Pyrazoleamide-treated P. falciparum cultures

Extended culturing of P. falciparum within the presence of a sublethal pyrazoleamide dose selects for resistance-conferring mutations in its metabolic enzyme PfATP4^7,10. To know the preliminary metabolic responses of P. falciparum within the presence of a pyrazoleamide, we first carried out self sufficient experiments in quadruplicate the usage of more than a few doses of PA21A092 to decide the optimum focus of the drug that can impact parasite enlargement with out killing the parasites upfront (between 30 and 70% inhibition can be optimum). We handled extremely synchronized cultures of ring-stage parasites with other concentrations of PA21A092 for 40 h. We then assessed the facility of those parasites to duplicate via slicing the cultures via an element of 10 with recent erythrocytes and drug-free medium. Determine 1A displays that 12 nM PA21A092 achieves the specified stage of inhibition, subsequently, we used this focus for data-collection experiments.

Experiments to decide a sublethal dose of PA21A092, a pyrazoleamide drug. (A) Parasitemia at ~ 70 h of experiment after medication with the notated doses of PA21A092. We handled extremely synchronized parasite cultures with 0, 8, 12, 16, or 24 nM PA21A092 in quadruplicate-independent experiments for 40 h. We then assessed the facility of those parasites to duplicate via slicing the cultures via an element of 10 with recent erythrocytes and drug-free medium. The mistake bars denote one usual deviation from imply parasitemia. (B) Inhibition noticed within the 4 pyrazoleamide-treated cultures used for transcriptomic and metabolomic pattern series. The ordinate displays the share of inhibition noticed in every mirror after medication with 12 nM PA21A092. For every mirror, the mistake bars denote variability in triplicate flow-cytometry measurements. The horizontal dotted line denotes the common of proportion inhibition noticed throughout all measurements. (C) Consultant Giemsa-stained pictures of untreated (CTL) and drug-treated (PYZ) parasites on the 0-, 8-, 16-, 24-, 32-, and 40-h time issues of the experiment. Be aware that the 0-h time level represents the illusion of > 95% rings within the parasite tradition and corresponds to ~ 0–2 h of the experiment. Presently (0 h), we added recent medium containing the drug (PYZ) or no drug (CTL). (D) Choice of merozoites in keeping with schizont within the untreated and drug-treated parasite cultures on the 40-h time level of the experiment. We discovered {that a} 40-h medication via 12 nM PA21A092 (PYZ) led to an important lower in daughter merozoites in keeping with schizont. Magenta dotted strains constitute the median of fifty observations underneath untreated (CTL) and drug-treated prerequisites. The black dotted strains denote the 25% and 75% quartiles. The asterisks denote that the variation between median collection of merozoites/schizont underneath the 2 prerequisites is statistically vital (p < 0.0001; Wilcoxon rank-sum take a look at).

Previous to pattern series for transcriptomic and metabolomic information profiling, we once more quantified the volume of parasite enlargement inhibition and in addition monitored the morphologies of untreated and drug-treated parasites throughout the IDC. Determine 1B quantifies the inhibition of parasite proliferation, as a proportion of parasitemia noticed in untreated parasite cultures, confirming that the recognized PA21A092 dosage led to an inhibition throughout the desired vary (30–70%).

To observe morphological adjustments in parasites because of the drug medication, we carried out Giemsa staining each 8 h throughout the IDC. Determine 1C displays consultant pictures of parasite-infected erythrocytes within the absence and presence of 12 nM PA21A092 at 0, 8, 16, 24, 32, and 40 h of the experiment. Those pictures counsel that the recognized dose of PA21A092 didn’t perturb parasite morphologies considerably till the 40-h time level of the experiment, then again, it nonetheless led to ~ 35% of the drug-treated parasites to fail to proliferate within the drug-free medium after 40 h (Fig. 1B). We additionally quantified the collection of merozoites in keeping with schizont on the 40-h time level underneath untreated (CTL) and drug-treated (PYZ) prerequisites. Determine 1D displays that 12 nM PA21A092 led to an important lower within the collection of daughter merozoites in keeping with schizont (CTL: 12; PYZ: 10), suggesting that the drug negatively affected parasite construction.

