Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA

Background Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages. Results We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His, while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and their impact on drug responses require investigations using reverse genetics techniques in malaria parasites.


Introduction
Malaria is a leading cause of death across many tropical and subtropical countries 1 .Of the six species that infect humans, Plasmodium falciparum is the most virulent and significant cause of the majority of deaths 2 .In 2020, at least 241 million malaria cases and 627,000 deaths were reported globally, with 95% in Africa 1 .The disease disproportionally affects children under five and accounts for 77% of malaria-associated deaths 1 .Currently, the recommended treatment for uncomplicated Plasmodium falciparum malaria involves using artemisinin-based combination therapies (ACTs), comprising two components: a short-acting artemisinin derivative and a long-acting partner drug 1 .In Africa, three main ACTs are advocated: lumefantrine (LM)-artemether (ATM), piperaquine (PQ)-dihydroartemisinin (DHA), or Artesunate (ASN)-amodiaquine (AQ) 1 .The artemisinin derivative rapidly eliminates the bulk of the parasites in two hours, while the long-acting drug kills the residual parasites over weeks 3 .
Over the last two decades, the use of ACTs against Plasmodium falciparum has significantly decreased malaria-related deaths worldwide.However, the emergence of resistance to frontline artemisinins and partner drugs in Southeast Asia and Africa [4][5][6][7] , has threatened these gains made by the ACTs treatments.The stable parasite transmissions in many African countries, coupled with the extensive use of the ACTs, have consistently submitted prime pressure for selecting drug-resistant parasites, primarily against the long-acting drugs LM, PQ, and AQ 8 .This has spurred a new impetus to interrogate shared and unique resistance mechanisms against LM, PQ, and AQ.Like chloroquine, AQ and PQ are aminoquinoline drugs predicted to exert their action within the parasite digestive vacuole by inhibiting toxic heme polymerization 9 .However, PQ and AQ are active against selected CQ-resistant parasites, suggesting that these three drugs may differ in some modes of action and resistance mechanisms.These studies also indicate that the mechanisms of AQ, PQ, and LM may involve other unexplored essential parasite proteins.LM is an aryalcohol drug, predicted to share mechanisms of action with 4-aminoquinoline 9 .However, recent studies suggest reciprocal polymorphisms between 4-aminoquinoline drugs and LM 10,11 .These studies indicate that while LM, PQ, and AQ may share some resistance mechanisms, unique drug-specific mechanisms also need further investigation.Several genes, associated mutations, and resistance mechanisms against LM, PQ, and AQ have been identified and validated 8 .The shared genes between the LM, PQ, and AQ are polymorphisms or amplification of the chloroquine resistance transporter (crt) and multi-drug resistance 1 gene (mdr1) 12,13 .Recent studies have also identified new markers potentially unique for LM, PQ, and AQ.For instance, amplification and SNPs in Plasmepsin 2/3 in Plasmodium falciparum are associated with PQ resistance 14,15 .Also, decreased susceptibility to PQ and recrudescence following DHA/PQ treatment is associated with an E415G substitution in the exonuclease 16 .On the other hand, a mutation in cysteine desulfurase is associated with reducing susceptibility to LM in field isolates 17 .These studies reveal new and unprecedented genes that directly or indirectly mediate LM, PQ, and AQ efficacy.
Studies on the discovery of antimalarial drugs with transcending action to the different life cycle stages of the malaria parasites have validated protein kinases in mediating drug action and resistance [18][19][20][21][22] .In Plasmodium species, kinases control signal transduction pathways that regulate essential cellular processes such as growth, development, and reproduction 23,24 .While a link between kinases and AQ, LM, or PQ in Plasmodium remains unclear, the effect of aminoquinoline drugs; chloroquine (CQ), and AQ on the function of kinases in other eukaryotic and cancer cells is well documented [25][26][27] .For instance, CQ and AQ significantly enhance the activity of cyclin-dependent kinase inhibitors in rheumatoid arthritis 26 .As an anti-inflammatory drug, CQ affects the phosphorylation of extracellular signal-regulated kinases and the mitogenactivating protein kinase 28 .There are divergences in protein kinases between other eukaryotes and Plasmodium species 19,29 .However, the ability of aminoquinoline drugs CQ and AQ to inhibit phosphorylation suggests a link between protein kinases and the action of the aminoquinoline antimalarial drugs.In Plasmodium, protein kinase C involved in the growth, maturation, and differentiation of all the asexual blood stages can be inhibited by CQ, but only in the CQ-sensitive Plasmodium falciparum 30 , suggesting a possible association between the kinase with CQ susceptibility in the CQ resistant P. falciparum.In a separate study, CQ could competitively inhibit protein tyrosine kinase activity by blocking the protein substrate binding site 31 .The ability of CQ to inhibit key kinase proteins implies that closely related antimalarial drugs, such as AQ and PQ, may exert their action through similar mechanisms.
In Plasmodium species, the protein kinases are drug activators, potential targets for the drug metabolites, and mediators of drug resistance 22,[32][33][34] .For instance, artemisinin inhibits parasite growth by blocking the Plasmodium falciparum phosphatidylinositol-3-kinase (PfPI3K), while an increase in PfPI3K levels confers artemisinin resistance 35 .Polymorphisms in PANK1, the first enzyme in the coenzyme A (CoA) biosynthesis pathway, are associated with resistance to newly developed pathothenamides drugs 22,36 .Other kinase based-inhibitors that have progressed to human clinical trials are phosphatidylinositol 4-kinase (PI4K), with inhibitor 37 (MMV048) 23,37 .New multi-stage target drugs such as Imidazopyrazines block the ATP-binding pocket of PI4K, altering the intracellular distribution of phosphatidylinositol 4-phosphate 38 .Commonly, genes and mutations mediating resistance to one class may be associated with cross-resistance to drugs of unrelated chemical classes and modes of action.
Here, we used in silico computational analysis, PCR, sequencing, and qPCR analysis to interrogate the association of five selected kinases with LM, AQ, and PQ resistance in Plasmodium berghei ANKA.The stable drug-resistant parasites used in this study had previously been selected by submitting P. berghei ANKA parasites separately to LM, AQ, or PQ for at least 40 mechanical passages [39][40][41] .We reveal nonsynonymous mutations in Pantothenate kinase 1 (PANK1) (PBANKA_ 1022600), Diacylglycerol kinase (DAGK) (PBANKA_1334600), and Phosphatidylinositol 4-kinase beta (PI4Kβ) (PBANKA_ 1109400), only in the AQr parasites.The mutations either occurred in ligand binding motifs or within active sites of the enzymes and thus may impact the function of the kinases.We further report differential mRNA expression profiles of the PI4Kβ, PANK1, and DAGK across the LM, AQ, and PQ resistant parasites.

