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Novel antimalarial compounds targeting both the pathogenic and transmissible stages of the human malaria parasite, Plasmodium falciparum, would greatly benefit malaria elimination strategies. However, most compounds affecting asexual blood stage parasites show severely reduced activity against gametocytes. The impact of this activity loss on a compound's transmission-blocking activity is unclear. Here, we report the systematic evaluation of the activity loss against gametocytes and investigate the confounding factors contributing to this. A threshold for acceptable activity loss between asexual blood stage parasites and gametocytes was defined, with near-equipotent compounds required to prevent continued gametocyte maturation and onward transmission. Target abundance is not predictive of gametocytocidal activity, but instead, lipoidal uptake is the main barrier of dual activity and is influenced by distinct physicochemical properties. This study provides guidelines for the required profiles of potential dual-active antimalarial agents and facilitates the development of effective transmission-blocking compounds., Competing Interests: Competing interests D.L. is a senior director in drug discovery at the Medicines for Malaria Venture (M.M.V.). M.M.V. funded part of this study. The other authors declare no competing interest., (© 2024. The Author(s).)

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Inhibition of the lactate transporter PfFNT is a valid novel mode of action against malaria parasites. Current pyridine-substituted pentafluoro-3-hydroxy-pent-2-en-1-ones act as substrate analogs with submicromolar EC 50 in vitro, and >99.7% activity in mice. The recently solved structure of a PfFNT-inhibitor complex visualized the binding mode. Here, we extended the inhibitor layout by series of amine- and anilide-linked pyridine p-substituents to generate additional interactions in the cytoplasmic vestibule. Virtual docking indicated hydrogen bonding to Tyr31 and Ser102. Fluorescence cross-correlation spectroscopy yielded respectively enhanced target affinity. Strikingly, the in vitro activity increased by 1 order of magnitude to 14.8 nM at negligible cytotoxicity. While p -amine substitutions were rapidly metabolized, the more stable p -acetanilide cleared 99.7% of parasites at 4 × 50 mg kg -1 in a mouse malaria model. Future stabilization of the p-substitution against metabolism may translate the gain in in vitro potency to the in vivo situation.

The Plasmodium falciparum mitochondrial electron transport chain (mETC) is responsible for essential metabolic pathways such as de novo pyrimidine synthesis and ATP synthesis. The mETC complex III (cytochrome bc 1 complex) is responsible for transferring electrons from ubiquinol to cytochrome c and generating a proton gradient across the inner mitochondrial membrane, which is necessary for the function of ATP synthase. Recent studies have revealed that the composition of Plasmodium falciparum complex III (PfCIII) is divergent from humans, highlighting its suitability as a target for specific inhibition. Indeed, PfCIII is the target of the clinically used anti-malarial atovaquone and of several inhibitors undergoing pre-clinical trials, yet its role in parasite biology has not been thoroughly studied. We provide evidence that the universally conserved subunit, PfRieske, and the new parasite subunit, PfC3AP2, are part of PfCIII, with the latter providing support for the prediction of its divergent composition. Using inducible depletion, we show that PfRieske, and therefore, PfCIII as a whole, is essential for asexual blood stage parasite survival, in line with previous observations. We further found that depletion of PfRieske results in gametocyte maturation defects. These phenotypes are linked to defects in mitochondrial functions upon PfRieske depletion, including increased sensitivity to mETC inhibitors in asexual stages and decreased cristae abundance alongside abnormal mitochondrial morphology in gametocytes. This is the first study that explores the direct role of the PfCIII in gametogenesis via genetic disruption, paving the way for a better understanding of the role of mETC in the complex life cycle of these important parasites and providing further support for the focus of antimalarial drug development on this pathway.