Transcriptomic adjustments because of a sublethal dose of a pyrazoleamide

To spot main coherent alterations in gene-expression information from the handled parasites, we carried out hierarchical clustering research (HCA). Determine 2A illustrates the common expression of five,851 parasite genes throughout the IDC within the absence and presence of PA21A092. We didn’t in finding any main alteration within the transcription program of the drug-treated parasites. To quantify the impact of PA21A092 medication on P. falciparum IDC development, we when put next gene-expression profiles from untreated and drug-treated parasites with the ones from hourly sampled gene-expression information spanning all of the IDC of P. falciparum Dd2 pressure¹³.

International research of transcriptomic information from untreated and pyrazoleamide-treated P. falciparum. (A) Hierarchical clustering research of transcriptomic information from the untreated and handled parasites. Every row corresponds to a parasite gene, and every column represents a sampled time level of the intraerythrocytic construction cycle (IDC). The gene-expression stage, underneath a given perturbation (untreated or drug handled), at a specific time level represents the common of 4 technical replicates. To facilitate inter-study comparisons, we normalized temporal expression of every gene via its 0-h expression worth (vertical grey bars). The untreated and drug-treated parasites seem to have equivalent expressions throughout the IDC, indicating a loss of alteration within the parasite’s gene-expression program because of drug medication. (B) IDC development of untreated and drug-treated parasites. To quantify IDC development, we computed the Pearson correlation between gene-expression profiles of every mirror from this be taught and the ones from the hourly sampled information of Llinas et al.¹³. The closed circles denote the imply of extreme correlation between the 2 datasets (untreated or handled) on the time issues indicated at the x-axis. (C) Moderate fold exchange in gene expression relative to its expression at 0 h (FC_0h) in untreated and drug-treated parasites. Plasmodium genes proven with crimson circles have fourfold (log₂ scale) other FC_0h after medication as in comparison to untreated prerequisites, whilst genes proven with grey circles have a lower than twofold (log₂ scale) distinction; all different genes are proven the usage of inexperienced circles. The open and closed circles denote genes with unknown and identified (or putative) purposes, respectively. h.p.i. hour put up an infection, PA21A092 a pyrazoleamide drug, PfEMP1 Plasmodium falciparum erythrocyte membrane protein 1.

Determine 2B demonstrates that IDC development of the drug-treated parasites (crimson curve) is most commonly related with that of the untreated parasites (black curve) for the primary 24 h, however differing after 32 h into the IDC. Since the expression profile of particular genes could have been affected because of the medication, we subsequent quantified the common exchange in expression, relative to 0 h (FC_0h), of every gene underneath untreated and drug-treated prerequisites. Determine 2C displays considerably altered genes in line with drug medication the usage of crimson circles and inexperienced circles. We discovered that most effective 8 genes (out of five,851) had FC_0h values that had been fourfold (log₂ scale) other in magnitude after medication as in comparison to untreated prerequisites (Fig. 2C, crimson circles). Of those 8 genes, 5 encode paralogs of 18S ribosomal RNA, two encode the erythrocyte membrane protein (PfEMP1), and one encodes a conserved Plasmodium protein of unknown serve as (PF3D7_1142900). The PfEMP1 proteins are encoded via a circle of relatives of roughly 60 var genes¹⁴. A person parasite most effective expresses a specific var gene at a time, whilst holding all different genes transcriptionally silent¹⁴. Due to this fact, the dramatically upper expression of 1 gene and decrease expression (PfEMP1, Fig. 2C) of the opposite is most probably because of var gene switching and now not a results of pyrazoleamide medication.

To additional establish treatment-specific perturbations within the expression of Plasmodium genes, we subsequent tested the useful annotation of the genes with a ≥ twofold distinction in FC_0h values (depicted via inexperienced circles in Fig. 2C), and recognized 66 (out of a complete of 102) genes with identified or putative purposes (Desk S1: Sheet 1). Amongst those had been carbonic anhydrase (PF3D7_1140000) and aquaglyceroporin (PF3D7_1132800). Carbonic anhydrase converts water and carbon dioxide into carbonic acid and protons¹⁵, whilst aquaglyceroporin is a water and ammonia transporter of the parasite¹⁶. The actions of carbonic anhydrase¹⁷ and aquaglyceroporin¹⁶ are pH dependent. PfATP4-associated medication at deadly concentrations are identified to change pH homeostasis in malaria parasites, and those effects counsel {that a} sublethal dose of a pyrazoleamide drug might also purpose alterations within the pH of the handled parasites.