Ethical consideration
This study was conducted at KEMRI, Nairobi, Kenya.Permission to use laboratory animals was granted under the approvals SERU No KEMRI/CTMDR/SERU/P092/4096 on 20 th November 2020 and KEMRI ACUC/02.06.2021.To ameliorate any suffering of the laboratory animals used in the study, we adhered to the ARRIVE Guidelines.Experimental mice were monitored daily for any severe illness or injury to alleviate pain and suffering and avoid mortality associated with parasite infection.No animal became severely ill before the experimental endpoint (day nine post parasite infection for drug assays).At the end of each study, mice were euthanized using carbon dioxide (CO 2 ) inhalation.CO 2 exposure was delivered from compressed gas cylinders using a gradual fill method at a displacement rate of 20% of the chamber volume per minute.ARRIVE Guidelines checklist and the detailed report on the use and care of laboratory animals used in the study are deposited in OSF 42 .
In silico computational analysis by multiple sequence alignment and I-TASSER PlasmoDB and UniProt were used to retrieve protein sequences for the CK, PANK1, DAGK, PI4Kβ, and CDPK1.The evaluated protein kinases were derived from six different parasite species; two species that infect humans; Plasmodium falciparum 3D7, Plasmodium vivax P01, two species that infect the primates: Plasmodium adleri G01, and Plasmodium reichenowi G01) and two species that infect rodents: Plasmodium berghei ANKA, Plasmodium chabaudi chabaudi).Multiple sequence alignments of the retrieved proteins were performed using CLUSTAL OMEGA.Mapping of conserved regions and prediction of structure, function, and ligand binding motifs were queried using the protein structure prediction software Alpha Fold 42 , MOTIF search, Phyre2 version 2.0 43 , and ITASSER version 5.1 44,45 .

Parasites, host, and compounds
Two parasite reference lines of P. berghei ANKA (the MRA-865 and MRA-868 reference lines obtained from MR4, ATCC ® Manassas, Virginia) were used as the drug-sensitive wild-type (WT) parasites.Also, stable multidrug-resistant parasites: the PQ-resistant (PQ R ) P. berghei ANKA parasites previously selected from the MRA-865 line, the LM-resistant (LM R ), and AQ-resistant (AQ R ) P. berghei ANKA parasites previously selected from MRA-868 line 39,41 , were utilized.Male Swiss albino mice weighing 20±2g outbred at KEMRI Animal house Nairobi were employed, housed, and fed as previously described 46 .LM, PQ and AQ were donated from Universal Corporation, Kikuyu, Kenya.All drugs, PQ, LM, and AQ were freshly prepared and administered as previously described 39 .

Baseline drug profiles of the resistant parasites
The drug susceptibility profiles of the WT, LM R , AQ R , and PQ R parasites were assessed in the standard 4-day suppressive test (4-DT) 47 .Infection, randomization of experimental mice, drug administration, estimation of parasite densities, and calculation of the percentage of drug killing was performed and determined as previously described 46,48 .At least three mice were used for each drug dosage and placebo-treated control.On day zero (D0), the mice were randomly infected with 1×10 6 parasitized red blood cells (PRBCs); the mice were then orally treated with either 2.5, 5, 10, 20, 40, or 50mg/kg of drug concentrations at 4 hrs, 24hrs, 48hrs, and 72hrs post parasite inoculation.Each mouse's percentage of parasite density was estimated using a microscope ×100 magnification.Briefly, 10 different fields per smear on a slide from each mouse were counted with each field consisting of approximately 100 red blood cells, translating to coverage of 1000 total red blood cells per smear.Each drug dosage's percentage of drug killing ability was calculated as previously described 47 .
Extraction of DNA, PCR amplification, and sequencing DNA was extracted from WT, LM R , AQ R , and PQ R parasites in at least 1 ml of the infected blood isolated from a mouse with a parasitemia of 5 -10%.Blood was spun at room temperature for 2 minutes at 8,000xg to pellet the infected red blood cells.The supernatant was discarded, and the pellet was resuspended in a 30 ml volume of cold 1× Red blood cells lysis buffer (Thermo Fischer Scientific™ Cat No 00-4333-57) for 30 minutes, followed by spinning at 4,000 rpm for 10 min at room temperature.The parasite pellet was washed twice with 30 ml 1×PBS with centrifugation at 4,000 rpm for 5 min at 4°C.Parasite genomic DNA (gDNA) was extracted using a commercial GeneJET Whole Blood Genomic DNA Purification Mini Kit (Thermo Fischer Scientific TM Cat No K0781) following the instructions from the manufacturer.Target regions in selected kinases were PCR amplified using the primers listed in Table 1.Briefly, 2 µl of pgDNA, 0.2 pmol/µl forward, and reverse primers were used in 25 µl reactions and amplified using the Q5 High Fidelity Master Mix (NEB™ Cat No M0494S).PCR products were analyzed in 2% agarose gel, purified using the AddBio PCR Purification Kit (addbio™ Cat No 10078), and sequenced based on BigDye v3.1 using a 3730xl sequencer based on BigDye v3.1.
Sequence analysis and prediction of protein structure DNA sequences were analyzed using CodonCode Aligner, Lasergene 11 Core Suite, and CLUSTAL Omega, available Extraction of RNA and synthesis of cDNA Total RNA was prepared from approximately 1×10 6 fresh parasites pellet.The pellets were first thawed and then resuspended by vortexing for 10 minutes at room temperature.This was followed by centrifugation for 1 minute at 4,000 rpm at 4°C to remove debris.The supernatant was transferred to a clean Eppendorf tube.To each sample, 200 µl of chloroform (Sigma-Aldrich™ Cat No C2432) was added, vortexed for up to 30 seconds, and left at room temperature for 10 minutes.Centrifugation followed for 10 minutes at 13,000x g at 4°C to separate the phases.The upper aqueous phase was transferred to a fresh tube, and 500 µl of isopropanol (Sigma-Aldrich™ Cat No SKU 19516) was added.The samples were allowed to precipitate at -20°C for 1 h.The RNA was pelleted by centrifugation at 4,000 rpm for 15 min at 4°C.The supernatant was decanted, the pellet washed with 1 ml of 70% ethanol, and then spun again for 10 minutes at 13,000x g at 4°C.
The supernatant was decanted, and the pellet was dried on a hot plate for 3 -5 min.mixed gently.The reaction mix was incubated at 65°C for 30 min, then at 85°C for 5 min to terminate the reaction, and finally chilled on ice.The synthesized cDNA was used as a template for qPCR assays.