Emerging Artemisinin (ART) resistance in Plasmodium falciparum (Pf) poses challenges for the discovery of novel drugs to tackle ART-resistant parasites. Concentrated efforts toward the ART resistance mechanism indicated a strong molecular link of ART resistance with upregulated expression of unfolded protein response pathways involving Prefoldins (PFDs). However, a complete characterization of PFDs as molecular players taking part in ART resistance mechanism, and discovery of small molecule inhibitors to block this process have not been identified to date. Here, we functionally characterized all Pf Prefoldin subunits (PFD1-6) and established a causative role played by PFDs in ART resistance by demonstrating their expression in intra-erythrocytic parasites along with their interactions with Kelch13 protein through immunoprecipitation coupled MS/MS analysis. Systematic biophysical interaction analysis between all subunits of PFDs revealed their potential to form a complex. The role of PFDs in ART resistance was confirmed in orthologous yeast PFD6 mutants, where PfPFD6 expression in yeast mutants reverted phenotype to ART resistance. We identified an FDA-approved drug "Biperiden" that restricts the formation of Prefoldin complex and inhibits its interaction with its key parasite protein substrates, MSP-1 and α-tubulin-I. Moreover, Biperiden treatment inhibits the parasite growth in ART-sensitive Pf3D7 and resistant Pf3D7k13 R539T strains. Ring survival assays that are clinically relevant to analyze ART resistance in Pf3D7k13 R539T parasites demonstrate the potency of BPD to inhibit the growth of survivor parasites. Overall, our study provides the first evidence of the role of PfPFDs in ART resistance mechanisms and opens new avenues for the management of resistant parasites., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be 'hijacked' for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance., (© 2024. The Author(s).)

With resistance to most antimalarials increasing, it is imperative that new drugs are developed. We previously identified an aryl acetamide compound, MMV006833 (M-833), that inhibited the ring-stage development of newly invaded merozoites. Here, we select parasites resistant to M-833 and identify mutations in the START lipid transfer protein (PF3D7_0104200, PfSTART1). Introducing PfSTART1 mutations into wildtype parasites reproduces resistance to M-833 as well as to more potent analogues. PfSTART1 binding to the analogues is validated using organic solvent-based Proteome Integral Solubility Alteration (Solvent PISA) assays. Imaging of invading merozoites shows the inhibitors prevent the development of ring-stage parasites potentially by inhibiting the expansion of the encasing parasitophorous vacuole membrane. The PfSTART1-targeting compounds also block transmission to mosquitoes and with multiple stages of the parasite's lifecycle being affected, PfSTART1 represents a drug target with a new mechanism of action., (© 2024. The Author(s).)

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm 5 s 2 U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s 2 U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s 2 U reprogramming that ART-resistant parasites may employ to survive ART-induced stress., (© 2024. The Author(s).)

Malaria tropica , caused by the parasite Plasmodium falciparum ( P. falciparum ), remains one of the greatest public health burdens for humankind. Due to its pivotal role in parasite survival, the energy metabolism of P. falciparum is an interesting target for drug design. To this end, analysis of the central metabolite adenosine triphosphate (ATP) is of great interest. So far, only cell-disruptive or intensiometric ATP assays have been available in this system, with various drawbacks for mechanistic interpretation and partly inconsistent results. To address this, we have established fluorescent probes, based on Förster resonance energy transfer (FRET) and known as ATeam, for use in blood-stage parasites. ATeams are capable of measuring MgATP 2- levels in a ratiometric manner, thereby facilitating in cellulo measurements of ATP dynamics in real-time using fluorescence microscopy and plate reader detection and overcoming many of the obstacles of established ATP analysis methods. Additionally, we established a superfolder variant of the ratiometric pH sensor pHluorin (sfpHluorin) in P. falciparum to monitor pH homeostasis and control for pH fluctuations, which may affect ATeam measurements. We characterized recombinant ATeam and sfpHluorin protein in vitro and stably integrated the sensors into the genome of the P. falciparum NF54 attB cell line. Using these new tools, we found distinct sensor response patterns caused by several different drug classes. Arylamino alcohols increased and redox cyclers decreased ATP; doxycycline caused first-cycle cytosol alkalization; and 4-aminoquinolines caused aberrant proteolysis. Our results open up a completely new perspective on drugs' mode of action, with possible implications for target identification and drug development., Competing Interests: The authors declare no conflict of interest.