To represent the consequences of PA21A092 medication on Plasmodium body structure, we carried out an enrichment research the usage of gene units to be had within the Malaria Parasite Metabolic Pathway (MPMP) database¹⁸. We integrated prior to now revealed transcriptomic information acquired from P. falciparum maintained underneath 3 other perturbations^11,12,19 to isolate treatment-specific results. Desk 1 lists gene units altered most effective in pyrazoleamide-treated parasites. Our research printed a considerable downregulation of genes encoding protein kinases (Cellular cycle legislation in Desk 1) and phosphatases (Phosphatase-related proteins in Desk 1). Each protein kinases and phosphatases play crucial roles in phosphorylation and dephosphorylation of regulatory proteins throughout the IDC²⁰.

Despite the fact that indirectly associated with our findings, a lack of dephosphorylation processes in P. falciparum leads to blockage of parasite egress throughout IDC²¹, suggesting {that a} sublethal dose of PA21A092 may also be affecting parasite egress throughout the IDC. Curiously, a prior be taught confirmed untimely schizogony-like occasions in parasites uncovered to deadly doses of PfATP4 inhibitors⁶. With a dose of 12 nM PA21A092, we didn’t apply any untimely unencumber of merozoites at any of the time issues in our be taught. Desk S2 items the share of altered genes in the entire gene units acquired from the MPMP database¹⁸.

Metabolomic information from untreated and pyrazoleamide-treated cultures

We carried out HCA at the metabolomic information from untreated and drug-treated cultures of uninfected erythrocytes (uRBC) and parasite-infected erythrocytes (iRBC) to spot drug-induced alterations in P. falciparum metabolism. Determine 3A illustrates the common abundance of metabolites detected in quadruplicate samples of uRBC and iRBC cultures. Strangely, HCA printed uRBC metabolites with considerably other abundances underneath untreated and drug-treated prerequisites (annotated 1 and a couple of, Fig. 3A).

International research of metabolomic information from untreated and pyrazoleamide-treated cultures of uninfected and parasite-infected erythrocytes. (A) Hierarchical clustering research of metabolomic information from untreated and drug-treated cultures of uninfected (uRBC) and parasite-infected (iRBC) erythrocytes. Every row denotes a metabolite and every column represents a sampled time level throughout the experiment. The abundance of a metabolite at every time level, underneath a specific situation (uRBC or iRBC) and perturbation (untreated or drug handled), represents the common of 4 technical replicates. To facilitate comparisons between other prerequisites and perturbations, we normalized the abundance of every metabolite via its abundance at 0 h. The vertical grey bars denote the 0-h time level underneath every situation and perturbation. Clades annotated “1” and “2” counsel alteration in abundance of uRBC metabolites because of the drug medication. (B) Fold exchange, relative to 0 h (FC_0h), in abundance of uRBC metabolites underneath untreated and drug-treated prerequisites. The uRBC metabolites with |Δ|≥ 4 are proven the usage of crimson circles, the place Δ = ({textual content{F}}{textual content{C}}_{textual content{0h}}^{textual content{PYZ}}/{textual content{F}}{textual content{C}}_{textual content{0h}}^{textual content{CTL}}). ({textual content{F}}{textual content{C}}_{textual content{0h}}^{textual content{PYZ}}) and ({textual content{F}}{textual content{C}}_{textual content{0h}}^{textual content{CTL}}) denote FC_0h of an uRBC metabolite within the presence and shortage of the drug, respectively. The grey circles denote metabolites with |Δ|< 2, whilst inexperienced circles constitute metabolites with |Δ|≥ 2 however lower than 4. The open and closed circles, respectively, denote metabolites that modify considerably and minimally over a 2-day length²². (C) Volcano plot appearing considerably altered metabolites (p < 0.001) because of parasite an infection in untreated cultures. The crimson markers denote considerably altered metabolites with FC_IDC ≤ 0.5, whilst inexperienced markers constitute considerably altered metabolites with FC_IDC ≥ 2; right here, FC_IDC denotes fold exchange in moderate abundance of a metabolite in iRBC cultures relative to uRBC cultures. (D) Volcano plot appearing considerably altered metabolites (p < 0.001) because of parasite an infection in drug-treated cultures. The crimson and inexperienced markers, respectively, denote considerably altered metabolites with FC_IDC ≤ 0.5 and FC_IDC ≥ 2.