qPCR assays
Using the Pbβ-actin I as the reference gene, the relative expression of pank1, dagk, and pi4kβ was performed in a final volume of 20 µl using HOT FIREPol ® EvaGreen qPCR Mix Plus (ROX), 5x (Solis Biodyne Cat No: 08-24-0000S).Briefly, 4 µl of 5x HOT FIREPol ® mix and 14 µl of water were mixed with 0.5 µl of forward and reverse primers each (Table 2) and 1µl cDNA.The reaction mix was run for initial activation at 95°C for 12 min; denaturation at 95°C for 15 secs; annealing at 60°C for 20 secs for 40 cycles, and elongation at 72°C for 20 secs.The samples were run in triplicates using QuantStudio™ 5 System qPCR machine serial number: 272520210 (Thermo Fischer Scientific).

Statistical analysis
The percentage of parasitemia and drug killing in the different drug concentrations were analyzed using the nonparametric Mann-Whitney U test in the R statistical software version RStudio 2022.07.0 with a p-value set at 0.05.The means for the gene expression levels from three independent experiments and triplicate assays were compared using the Mann-Whitney U test with a p-value set at 0.05.The relative expression data were normalized using β-actin I as the reference gene based on the formula 2 ΔΔCT49 .

Results and discussion
In silico bioinformatics analysis reveals unique binding sites Here, we have analyzed five kinases: choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase, putative (DAGK), phosphatidylinositol 4-kinase beta (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1), and their association with AQ, LM and PQ resistance in Plasmodium berghei (Figure S1) 42 .We identified the likely functional regions in the five kinases using multiple sequence analysis (MSA), ITASSER tools, and MOTIF finder.We prioritized these sites for targeted PCR amplification and sequencing.
Our MSA of the kinase protein reveals unique conservation of motifs across the different species suggesting a likely association with function.Conserved motifs are enriched enzyme's active sites, ligand binding sites, functional groups, and plausible drug targets 44,45 .We further reasoned that the credible kinases that may associate with LM, PQ, and AQ resistance and drug responses are expressed within the ABS, the predicted sites of action of the drugs.The query on the expression profile of the five kinases from PlasmoDB shows a consistent expression across the different stages of the parasite's lifecycle, notwithstanding variation in the levels.To validate in silico bioinformatics analysis, we searched the previous link for each of the kinases as a drug target, drug resistance, or activator of the drug in malaria parasites.Several studies have validated the five kinases as drug targets in the malaria parasites 34,[50][51][52] .
Mutations in regulatory and functional motifs and upregulation of kinases modify drug responses in malaria parasites 22,35,53 .By querying the COACH and COFACTOR tools on the ITASSER platform using sequences from Plasmodium berghei, we identified the predicted functional, active sites, and ligand binding sites of the CK, PANK1, DAGK, PI4Kβ, and CDPK1 (Table 3).ATP binding sites, present in all kinases, are highly druggable, and the structural requirements for designing potent ATP-competitive kinase inhibitors 54 .
To identify critical regions in the target kinases, we used reference protein sequences from Plasmodium berghei ANKA.
Using the MOTIF finder, we found three predicted motifs in CK (Table 3).As expected, the choline kinase motif was identified from amino acids 139 -350 of the protein.The other motifs were APH and TCAD9, corresponding to amino acids positions 160 -324 and 281 -324, respectively.We then located binding motifs from amino acids 184 -190 and 284 -305 using the ITASSER tools.Here, we PCR amplified targeting positions 221 -318 region comprising the protein's predicted binding and active sites.The target region of interest included four ligand-binding sites with the ADP-binding site and an essential ATP synthesis site at position 288 (Table 3).
As the first enzyme in the CDP-choline pathway or Kennedy pathway, CK catalyzes the initial step of the CDP-choline pathway and can regulate phosphatidylcholine, the most abundant phospholipid in Plasmodium falciparum 55 .The binding of small molecules at the active site was shown to inhibit the function of CK, resulting in parasite killing 52 .Therefore, it is likely that for parasites to escape killing by drugs and drug metabolites targeting the CK, mutations within the active site or its close proximity may modify binding affinities and offer protection from such drugs.
When we focused our analysis on PANK1, using the MOTIF finder, we located two fumble motifs (Table 3).A functional fumble motif is critical for cell division and cell membrane synthesis 56 and thus an important region for mapping potential polymorphisms that may occur in resistant parasites.PANK1 is a proven drug activator 57 and catalyzes the first step in coenzyme A biosynthesis, which involves the phosphorylation of pantothenate 21 .Mutations in PANK1 alter its activity and responses to new antiplasmodial pantothenate analogs 22 .Our analysis using ITASSER tools predicted four ligand-binding sites of importance in acetyl coenzyme A and adenosine 5-diphosphate.The region of interest in this study was within the predicted motifs (303 -316) located in the fumble domain.
We found consensus binding residues from 311 -330 in all the top Protein Database (PDB) hits based on the COACH -ITASSER results, suggesting the essentiality of these residues in the functioning of the PANK1 protein.Acquisition of mutations within the fumble domain results in inactivation of the domain and significantly alters phospholipids biosynthesis and cell division 56 , supporting our search for polymorphisms within this region.
Our in silico analysis of DAGK using the MOTIF finder predicted two motifs (Table 3).As expected, we located the Diacylglycerol kinase accessory domain (249 -433), and Diacylglycerol kinase catalytic domain (120 -179).DAGK has catalytic and accessory domain motifs.The enzyme converts diacylglycerol to phosphatidic acid, which regulates exocytosis in apicomplexan, including Plasmodium species 58 .Here, we mapped our target motif at positions 129 to 178 of the DAGK, which is within the highly conserved catalytic domain as predicted by the MOTIF search on the genome.jpdatabase (Table 3).Further analysis using COACH and COFACTOR tools on the ITASSER platform revealed an adenosine 5-diphosphate ligand-binding site with several potential enzyme active site residues in these regions (Table 3).
By multiple sequence alignment of PI4Kβ protein from the different plasmodium species, we found a conserved region starting position 1254 to 1552 of the PI4Kβ.Further interrogation using the MOTIF finder traced three plausible motifs.These are PI3Ka, Peptidase_S26, and two PI3_PI4_kinase motifs.
Here, we targeted PI4Kβ from position 1280 to 1540 of the protein by PCR amplification which localizes the conserved ligand-binding site at position 1294 -1370 and houses the two PI3_PI4_kinase motifs and the catalytic domain.Several chemical inhibitors such as MMV 0048 and KDU691 kill Plasmodium falciparum by targeting the conserved region of the PI4Kβ 59 .Acquisition of mutations within the catalytic site of the PI4Kβ decreases parasite susceptibility to chemical compounds MMV 0048 and KDU691 59 , supporting our focus on the 1280 to 1540 position of the protein in this study.
Our MOTIF search on CDPK1 showed 14 predicted motifs: the expected Pkinase, PK_Tyr_Ser_Thr, and Kinase-like motifs.Interestingly, the proteins comprise 19 EF-hand motifs, four EF-hand_1, four EF-hand_6, four EF-hand_8, three EF-hand_5, two EF-hand_1, one EF-hand_4, and one EF-hand_9, suggesting high conservation of the protein.Unsurprisingly EF-hand motifs reside in the most conserved carboxyl-terminal end of the protein and show 90% conservation across the six malaria parasite species, suggesting the important function of the motifs in the CDPK1.The ITASSER query showed ligand-binding motifs at positions 145 -263 which is within our PCR amplification target region (Table 3).