The molecular chaperone heat shock protein 90 (Hsp90) has an essential but largely undefined role in maintaining proteostasis in Plasmodium falciparum, the most lethal malaria parasite. Herein, we identify BX-2819 and XL888 as potent P. falciparum (Pf)Hsp90 inhibitors. Derivatization of XL888's scaffold led to the development of Tropane 1, as a PfHsp90-selective binder with nanomolar affinity. Hsp90 inhibitors exhibit anti-Plasmodium activity against the liver, asexual blood, and early gametocyte life stages. Thermal proteome profiling was implemented to assess PfHsp90-dependent proteome stability, and the proteasome-the main site of cellular protein recycling-was enriched among proteins with perturbed stability upon PfHsp90 inhibition. Subsequent biochemical and cellular studies suggest that PfHsp90 directly promotes proteasome hydrolysis by chaperoning the active 26S complex. These findings expand our knowledge of the PfHsp90-dependent proteome and protein quality control mechanisms in these pathogenic parasites, as well as further characterize this chaperone as a potential antimalarial drug target., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

New drugs with novel modes of action are needed to safeguard malaria treatment. In recent years, millions of compounds have been tested for their ability to inhibit the growth of asexual blood-stage Plasmodium falciparum parasites, resulting in the identification of thousands of compounds with antiplasmodial activity. Determining the mechanisms of action of antiplasmodial compounds informs their further development, but remains challenging. A relatively high proportion of compounds identified as killing asexual blood-stage parasites show evidence of targeting the parasite's plasma membrane Na + -extruding, H + -importing pump, PfATP4. Inhibitors of PfATP4 give rise to characteristic changes in the parasite's internal [Na + ] and pH. Here, we designed a "pH fingerprint" assay that robustly identifies PfATP4 inhibitors while simultaneously allowing the detection of (and discrimination between) inhibitors of the lactate:H + transporter PfFNT, which is a validated antimalarial drug target, and the V-type H + ATPase, which was suggested as a possible target of the clinical candidate ZY19489. In our pH fingerprint assays and subsequent secondary assays, ZY19489 did not show evidence for the inhibition of pH regulation by the V-type H + ATPase, suggesting that it has a different mode of action in the parasite. The pH fingerprint assay also has the potential to identify protonophores, inhibitors of the acid-loading Cl - transporter(s) (for which the molecular identity(ies) remain elusive), and compounds that act through inhibition of either the glucose transporter PfHT or glycolysis. The pH fingerprint assay therefore provides an efficient starting point to match a proportion of antiplasmodial compounds with their mechanisms of action.

