-A is 2-fold more resistant (95% CI exclude 0).The sensitivity of PanOH-B to N-PE-αMe-PanAm was statistically indistinguishable from that of the Parent line (95% CI = -0.046 to 0.005; Figure 10 b and Table 3, left side).These results indicate that PfPanK1 can influence the sensitivity of the parasite to multiple antiplasmodial pantothenate analogues.Remarkably, the mutation at position 507 of the PfPanK1 in PanOH-A makes the parasite resistant to the antiplasmodial activity of certain pantothenate analogues (PanOH and CJ-15,801) whilst at the same time hyper-sensitises the parasite to pantothenate analogues of a different class (modified pantothenamides, N5-trz-C1-Pan and N-PE-αMe-PanAm).
phosphorylated N5-trz-C1-Pan ([M-H] -m/z 378.1668), a dephospho-CoA analogue of N5-trz-C1-Pan ([M-2H] 2-m/z 353.6099) and a CoA analogue of N5-trz-C1-Pan ([M-H] - m/z 787.1858) as shown in Figure S2.This is consistent with N5-trz-C1-Pan being metabolised within infected erythrocytes to generate a CoA antimetabolite.Lastly, we investigated the ability of the different pantothenate analogues to inhibit the phosphorylation of [ 14 C]pantothenate by parasite lysates prepared from the various mutant clones.These data are shown in Figure 10 c -f and Table a and b) -and is starkly different to those of PanOH and CJ-15,801 (Figure 2).These are congruent with (i) the antiplasmodial mechanism of action of N-PE-αMe-PanAm being similar to that of N5-trz-C1-Pan and (ii) the antiplasmodial mechanism of action of N-PE-αMe-PanAm and N5-trz-C1-Pan being different to that of PanOH and CJ-15,801.

Figure 1 .
Figure 1.Structures of pantothenate and the pantothenate analogues used in this study.

Figure 2 .Table 1 . 2 Table 2 .
Figure 2. Percentage proliferation of parasites from the Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) lines in the presence of (a) PanOH or (b) CJ-15,801.Drug-pressured lines were generated by exposing Parent line parasites to 11 − 13 weeks of continuous drug-pressuring with either PanOH (for PanOH-A and PanOH-B) or CJ-15,801 (for CJ-A), followed by limiting dilution cloning.Values are averaged from ≥ 4 independent experiments, each carried out in triplicate.All error bars represent SEM.Error bars are not shown if smaller than the symbols.

Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 . 1 Figure 10 .Table 3 .
Figure 3.A three-dimensional homology model of the PfPanK1 protein (pink) based on the solved structure of human PanK3 (PDB ID: 5KPR), overlaid on the human PanK3 structure in its active conformation (blue), with an ATP analogue (AMPPNP; carbon atoms coloured green) and pantothenate (carbon atoms coloured yellow) bound.Red spheres indicate the residues (G95 and D507) affected by the mutations in the parasite proteins.Human PanK3 has been shown to exist as a dimer.Here, individual monomers are shown in different shades of pink and blue.

Figure 11 .
Figure 11.The proposed antiplasmodial mechanisms of action of various pantothenate analogues in Plasmodium falciparum.PanOH and CJ-15,801 (orange) are phosphorylated by PfPanK and their phosphorylated forms accumulate (indicated by the bigger font) and then competitively inhibit PfPPCS.The step mediated by PfPPCS has been hypothesised to be a pathway flux control point (illustrated by the red box) in the CoA biosynthesis pathway.On the other hand, we propose that although N-PE-αMe-PanAm and N5-trz-C1-Pan (cyan) are also phosphorylated by PfPanK, they then bypass PfPPCS and PfPPCDC (as shown previously in bacteria) and rejoin the pathway as substrates of PfPPAT and then PfDPCK, resulting in the generation and accumulation of CoA antimetabolites.These CoA antimetabolites exert their antiplasmodial activity by inhibiting CoA-dependent processes.

Figure S1 .Figure S2 . 3 Figure S3 .
Figure S1.The attempt at knocking-out Pfpank1 in wild-type 3D7 parasites through homologous integration.(a) Schematic representations of the DPfpank1-pCC-1 construct and the wild-type Pfpank1 gene locus before and after a gene disruption attempt.These gene disruptions were expected to involve either a homologous double crossover integration of the hdhfr cassette of DPfpank1-pCC-1 or a 5'/3' single crossover homologous integration of the DPfpank1-pCC-1 construct.The positions of AflII restriction sites are indicated.The 5' Pfpank1 and 3' Pfpank1 homologous flanks are indicated by translucent blue boxes.(b) Southern blot of AflII-digested gDNA extracted from wild-type parasites and from DPfpank1-pCC-1-transfectants resistant to WR99210 or both WR99210 and 5-fluorocytosine (5-FC) after one or two rounds of WR99210 cycling.(i) and (ii) represent independently selected drug-resistant cultures.The blot was probed with the 3' Pfpank1 flank (as indicated by the black bar in (a)).The probe hybridised to fragments that correspond to the 3D7 wild-type (3.3 kb) and the plasmid (8.9 kb).The fragment that is consistent with a homologous double crossover disrupted locus (3.8 kb) was not detected in either independent culture.hdhfr: human dihydrofolate reductase -conveys resistance to WR99210.Scfcu: Saccharomyces cerevisiae cytosine deaminase/phosphoribosyl transferase -conveys sensitivity to 5-FC.