To quantify the impact of pyrazoleamide medication on uRBC metabolite abundances, we computed moderate fold exchange in abundance of every metabolite relative to 0 h (FC_0h) for the untreated and drug-treated datasets. Determine 3B displays metabolites with considerably other FC_0h between untreated and drug-treated prerequisites the usage of crimson and inexperienced circles. In a contemporary effort, we quantified the anticipated variance in abundance of uRBC metabolites detected at the same time as in distinct donors²². The usage of those anticipated variances in uRBC metabolite abundances²², we discovered that many of the considerably other metabolites, denoted via open crimson circles in Fig. 3B, tended to alter in abundance without reference to a perturbation. Deoxythreonate, proven the usage of closed crimson circles in Fig. 3B, used to be essentially detected in urine samples of people^23,24 and used to be now not detected robustly (uncooked depend > 1,000; see “Fabrics and techniques”) in our prior to now analyzed metabolomic datasets²². Moreover, Chen et al.²⁵ demonstrated that serum deoxythreonate ranges correlate with age of blood donors. We don’t seem to be acutely aware of the ages of the donors for the self sufficient experiments carried out underneath untreated and drug-treated prerequisites, however traditionally the age of the donors for our blood-collection protocol has been between 18 and 60 years.

As a result of uRBC metabolite abundances can range between other donors, we normalized the metabolomic information from untreated and drug-treated cultures via an interior usual (see “Fabrics and techniques”) previous to computing fold exchange in moderate abundance of metabolites because of parasite an infection (FC_IDC). Determine 3C,D, respectively, display metabolites in untreated and drug-treated cultures which are perturbed considerably (p < 0.001) because of parasite an infection. Deoxythreonate seems to be depressed (Fig. 3C) or increased (Fig. 3D) in untreated and drug-treated prerequisites, respectively, however that is in large part because of the adaptation in its abundance (Fig. 3B) within the uRBC cultures of the 2 self sufficient experiments.

Desk 2 lists FC_IDC values of considerably altered metabolites because of the pyrazoleamide medication. The presence of considerable abundance permutations of a selected metabolite in uninfected cultures can both conceal or reinforce any obvious impact in this metabolite because of the drug medication²². Due to this fact, along with acting two-way research of variance (ANOVA), we additionally computed z-scores of variation in abundances of uRBC metabolites, within the absence and presence of the drug, the usage of their anticipated permutations reported in Tewari et al.²². This allowed us to spot uRBC metabolites having considerably other (p < 0.001) variation in abundance as in comparison to their anticipated variation²². In Desk 2, we annotated such uRBC metabolites the usage of asterisks. For instance, creatinine and hippurate have FC_IDC values more than 3 within the presence of the pyrazoleamide drug, then again, the temporal abundance of each those metabolites within the drug-treated uRBC cultures used to be considerably other than their anticipated variation in uRBC cultures²². Curiously, hippurate is a blood metabolic biomarker of intestine microbiome variety in people²⁶.

The FC_IDC of sedoheptulose-7-phosphate, a pentose phosphate pathway (PPP) metabolite, used to be smaller within the drug-treated cultures than within the untreated cultures, suggesting a decrease manufacturing of diminished nicotinamide adenine dinucleotide phosphate (NADPH) because of the medication. The FC_IDC of lactate, a manufactured from the ATP-generating section of glycolysis, used to be upper within the drug-treated cultures as in comparison to the untreated cultures. To additional examine the impact of drug on glycolysis, we when put next the temporal profile of glucose and 5 glycolysis intermediates on the sampled time issues between the 2 prerequisites. We discovered that the abundance of those metabolites confirmed equivalent alterations as much as the 24-h time level, however the values differed significantly afterwards (Desk S3). For the untreated cultures, glycolytic intermediates are most effective 3% in their preliminary abundances on the 40-h time level, whilst for the drug-treated cultures, they’re 32% in their preliminary abundances, indicating reduced intake of those intermediate metabolites (Desk S3). In a similar way, glycolytic merchandise (pyruvate and lactate) reached 748% in their preliminary abundance within the untreated cultures, however most effective 274% in their preliminary abundance within the drug-treated parasite cultures. Taken in combination, those alterations counsel a modulation of carbohydrate metabolism in pyrazoleamide-treated parasites, which is in keeping with partial inhibition of parasite proliferation throughout the primary IDC.