Malaria is a parasitic disease that remains a global concern and the subject of many studies. Metabolomics has emerged as an approach to better comprehend complex pathogens and discover possible drug targets, thus giving new insights that can aid in the development of antimalarial therapies. However, there is no standardized method to extract metabolites from in vitro Plasmodium falciparum intraerythrocytic parasites, the stage that causes malaria. Additionally, most methods are developed with either LC-MS or NMR analysis in mind, and have rarely been evaluated with both tools. In this work, three extraction methods frequently found in the literature were reproduced and samples were analyzed through both LC-MS and 1 H NMR, and evaluated in order to reveal which is the most repeatable and consistent through an array of different tools, including chemometrics, peak detection and annotation. The most reliable method in this study proved to be a double extraction with methanol and methanol/water (80:20, v/v). Metabolomic studies in the field should move towards standardization of methodologies and the use of both LC-MS and 1 H NMR in order to make data more comparable between studies and facilitate the achievement of biologically interpretable information., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plasmodium multi-resistance, including against artemisinin, seriously threatens malaria treatment and control. Hence, new drugs are urgently needed, ideally targeting different parasitic stages, which are not yet targeted by current drugs. The SUB1 protease is involved in both hepatic and blood stages due to its essential role in the egress of parasites from host cells, and, as potential new target, it would meet the above criteria. We report here the synthesis as well as the biological and structural evaluation of substrate-based α-ketoamide SUB1 pseudopeptidic inhibitors encompassing positions P4-P2'. By individually substituting each position of the reference compound 1 (MAM-117, Ac-Ile-Thr-Ala-AlaCO-Asp-Glu (Oall)-NH 2 ), we better characterized the structural determinants for SUB1 binding. We first identified compound 8 with IC 50 values of 50 and 570 nM against Pv- and PfSUB1, respectively (about 3.5-fold higher potency compared to 1). Compound 8 inhibited P. falciparum merozoite egress in culture by 37% at 100 μM. By increasing the overall hydrophobicity of the compounds, we could improve the PfSUB1 inhibition level and antiparasitic activity, as shown with compound 40 (IC 50 values of 12 and 10 nM against Pv- and PfSUB1, respectively, IC 50 value of 23 μM on P. falciparum merozoite egress). We also found that 8 was highly selective towards SUB1 over three mammalian serine peptidases, supporting the promising value of this compound. Finally, several crystal 3D-structures of SUB1-inhibitor complexes, including with 8, were solved at high resolution to decipher the binding mode of these compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Global efforts to eradicate malaria are threatened by multiple factors, particularly the emergence of antimalarial drug resistant strains of Plasmodium falciparum. Heat shock proteins (HSPs), particularly P. falciparum HSPs (PfHSPs), represent promising drug targets due to their essential roles in parasite survival and virulence across the various life cycle stages. Despite structural similarities between human and malarial HSPs posing challenges, there is substantial evidence for subtle differences that could be exploited for selective drug targeting. This review provides an update on the potential of targeting various PfHSP families (particularly PfHSP40, PfHSP70, and PfHSP90) and their interactions within PfHSP complexes as a strategy to develop new antimalarial drugs. In addition, the need for a deeper understanding of the role of HSP complexes at the host-parasite interface is highlighted, especially heterologous partnerships between human and malarial HSPs, as this opens novel opportunities for targeting protein-protein interactions crucial for malaria parasite survival and pathogenesis., Competing Interests: Declarations of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Tanzania is currently implementing therapeutic efficacy studies (TES) in areas of varying malaria transmission intensities as per the World Health Organization (WHO) recommendations. In TES, distinguishing reinfection from recrudescence is critical for the determination of anti-malarial efficacy. Recently, the WHO recommended genotyping polymorphic coding genes, merozoite surface proteins 1 and 2 (msp1 and msp2), and replacing the glutamate-rich protein (glurp) gene with one of the highly polymorphic microsatellites in Plasmodium falciparum to adjust the efficacy of antimalarials in TES. This study assessed the polymorphisms of six neutral microsatellite markers and their potential use in TES, which is routinely performed in Tanzania., Methods: Plasmodium falciparum samples were obtained from four TES sentinel sites, Kibaha (Pwani), Mkuzi (Tanga), Mlimba (Morogoro) and Ujiji (Kigoma), between April and September 2016. Parasite genomic DNA was extracted from dried blood spots on filter papers using commercial kits. Genotyping was done using six microsatellites (Poly-α, PfPK2, TA1, C3M69, C2M34 and M2490) by capillary method, and the data were analysed to determine the extent of their polymorphisms and genetic diversity at the four sites., Results: Overall, 83 (88.3%) of the 94 samples were successfully genotyped (with positive results for ≥ 50.0% of the markers), and > 50.0% of the samples (range = 47.6-59.1%) were polyclonal, with a mean multiplicity of infection (MOI) ranging from 1.68 to 1.88 among the four sites. There was high genetic diversity but limited variability among the four sites based on mean allelic richness (R S  = 7.48, range = 7.27-8.03, for an adjusted minimum sample size of 18 per site) and mean expected heterozygosity (H e  = 0.83, range = 0.80-0.85). Cluster analysis of haplotypes using STRUCTURE, principal component analysis, and pairwise genetic differentiation (F ST ) did not reveal population structure or clustering of parasites according to geographic origin. Of the six markers, Poly-α was the most polymorphic, followed by C2M34, TA1 and C3M69, while M2490 was the least polymorphic., Conclusion: Microsatellite genotyping revealed high polyclonality and genetic diversity but no significant population structure. Poly-α, C2M34, TA1 and C3M69 were the most polymorphic markers, and Poly-α alone or with any of the other three markers could be adopted for use in TES in Tanzania., (© 2024. The Author(s).)