Drug response profiles confirmed the stability of the LMr, AQr, and PQr parasites We first generated baseline drug susceptibility profiles of the LMr, PQr, and AQr parasites against respective drugs for which each line was initially selected.As expected, the untreated LMr or WT grew normally, with parasitemia rising to above 5% by day 4-post parasite inoculation.Submission of WT 5mg/kg cleared the parasite to undetectable levels by microscopy or less than 0.01% parasitemia.LM at 50mg/kg failed to kill the parasite suggesting retention of a stable resistant phenotype.The administration of 2.5mg/kg of AQ or PQ allowed modest growth of the WT parasites to an average parasitemia of 1% throughout the four days of drug administration.However, the WT parasites rose drastically to 9.46% by day 7, three days after cessation of the drug treatment.The WT parasite remained suppressed by 2.5mg/kg of PQ across the entire follow-up period of 9 days post parasite infection, with the highest parasitemia yielding 0.99%.AQ or PQ at 5mg/kg suppressed the WT parasites to below 0.5%, confirming the expected susceptibility of the WT parasites (Figure 1).We extended our investigation to PQr parasites by submitting them to 20mg/kg or 40mg/kg of PQ; our results show a general consistent rise in parasite densities despite the continuous drug pressure from day 0 to day 3-post parasite inoculation, with an expected sharp rise in parasitemia as recorded on day 7 and day 9-post parasite infection.AQr parasite yielded a steady increase in growth despite administration of 10 or 20 mg/kg (Figure 1).We found no significant distinction between AQr responses to 10 and 20 mg/kg, which could be attributed to varying AQ/DEAQ metabolism.Overall, the drug response profile shows the susceptibility of the WT to LM, PQ, and AQ.At the same time, their respective resistant parasites had retained resistance phenotypes, suggesting that the resistant mechanisms were encoded within the parasite's genome.
Mutations in DAGK, PANK1, and PI4Kβ in the amodiaquine resistant parasites To investigate possible SNPs in the kinases, target regions in ck, pank1, dagk, pi4kβ, and cdpk1 genes were PCR amplified, and the expected PCR products were obtained (Figure 2).Optimized PCR amplification conditions of the PCR experiments are shown in Table 4. Analysis of the PCR amplified, and sequenced motifs revealed that only DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations.Surprisingly, the mutations occurred only in the AQr parasites (Table 5).Parasites mechanically passed for several generations may acquire mutations independent of the drug pressure 60 .The WT parasites passaged a similar number of mechanical passages and did not acquire the mutations thus, we rule out the possibility of long-term mechanical passages inducing the nonsynonymous mutations observed in DAGK, PANK1, and PI4Kβ.We have also interrogated LMr and PQr parasites selected using a similar protocol but failed to select mutations in the DAGK, PANK1, and PI4Kβ; therefore, we argue that the mutations observed in AQr are derived from AQ pressure.This suggests that AQ or its long-acting metabolite, DEAQ, may submit selective pressure on PANK1, DAGK, and PI4Kβ proteins.We thus propose that submitting AQ pressure on Plasmodium berghei ANKA is accompanied by the acquisition of mutation in PANK1, DAGK, and PI4Kβ.
We found one nonsynonymous mutation Asn394His in PANK1 in the AQr parasites (Figure 3), but LMr and PQr parasites did not have any mutations in PANK1 (Table 5).We then used AlphaFold and ITASSER tools to predict the impact of the mutation on PANK1 protein, and we show Asn394 interacts with His450 (Figure 3A, 3B).The Asn394His mutation eliminates the Asn394 -His450 interaction suggesting a possible alteration of protein structure.Histidine has an imidazole side chain, which has a pKa of 6.8, the pH of the cytoplasm 61 .As a result, small shifts in cellular pH may change the charge of histidine side chains between acid and basic pH.The activities of many proteins are modulated by pH through the protonation of histidine side chains.Asparagine is uncharged but has polar amide groups with extensive hydrogen-bonding capacities.As a result, slight shifts in cellular pH may alter the charge of histidine side chains 61 .Although Asn and His are polar amino acids, Asn is neutral while His is a basic amino acid, suggesting alteration of the hydrophilicity of the side chain.Also, based on protein structure analysis using Alpha-Fold of the AQr mutant structure, the amino acid change at position 394 resulted in the introduction of a hydrogen    bond and a new interaction with Glu, an interaction illustrated as (394|O-Glu 398|N); this is due to the presence of amide groups with extensive hydrogen-bonding capacities.
Within the targeted motifs of the CK and CDPK1, we found no mutation in AQr, LMr, or PQr resistant parasites (Table 5).A mutation that impacts a significant effect on phenotype may Several studies have interrogated the possibility of motif's druggability since significant motifs are composed of the regulatory interplays between phosphorylation and proteolysis 62,63 .These studies discovered that motifs are significantly enriched with drug targets, suggesting the possibility of exploring these conserved motifs as potential drug targets 63 and thus likely mediators of drug responses.Here we only investigated the highly likely motifs of the CK and CDPK1.Therefore, mutation acquisition in other sections of the proteins may only have subtle changes in altering drug susceptibility.
We then probed PI4K and identified four nonsynonymous SNPs in the AQr parasite and none in PQr and LMr parasites.These are Asn1366Asp, Ser1367Arg, Tyr1394Asn, and Tyr1423Asn (Table 5).I-TASSER results of PI4Kβ wild type and AQr show a new ligand-binding site for trifluoromagnesate (MGF) at Asp1451Asn.Asp has a carboxylic acid side chain that forms ionic bonds and can also function as hydrogen bond acceptors.These changes might alter the binding efficiency and the protein function.Many proteins that bind metal ions for structural or functional purposes possess metal-binding sites containing aspartate 64 .Asn has amide R groups, and the amino group (NH 2 ) functions as a hydrogen bond donor.I-TASSER also predicted the modified protein structure due to the acquired mutations, further corroborated by AlphaFold protein structure prediction (Figure 4).AlphaFold prediction on amino acid interactions of the mutated protein showed that the interaction at position 1366 of the protein between Asp and Lys (1366|O-LYS1360|NZ) was retained despite the amino acid change.At position 1367Arg, the mutant protein obtained a new amino acid interaction Thr, illustrated as (1367|0-THR 1365|OG1), and lost two other amino acid interactions (1367|O-ILE 1371|N: 1367|OG-THR 1370|N) but retained (1367|O-LYS 1365|OG1).On the other hand, 1451Asn resulted in two new amino acid interactions between Asn and Gly and Asn and Arg; (1451|OD1-GLY 1453|N: 1451|O-ARG 1434|NH 2 ) (Figure 4).The changes in the interaction of the amino acids and protein structure changes suggest a possible interference with protein function.The role of these changes in mediating AQ resistance requires further investigation.
Analysis of DAGK sequence illustrated amino acid change; Lys270Arg and Lys292Arg in the AQr parasites (Figure 5).Arginine and lysine are basic amino acids and did not attract changes in new amino acid interactions as predicted by Alpha-Fold (270|O-ASN 276|ND2 and 292|N-SER 290|OG).Lysine  Differential gene expression of PANK1, PI4Kβ, and DAGK genes in AQr parasite lines Parasites may adapt to drug pressure through upregulation or downregulation of essential drug targets or drug modifying proteins 65 .To evaluate whether gene expression changes accompanied the mutation's acquisition, we measured the mRNA amount of PANK1, PI4Kβ, and DAGK.We argued that maximal differential expression would be expected at the trophozoites and early schizonts, the stages at which AQ and DEAQ are predicted to exert maximal action 66 .We reveal a differential gene expression in PANK1, PI4Kβ, and DAGK at trophozoites and early schizont stages between AQr and drug-sensitive parasites 42 .All three genes were significantly upregulated in the schizont stages and downregulated in the trophozoite stages (Figure 6).The expression level of PANK1 at the trophozoite stages was not significantly altered despite showing marginal downregulation by 1.3-fold (p<0.4815)but was significantly upregulated by 7.01-fold (p<0.001) during the schizont stages.In a similar trend, DAGK showed 1.2-fold downregulation at the trophozoite (p<0.086) and 5.4-fold (p<0.001)upregulation at the schizonts.Interestingly, PI4Kβ had the highest differential gene expression yielding a 7.1-fold (p<0.001)downregulation at the trophozoite and 9.0-fold (p<0.0035)upregulation at the schizont stages.The impact of the differential expression of PANK1, PI4Kβ, and DAGK genes in mediating drug responses requires validation.