Despite the fact that the tricarboxylic acid (TCA) cycle isn’t very important throughout the IDC²⁷, we discovered vital alterations in abundance of a number of TCA cycle metabolites (α-ketoglutarate, citrate, fumarate, and malate). We additionally discovered that FC_IDC of sphinganine-1-phosphate (Sa1P), an analogue of sphingosine-1-phosphate (S1P), within the handled cultures used to be virtually two times as huge as that within the untreated cultures. An larger abundance of S1P is related to an build up in glycolysis in iRBCs²⁸ and, therefore, rationalizes the roughly twofold build up in FC_IDC of lactate within the handled cultures as in comparison to the untreated cultures. Then again, those effects don’t provide an explanation for how an larger lactate abundance is related to a discounted PPP flux within the handled parasites.

Due to this fact, to spot mechanistic penalties of pyrazoleamide medication in P. falciparum, we built-in the time-resolved transcriptomic and metabolomic information from the handled parasites with a genome-scale metabolic mannequin of P. falciparum^11,12. In parallel, we additionally coupled transcriptomic and metabolomic information from untreated parasites with the metabolic mannequin. The model-predicted metabolism of untreated and drug-treated parasites allowed us to achieve further insights into pyrazoleamide-induced metabolic changes in P. falciparum.

Metabolism of pyrazoleamide-treated P. falciparum

The combination of condition-specific and time-resolved information (transcriptomic and metabolomic) with the P. falciparum metabolic community mannequin supplies a useful description of the adjustments happening in untreated and drug-treated parasites throughout the IDC. Determine 4A–F display charges of macromolecules synthesized throughout the IDC of untreated (black markers and curves) and drug-treated (crimson markers and curves) parasites. Our mannequin predicted an inhibition of RNA, protein, and polyamine synthesis within the handled parasites (Fig. 4A–C). In contrast, our mannequin predicted an build up in charges of lipid synthesis, cofactor synthesis, and inorganic ion era within the handled parasites (Fig. 4D–F).

Style-predicted charges of macromolecule syntheses in untreated and pyrazoleamide-treated P. falciparum. (A) Price of RNA synthesis. (B) Price of protein synthesis. (C) Price of polyamine synthesis. (D) Price of lipid synthesis. (E) Price of cofactor synthesis. (F) Price of inorganic ion era. The mistake bars denote one usual error of the imply for 10 simulations carried out after including Gaussian noise, with 0 imply and 10% usual deviation within the transcriptomic information underneath untreated and drug-treated prerequisites. CTL parasites maintained in natural RPMI medium, gDW gram dry weight of the parasite, PYZ parasites maintained in RPMI medium with a sublethal dose of a pyrazoleamide, RPMI, Roswell Park Memorial Institute.

To quantify internet accumulation or depletion of very important macromolecules and metabolites underneath each prerequisites, we numerically built-in their synthesis charges within the absence and presence of the drug, after which computed a ratio for every metabolite (and macromolecule) the usage of the values acquired for untreated and drug-treated parasites (denoted via Δm). Desk 3 lists Δm of choose very important macromolecules and metabolites. The mannequin predicted a internet accumulation of roughly 30% and 50% in DNA and phospholipids, respectively, within the handled parasites (Desk 3). By contrast to DNA and phospholipid accumulation, the mannequin predicted a internet depletion of roughly 40% and 15% in RNA and protein macromolecules, respectively, within the handled parasites (Desk 3). Desk S4 tabulates Δm of all very important macromolecules and metabolites.

Vaidya et al.¹⁰ discovered that medication of malaria parasites via deadly doses of pyrazoleamide compounds disrupts its sodium ion homeostasis, which is very important for phosphate uptake by way of PfPiT²⁹. Our mannequin predicted inhibition of PfATP4 and PfPiT actions within the pyrazoleamide-treated parasites (Fig. S1). As in comparison to untreated prerequisites, our built-in information and mannequin research predicted considerably much less efflux of cytosolic protons from the pyrazoleamide-treated parasites underneath steady-state prerequisites (Fig. S1). Curiously, a number of experiments^5,7,10 carried out the usage of deadly doses of sodium ion homeostasis disruptors have noticed an build up in cytosolic pH within the handled parasites. Be aware that our mannequin predictions don’t counsel that cytosolic pH decreases in parasites uncovered to sublethal doses of PA21A092, however reasonably point out that delivery of protons is minimum around the parasitic plasma membrane underneath steady-state prerequisites. In combination, those effects counsel an alteration within the homeostasis of sodium ions and protons in parasites handled with a sublethal dose of PA21A092.