Severe malaria is a life-threatening condition that is associated with a high mortality. Severe Plasmodium falciparum infections are mediated primarily by high parasitemia and binding of infected red blood cells (iRBCs) to the blood vessel endothelial layer, a process known as sequestration. Here, we show that including the 5-amino-2-methoxybenzenesulfonate (AMBS) chemical modification in soluble biopolymers (polyglutamic acid and heparin) and poly(acrylic acid)-exposing nanoparticles serves as a universal tool to introduce a potent parasite invasion inhibitory function in these materials. Importantly, the modification did not add or eliminated (for heparin) undesired anticoagulation activity. The materials protected RBCs from invasion by various parasite strains, employing both major entry pathways. Two further P. falciparum strains, which either expose ligands for chondroitin sulfate A (CSA) or intercellular adhesion molecule 1 (ICAM-1) on iRBCs, were tested in antisequestration assays due to their relevance in placental and cerebral malaria, respectively. Antisequestration activity was found to be more efficacious with nanoparticles vs gold-standard soluble biopolymers (CSA and heparin) against both strains, when tested on receptor-coated dishes. The nanoparticles also efficiently inhibited and reversed the sequestration of iRBCs on endothelial cells. First, the materials described herein have the potential to reduce the parasite burden by acting at the key multiplication stage of reinvasion. Second, the antisequestration ability could help remove iRBCs from the blood vessel endothelium, which could otherwise cause vessel obstruction, which in turn can lead to multiple organ failure in severe malaria infections. This approach represents a further step toward creation of adjunctive therapies for this devastating condition to reduce morbidity and mortality.

The rise of Plasmodium falciparum resistance to Artemisinin-based combination therapies (ACTs) is a significant concern in the fight against malaria. This situation calls for the search for novel anti-malarial candidates. 1-deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) is a potential target involved in various cellular processes in P. falciparum (Pf). We screened ∼0.69 billion novel compounds from the ZINC20 library and repurposed ∼1400 FDA drugs using computational drug discovery methods against PfIspC. Following our computational pipeline, we found five novel ZINC20 compounds (Z-2, Z-3, Z-10, Z-13, and Z-14) and three FDA drugs (Aliskiren, Ceftolozane, and Ombitasvir) that showed striking docking energy (ranging from -8.405 to -10.834 kcal/mol), and strong interactions with key binding site residues (Ser269, Ser270, Ser306, Asn311, Lys312, and Met360) of PfIspC. The novel anti-malarial compounds also exhibited favorable pharmacokinetics and physicochemical properties. Furthermore, through molecular dynamics simulation, we observed the stable dynamics of PfIspC-inhibitor complexes and the influence of inhibitor binding on the protein's conformational arrangements. Notably, the binding free energy estimation confirmed high binding affinity (varied from -11.68 to -33.16 kcal/mol) of these compounds for PfIspC. Our findings could contribute to the ongoing efforts in combating malaria and invite experimental-lab researchers for validation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Many natural products have been shown to possess antiplasmodial activities, but their protein targets are unknown. This work employed molecular docking and molecular dynamics simulations to explore the inhibitory activity of some antiplasmodial natural products against wild-type and mutant strains of Plasmodium falciparum dihydrofolate reductase ( Pf DHFR). From the molecular docking study, 6 ligands preferentially bind at the active site of the DHFR domain with binding energies ranging from -6.4 to -9.5 kcal/mol. Interactions of compounds with MET55 and PHE58 were mostly observed in the molecular docking study. From the molecular dynamics study, the binding of 2 of the ligands-nitidine and oplodiol-was observed to be stable against all tested strains of Pf DHFR. The average binding free energy of oplodiol in complex with the various Pf DHFR strains was -93.701 kJ/mol whereas that of nitidine was -106.206 kJ/mol. The impressive in silico activities of the 2 compounds suggest they could be considered for development as potential antifolate agents.Communicated by Ramaswamy H. Sarma.