Conclusion
Here, we show the selection of nonsynonymous mutations in malaria parasites' druggable kinases: the PANK1, DAGK, and PI4Kβ (Figure 7).The mutation occurred within or close to ligand binding sites or predicted active sites.Selectively acquiring these mutations in AQr resistant and not in the LMr or PQr resistant parasites may signify proteins under AQ or metabolite DEAQ pressure.We further show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites.Future studies employing reverse genetics techniques to determine and validate the impact of the mutations in modifying drug susceptibility may reveal important insights into understanding the mechanisms of AQ resistance in malaria parasites.
This project contains the following underlying data: • Amodiaquine_baseline_profile.xlsx (Amodiaquine-resistant and wild-type parasite responses to amodiaquine drug).
• Figure S1: A flow chart summary of selected protein kinases as mediators of drug target, action, locatlization and resistance.
Data are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).Introduction The introduction section is well written and concise.I would suggest that the full form of CQ (chloroquine) should be provided first, followed by referring to it as CQ (in the second paragraph).

Methods
Either use the unit g or RPM. 1.
The concentration of DNA used for PCR reactions is missing.It's written as 2µl of pg DNA. 2.

Results and Discussion
In the 2 nd paragraph, mention what's ABS? 1.

Major comments:
In their RNA extraction protocol, authors mentioned that they dried RNA on a hot plate for 3-5min.This seems very absurd as this is a normalized protocol for drying DNA, but RNA is highly degradable.Did they measure the ratio of A260/A280?This seems to be a major concern because in Fig 6, the standard deviation is way too high.As the number of independent experiments is less, the difference may not be conclusive.Although the overall values are statistically significant, the authors might want to repeat this experiment to increase the number of independent experiments which might lead to a firm conclusion.Also, please mention the statistical significance of the data in the figure itself.Authors have used alpha fold and ITASSER to predict the structure of the kinases and MOTIF finder to find the motif.However, from the ribbon model it is hard to determine the tertiary structure of the proteins and hence the active motifs.Authors may replace/add the ribbon models with tertiary structures (sphere models).Also, authors might consider verifying the results of motif finder with other freely available software and energy minimization algorithms.