Pyrazoleamide medication led to temporal upregulation in expression of the gene encoding aquaglyceroporin (Desk S1: Sheet 1), which transports ammonia (or water) around the parasite’s plasma membrane¹⁶, suggesting an accumulation of ammonia (or water) within the handled parasites. Consistent with those observations, our mannequin predicted an accumulation of ammonium, the ionized type of ammonia, within the handled parasites (Δm = 3.22, Desk 3). The PfPdx1 enzyme of P. falciparum synthesizes pyridoxal 5-phosphate (PLP) from ammonia, glyceraldehyde 3-phosphate, and ribose 5-phosphate³⁰, which could also be captured in our mannequin simulations (Δm = 1.76, Desk 3). Particularly, in parasites handled with a sublethal dose of PA21A092, the synthesis of PLP larger 30 h put up an infection, previous to which the PfPdx1 task used to be minimum as in comparison to the untreated parasites.

Metabolic penalties of a sublethal pyrazoleamide dose in P. falciparum

Determine 5 displays metabolic changes and variations in pyrazoleamide-treated P. falciparum as predicted via the built-in metabolic community mannequin. We discovered {that a} sublethal dose of PA21A092 considerably inhibited PfATP4 task throughout the overdue ring and early trophozoite phases of the IDC (Fig. S1). As a result of PfATP4 task maintains the sodium ion gradient around the P. falciparum plasma membrane⁵, those effects counsel that sublethal doses of pyrazoleamides additionally disrupt sodium ion homeostasis in P. falciparum. The mannequin simulations predicted an inhibition of amino acid uptake within the handled parasites. Despite the fact that P. falciparum obtains maximum amino acids from hemoglobin degradation, it is dependent upon isoleucine uptake from its host to care for enlargement throughout the IDC³¹. Accordingly, the relief of isoleucine uptake within the handled parasites is appropriate with the model-predicted 15% relief in protein synthesis of the handled parasites (Desk 3).

Results of pyrazoleamide medication in P. falciparum. Our built-in mannequin research suggests {that a} sublethal dose of pyrazoleamide (PYZ) led to a discount in PfATP4 flux, disrupting the homeostasis of sodium ions (Na⁺) and protons (H⁺) and, in the end, lowering the osmotic force. The mannequin additionally indicated diminished uptake of amino acids (AA), comparable to isoleucine, and a reduced price of protein synthesis in drug-treated parasites. We discovered that the parasites alter carbohydrate metabolism to provide an osmolyte (MI) that may counter the consequences of drug medication. Then again, this metabolic adjustment leads to reduced flux by way of PPP, because of this reducing NADPH and PRPP, and exacerbating the consequences of the drug via expanding oxidative pressure. Therefore, our effects counsel that downstream penalties of metabolic changes give a contribution to the efficacy of pyrazoleamides in P. falciparum. AA amino acid, ADP adenosine diphosphate, ATP adenosine triphosphate, G6P glucose 6-phosphate, GLC D-glucose, Hb hemoglobin, MI myoinositol, NADPH nicotinamide adenine dinucleotide phosphate, Pi inorganic phosphate, PPP pentose phosphate pathway, PRPP 5-phosphoribosyl 1-pyrophosphate, tRNA switch RNA.

We discovered 49% extra accumulation of phospholipids in pyrazoleamide-treated parasites as in comparison to untreated parasites (Desk 3). Upon investigation of the metabolic community, we discovered that the handled parasites redirected flux by way of glucose 6-phosphate (G6P, Fig. 5) to reinforce flux by way of myoinositol-3-phosphate synthase (MIPS, Fig. S2) and myoinositol-phosphate phosphatase (MIPP, Fig. S2). In combination, those enzymes shape the primary and 2d steps of de novo synthesis of myoinositol (MI, Fig. 5). Those effects counsel that during drug-treated parasites the synthesis of MI—a precursor for phosphatidylinositol (PtdIns) synthesis—is larger inflicting an enhanced phospholipid synthesis within the handled parasites (Fig. 4D). The mannequin simulations are expecting a downregulation in PPP flux as an immediate result of this metabolic adaptation. Additionally they confirmed that the downstream penalties of this metabolic adaptation, e.g., downregulated redox metabolism, larger oxidative pressure, downregulated hemoglobin degradation, and many others., are unfavorable to the parasite and, therefore, may just give a contribution to effectiveness of pyrazoleamides in P. falciparum.