Aim: To assess the functional relevance of a putative Major Facilitator Superfamily protein (PF3D7_0210300; ' Pf MFSDT') as a drug transporter, using Candida glabrata for orthologous protein expression. Methods: Complementary Determining Sequence encoding Pf MFSDT was integrated into the genome of genetically engineered C. glabrata strain MSY8 via homologous recombination, followed by assessing its functional relevance as a drug transporter. Results & conclusion: The modified C. glabrata strain exhibited plasma membrane localization of Pf MFSDT and characteristics of an Major Facilitator Superfamily transporter, conferring resistance to antifungals, ketoconazole and itraconazole. The nanomolar inhibitory effects of the drugs on the intra-erythrocytic growth of Plasmodium falciparum highlight their antimalarial properties. This study proposes Pf MFSDT as a drug transporter, expanding the repertoire of the currently known antimalarial 'resistome'.

Introduction: MKT-077 and its derivatives are rhodacyanine inhibitors that hold potential in the treatment of cancer, neurodegenerative diseases and malaria. These allosteric drugs act by inhibiting the ATPase action of heat shock proteins of 70 kDa (HSP70). MKT-077 accumulates in the mitochondria and displays differential activity against HSP70 homologs., Methods: The four Plasmodium falciparum HSP70s (PfHSP70) are present in various subcellular locations to perform distinct functions. In the present study, we have used bioinformatics tools to understand the interaction of MKT-077 at the ADP and HEW (2-amino 4 bromopyridine) binding sites on PfHSP70s. Our molecular docking experiments predict that the mitochondrial and endoplasmic reticulum PfHSP70 homologs are likely to bind MKT-077 with higher affinities at their ADP binding sites., Results: Binding analysis indicates that the nature of the identified interactions is primarily hydrophobic. We have also identified specific residues of PfHSP70s that are involved in interacting with the ligand., Conclusion: Information obtained in this study may form the foundation for the design and development of MKT-077-based drugs against malaria., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Solebo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Emerging resistance against artemisinin (ART) poses a major challenge in controlling malaria. Parasites with mutations in PfKelch13, the major marker for ART resistance, are known to reduce hemoglobin endocytosis, induce unfolded protein response (UPR), elevate phosphatidylinositol-3-phosphate (PI3P) levels, and stimulate autophagy. Nonetheless, PfKelch13-independent resistance is also reported, indicating extensive complementation by reconfiguration in the parasite metabolome and transcriptome. These findings implicate that there may not be a single 'universal identifier' of ART resistance. This review sheds light on the molecular, transcriptional, and metabolic pathways associated with ART resistance, while also highlighting the interplay between cellular heterogeneity, environmental stress, and ART sensitivity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Single amino acid changes in the parasite protein Kelch13 (K13) result in reduced susceptibility of P. falciparum parasites to artemisinin and its derivatives (ART). Recent work indicated that K13 and other proteins co-localising with K13 (K13 compartment proteins) are involved in the endocytic uptake of host cell cytosol (HCCU) and that a reduction in HCCU results in reduced susceptibility to ART. HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process. Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site. Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins. While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion. Overall, this work identified novel proteins functioning in endocytosis and at the K13 compartment. Together with comparisons of structural predictions it provides a repertoire of functional domains at the K13 compartment that indicate a high level of adaption of endocytosis in malaria parasites., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Ethiopia has the greatest burden of Plasmodium vivax in Africa, but little is known about the epidemiological landscape of parasites across the country. We analysed the genomic diversity of 137 P. vivax isolates collected nine Ethiopian districts from 2012 to 2016. Signatures of selection were detected by cross-country comparisons with isolates from Thailand (n = 104) and Indonesia (n = 111), representing regions with low and high chloroquine resistance respectively. 26% (35/137) of Ethiopian infections were polyclonal, and 48.5% (17/35) of these comprised highly related clones (within-host identity-by-descent > 25%), indicating frequent co-transmission and superinfection. Parasite gene flow between districts could not be explained entirely by geographic distance, with economic and cultural factors hypothesised to have an impact on connectivity. Amplification of the duffy binding protein gene (pvdbp1) was prevalent across all districts (16-75%). Cross-population haplotype homozygosity revealed positive selection in a region proximal to the putative chloroquine resistance transporter gene (pvcrt-o). An S25P variant in amino acid transporter 1 (pvaat1), whose homologue has recently been implicated in P. falciparum chloroquine resistance evolution, was prevalent in Ethiopia (96%) but not Thailand or Indonesia (35-53%). The genomic architecture in Ethiopia highlights circulating variants of potential public health concern in an endemic setting with evidence of stable transmission., (© 2023. The Author(s).)