Is the work clearly and accurately presented and does it cite the current literature? Yes
Is the study design appropriate and is the work technically sound?Partly

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.

Reviewer Expertise:
We confirm that we have read this submission and certain that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.

Summary:
The research article titled "Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA" by Jean Chepngetich and colleagues, investigates the association of protein kinases with resistance to amodiaquine, lumefantrine, and piperaquine in Plasmodium berghei ANKA.Using in silico tools, they identified conserved motifs and ligand binding sites in choline kinase, pantothenate kinase 1, diacylglycerol kinase, phosphatidylinositol 4-kinase beta, and calciumdependent protein kinase 1.They found nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol 4-kinase beta only in the amodiaquine-resistant parasites.The mutations occurred within or near ligand binding sites or active sites.The expression of pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol 4-kinase beta was downregulated in trophozoites but upregulated in schizonts of the amodiaquine-resistant parasites.The research concludes that the selective acquisition of mutations and differential gene expression in AQ-resistant parasites may indicate proteins under AQ pressure.

Major concerns:
The researchers concluded that the selective mutations and differential gene expression in the amodiaquine-resistant parasites may indicate proteins under amodiaquine pressure.However, this assumption is not supported by direct experimental evidence that would confirm the association between the identified mutations and amodiaquine resistance.If reverse genetics is out of the scope of this paper, perhaps the authors could use gene network analysis or identify genes that interact with gene partners that have already been associated with AQ resistance.I would recommend mining PlasmoDB to identify these gene partners. 1.
Alternatively, the authors could check if P. falciparum orthologs of the kinases have been associated with AQ selection/pressure.

2.
Minor concerns: Data Inclusion: Incorporate data comparing the viability of drug-sensitive versus drugresistant parasites.This could involve phenotypic evaluations or measurements of growth replication to support the findings.

1.
Figure Improvements: Include missing marker labels in figures, provide a figure illustrating key findings related to PANK1, PI4Kβ, and DAGK, and consider adding a figure to demonstrate the link between protein kinases and drug action/resistance.

2.
Strengthen qPCR Data Presentation: Include all necessary information in the qPCR data presentation, such as housekeeping gene data, statistical determinants, and the number of biological replicates. 3.

Is the work clearly and accurately presented and does it cite the current literature? Dina Coertzen
University of Pretoria, Pretoria, South Africa The manuscript: "Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA" evaluated the development of mutations to known ACT partner drugs in various key kinases in P. berghei ANKA parasites.From a biological perspective this link is interesting as the role of kinases and associated signaling cascades has not been investigated in P. falciparum drug resistant lines.There are some major points that I think the authors need to take into consideration, in order to substantiate their findings.
For the introduction section: I would propose a rewrite of the second sentence in the introduction as follows: "Of the six 1.
species that infect humans, Plasmodium falciparum is the most virulent, and is responsible for majority of deaths."The sentence "LM is an aryl alcohol drug, predicted to share mechanisms of action with 4aminoquinoline 9." Can be moved up after the "Like chloroquine, AQ and PQ are aminoquinoline drugs predicted to exert their action within the parasite digestive vacuole by inhibiting toxic heme polymerization."sentence.

2.
The authors need to include examples or references to the following statement: "These studies also indicate that the mechanisms of AQ, PQ, and LM may involve other unexplored essential parasite proteins."

3.
I am unsure what the authors mean by the term "protein kinases are drug activators"?Perhaps include a reference to this.

4.
Edit the following sentence as suggested: "In Plasmodium species, the protein kinases are drug activators, potential targets for the drug metabolites, and mediators of drug resistance,32-34."

5.
I think the bolded number "37" may be a typographical error: "Other kinase based-inhibitors that have progressed to human clinical trials are phosphatidylinositol 4-kinase (PI4K), with inhibitor 37 (MMV048)23,37."Edit the following sentence as suggested: "Commonly, Ggenes and mutations mediating resistance to one class may be associated with cross-resistance to drugs of unrelated chemical classes and modes of action."

8.
Include a ref to the following sentence: "Commonly, genes and mutations mediating resistance to one class may be associated with cross-resistance to drugs of unrelated chemical classes and modes of action."9.

Scientific results and methods
The observation that nonsynonymous mutations only occurred in AQr parasites is strange.This drug is of the same mechanism of action and chemical class as the PQ (8aminoquinolines), yet no kinase specific mutations were observed in the PQr line.The authors need to provide some justification as to this observation.This is in line with the following sentence in the manuscript:" We thus propose that submitting AQ pressure on Plasmodium berghei ANKA is accompanied by the acquisition of mutation in PANK1, DAGK, and PI4Kβ."The authors need to give some reference to any biological consideration that could justify this observation, and why specifically this occurs within a AQr line.

1.
Furthermore, the authors showed no justification or validation that mutations are specific to kinases in the drug resistant AQr line.There is no sequencing data of other genes presented in this paper.I think that if the authors want to emphasize the synonymous mutation of the specific kinases they need to show WGS full genomic sequence analysis, for at least a couple of parasites.Or at least include several other genes in the PCR experiment.

2.
The authors even state this in the paper: " The parasites likely possess mutations in other protein sections.Whole gene sequencing or whole-genome sequence of the parasites may reveal changes in other functional regions of the protein." The use of SI units and scientific nomenclature in the methods section is incorrect.Minutes should be min hour hour/hours are h, rpm's are not SI units and need to be converted to xg (note italics of g), ml and µl should be mL and µL, degrees should be °C, spaces should be included between all SI units.

3.
All text the manuscript must be justified.4.
I think the authors needed to include some data as the viability of drug sensitive vs drug resistant parasites, either as a phenotypic evaluation or measurement of growth replication.

5.
Most importantly to truly assess the effect of kinase specific mutations, drug shift assays with known kinase inhibitors with and without PQ, AQ and LM needed to be performed.This result is critical to this paper.

6.
The following sentence is unnecessary:" At the same time, their respective resistant parasites had retained resistance phenotypes, suggesting that the resistant mechanisms were encoded within the parasite's genome."All mutations incurred in parasite drug resistant lines occur on a genomic level.

7.
The following sentence needs to be rewritten: "The WT parasites passaged a similar number of mechanical passages and did not acquire the mutations thus, we rule out the possibility of 8.
long-term mechanical passages inducing the nonsynonymous mutations observed in DAGK, PANK1, and PI4Kβ." The authors need to include the number of technical/ biological replicates and statistical evaluation in figure legend 1. 1.
The pictures of the gels in Figure 2 can be condensed to only show the respective bands, with the rest of the gel being cropped out.The secondary maker lane can be removed.

2.
Tables with PCR primer sequences and PCR amplification conditions can be moved to a supplementary file.

3.
Mutations cannot be "toxic" to parasites, they can incur a cost to fitness phenotype or if the gene product is essential to the parasite, parasite growth will be refractory."CK and CDPK1 are essential for the growth of the asexual blood-stage parasites; thus, the acquisition of a mutation at the functional motif may be toxic for parasites".Furthermore, the authors also need to include a ref for the essentiality of CK and CDPK genes.

4.
I think that the following conclusion is unjustified: "and thus likely mediators of drug responses".This is based on the fact that the authors do not show any drug response effect with known kinase inhibitors.This statement should be verified with a reference at least.

5.
My last major concern is the context of the qPCR data.I do not think that that the paper provides sufficient proof or biological context to validate that the mutations of the specific kinases would influence their expression profiles, and how this relates to AQ resistance.Context can be provided if the expression profiles of genes conveying the AQ resistance was perhaps included, and this was provided in context with literature with some means of a biological explanation.The SD deviations provided by the qPCR is also more than >2/3 than the actual values, and the housekeeping gene data is not included.The statistical determinants are not shown on the graph and the number of biological replicates is not indicated in the figure legend.

6.
The manuscript: "Amodiaquine drug pressure selects nonsynonymous mutations in pantothenate kinase 1, diacylglycerol kinase, and phosphatidylinositol-4 kinase in Plasmodium berghei ANKA" evaluated the development of mutations to known ACT partner drugs in various key kinases in P. berghei ANKA parasites.From a biological perspective this link is interesting as the role of kinases and associated signaling cascades has not been investigated in P. falciparum drug resistant lines.There are some major points that I think the authors need to take into consideration, in order to substantiate their findings.
For the introduction section: I would propose a rewrite of the second sentence in the introduction as follows: "Of the six species that infect humans, Plasmodium falciparum is the most virulent, and is responsible for majority of deaths." 1.
The sentence "LM is an aryalcohol drug, predicted to share mechanisms of action with 4-aminoquinoline9."Can be moved up after the "Like chloroquine, AQ and PQ are aminoquinoline drugs predicted to exert their action within the parasite digestive vacuole by inhibiting toxic heme polymerization9."sentence.

2.
The authors need to include examples or references to the following statement: "These studies also indicate that the mechanisms of AQ, PQ, and LM may involve other unexplored essential parasite proteins."

3.
I am unsure what the authors mean by the term "protein kinases are drug activators"?Perhaps include a reference to this.

4.
Edit the following sentence as suggested: "In Plasmodium species, the protein kinases are drug activators, potential targets for the drug metabolites, and mediators of drug resistance22,32- I think the bolded number "37" may be a typographical error: "Other kinase based-inhibitors that have progressed to human clinical trials are phosphatidylinositol 4-kinase (PI4K), with inhibitor 37 (MMV048)23,37." The statement highlighted in point Edit the following sentence as suggested: "Commonly, Ggenes and mutations mediating resistance to one class may be associated with cross-resistance to drugs of unrelated chemical classes and modes of action."

8.
Include a ref to the following sentence: "Commonly, genes and mutations mediating resistance to one class may be associated with cross-resistance to drugs of unrelated chemical classes and modes of action." 9.

Scientific results and methods
The observation that nonsynonymous mutations only occurred in AQr parasites is strange.This drug is of the same mechanism of action and chemical class as the PQ (8aminoquinolines), yet no kinase specific mutations were observed in the PQr line.The authors need to provide some justification as to this observation.This is in line with the 1.
following sentence in the manuscript:" We thus propose that submitting AQ pressure on Plasmodium berghei ANKA is accompanied by the acquisition of mutation in PANK1, DAGK, and PI4Kβ."The authors need to give some reference to any biological consideration that could justify this observation, and why specifically this occurs within a AQr line.Furthermore, the authors showed no justification or validation that mutations are specific to kinases in the drug resistant AQr line.There is no sequencing data of other genes presented in this paper.I think that if the authors want to emphasize the synonymous mutation of the specific kinases they need to show WGS full genomic sequence analysis, for at least a couple of parasites.Or at least include several other genes in the PCR experiment.
The authors even state this in the paper: " The parasites likely possess mutations in other protein sections.Whole gene sequencing or whole-genome sequence of the parasites may reveal changes in other functional regions of the protein." 2.
The use of SI units and scientific nomenclature in the methods section is incorrect.Minutes should be min hour hour/hours are h, rpm's are not SI units and need to be converted to xg (note italics of g), ml and µl should be mL and µL, degrees should be °C, spaces should be included between all SI units.

3.
All text the manuscript must be justified.4.
I think the authors needed to include some data as the viability of drug sensitive vs drug resistant parasites, either as a phenotypic evaluation or measurement of growth replication.

5.
Most importantly to truly assess the effect of kinase specific mutations, drug shift assays with known kinase inhibitors with and without PQ, AQ and LM needed to be performed.This result is critical to this paper.

6.
The following sentence is unnecessary:" At the same time, their respective resistant parasites had retained resistance phenotypes, suggesting that the resistant mechanisms were encoded within the parasite's genome."All mutations incurred in parasite drug resistant lines occur on a genomic level.

7.
The following sentence needs to be rewritten: "The WT parasites passaged a similar number of mechanical passages and did not acquire the mutations thus, we rule out the possibility of 8.
long-term mechanical passages inducing the nonsynonymous mutations observed in DAGK, PANK1, and PI4Kβ." The authors need to include the number of technical/ biological replicates and statistical evaluation in figure legend 1. 1.
The pictures of the gels in Figure 2 can be condensed to only show the respective bands, with the rest of the gel being cropped out.The secondary maker lane can be removed.

2.
Tables with PCR primer sequences and PCR amplification conditions can be moved to a supplementary file.

3.
Mutations cannot be "toxic" to parasites, they can incur a cost to fitness phenotype or if the gene product is essential to the parasite, parasite growth will be refractory."CK and CDPK1 are essential for the growth of the asexual blood-stage parasites; thus, the acquisition of a mutation at the functional motif may be toxic for parasites".Furthermore, the authors also need to include a ref for the essentiality of CK and CDPK genes.

4.
I think that the following conclusion is unjustified: "and thus likely mediators of drug responses".This is based on the fact that the authors do not show any drug response effect with known kinase inhibitors.This statement should be verified with a reference at least.

5.
My last major concern is the context of the qPCR data.I do not think that that the paper provides sufficient proof or biological context to validate that the mutations of the specific kinases would influence their expression profiles, and how this relates to AQ resistance.

6.
Context can be provided if the expression profiles of genes conveying the AQ resistance was perhaps included, and this was provided in context with literature with some means of a biological explanation.The SD deviations provided by the qPCR is also more than >2/3 than the actual values, and the housekeeping gene data is not included.The statistical determinants are not shown on the graph and the number of biological replicates is not indicated in the figure legend.different drug concentrations, the resistant parasites grew and multiplied in the presence of the drug, while the drug-sensitive wild-type parasites were suppressed.These data confirmed that resistant parasites had retained the resistance phenotype.During the selection of amodiaquine-resistant parasites (Kiboi et al 2009; Ndungu et al 2018) and as stated in the results, the drug-sensitive wild-type parasites were passaged in mice without the drug pressure for an equivalent number of mechanical passages and did not acquire these non-synonymous mutations; therefore, we ruled out the possibility of long-term mechanical passages inducing the nonsynonymous mutations observed in DAGK, PANK1, and PI4Kβ.

Is
Competing Interests: No competing interests were disclosed.The manuscript is accepted for indexing with some minor modifications as follows: Abstract: Why were CK and CDPK1 missing from mRNA measurement despite probe for SNPs? 1.
I suggest to include a figure to illustrate the link between protein kinases in mediating drug action and resistance.
Give the details of the equipment used in the experiment which is missing in the manuscript.E.g.QuantStudio™ 5 System qPCR machine.

Results & Discussion:
There are missing marker labels in Figure 2. 1.
The authors should include a figure to explain the key findings from this research especially involved with PANK1, PI4Kβ, and DAGK.

Acknowledgment:
Provide the source of funding and declaration of conflict of interest among all authors.Reviewer Expertise: Molecular biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Drug activity profiles as assessed using the 4-day suppressive test.(A) Lumefantrine against the lumefantrine resistant (LMR) and the wild-type drug-sensitive (LMS) Plasmodium berghei ANKA parasites at 5mg/kg for wild-type drug-sensitive (WT) and 50mg/kg for LMR.(B) Piperaquine against the piperaquine resistant (PQR) and the WT Plasmodium berghei ANKA parasites at 2.5mg/kg and 5mg/kg for the PQS and 20mg/kg and 40mg/kg for the PQR.(C) Amodiaquine against amodiaquine resistant (AQR) and the WT Plasmodium berghei ANKA parasites at 2.5mg/kg and 5mg/kg for the AQS and 10mg/kg and 20mg/kg for the AQR.Percentage (%) parasite densities were monitored for a total of nine days post parasite inoculation and five days after cessation of drug treatment.

Figure 3 .
Figure 3.A section of pantothenate kinase 1 (PANK1) as predicted using AlphaFold (A) The linkage of Asn394 with His and Asn in the wildtype protein.(B) The mutation Asn394His abolishes the linkage.

Figure 5 .Figure 6 .
Figure 5. Diacylglycerol kinase (DAGK) as predicted using the Swiss Model showing the two Arg272 and Arg292 mutations acquired by the amodiaquine-resistant parasites.Figure 6.The relative gene expression of pantothenate kinase (pank1), diacylglycerol kinase (dagk), and phosphatidylinositol 4-kinase (pi4kβ) at late trophozoite (LT) and schizonts (SC) stages in the amodiaquine-resistant parasites relative to the drug-sensitive control parasites.The differential expression from a mean of at least three independent experiments was significantly upregulated for pank1, dagk, and pi4kβ at the schizont stage and downregulated at the trophozoite stage.and arginine have an overall charge of +1 at physiological pH.The guanidino group in arginine's side chain is the most basic of all R groups; this led to losing one hydrogen bond at position 270|N-ASN276|OD1.Based on AlphaFold, Lys -> Arg change resulted in modifying the secondary structure of the DAGK protein.To determine the effect of these mutations on the protein structure and specifically on the ligand and enzyme binding sites, we queried the mutant DAGK protein in ITASSER.This revealed different ligand binding sites on the mutant DAGK protein with altered enzyme active sites, such as an ADP ligand-binding site.

Figure 7 .
Figure 7.A summary of key findings of associated mutations and gene expression profiles of Choline kinase, Pantothenate kinase 1, Phosphatidylinositol 4-kinase beta, Diacylglycerol kinase, and Calcium-dependent protein kinase 1 in amodiaquineresistant Plasmodium berghei ANKA.

Figure 2 .
Figure 2. Gel electrophoresis profiles of PCR products: The authors could crop the extra ladder in the figure as it is redundant 4.

Version 1 Reviewer
Report 01 December 2022 https://doi.org/10.21956/openresafrica.14585.r29370© 2022 Ngit Shin L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Lai Ngit Shin Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, Gelugor, Malaysia

○
Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

Table 3 . A summary of the in-silico bioinformatics results from multiple alignments of the protein kinase sequences
, MOTIF Search using genome.jpdatabase (https://www.genome.jp/tools/motif/),ligand binding, and active sites search using ITASSER online tools.

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? No
Competing Interests: No competing interests were disclosed.Reviewer Expertise: Biochemistry, Parasitology, Molecular cell biology, in vitro cell culture, protein biochemistry, antimalarial drug discovery I