Nutrient import and waste efflux are critical dependencies for intracellular Plasmodium falciparum parasites. Nutrient transport proteins are often lineage specific and can provide unique targets for antimalarial drug development. P. falciparum nutrient transport pathways can be a double-edged sword for the parasite, not only mediating the import of nutrients and excretion of waste products but also providing an access route for drugs. Here we briefly summarise the nutrient acquisition pathways of intracellular P. falciparum blood-stage parasites and then highlight how these pathways influence many aspects relevant to antimalarial drugs, resulting in complex and often underappreciated interdependencies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plasmodium falciparum causes the most severe malaria and is exposed to various environmental and physiological stresses in the human host. Given that GCN5 plays a critical role in regulating stress responses in model organisms, we aimed to elucidate PfGCN5's function in stress responses in P. falciparum . The protein level of PfGCN5 was substantially induced under three stress conditions [heat shock, low glucose starvation, and dihydroartemisinin, the active metabolite of artemisinin (ART)]. With a TetR-DOZI conditional knockdown (KD) system, we successfully down-regulated PfGCN5 to ~50% and found that KD parasites became more sensitive to all three stress conditions. Transcriptomic analysis via RNA-seq identified ~1,000 up- and down-regulated genes in the wild-type (WT) and KD parasites under these stress conditions. Importantly, DHA induced transcriptional alteration of many genes involved in many aspects of stress responses, which were heavily shared among the altered genes under heat shock and low glucose conditions, including ART-resistance-related genes such as K13 and coronin . Based on the expression pattern between WT and KD parasites under three stress conditions, ~300-400 genes were identified to be involved in PfGCN5-dependent, general, and stress-condition-specific responses with high levels of overlaps among three stress conditions. Notably, using ring-stage survival assay, we found that KD or inhibition of PfGCN5 could sensitize the ART-resistant parasites to the DHA treatment. All these indicate that PfGCN5 is pivotal in regulating general and ART-resistance-related stress responses in malaria parasites, implicating PfGCN5 as a potential target for malaria intervention., Competing Interests: The authors declare no conflict of interest.

The nucleotidase ISN1 is a potential therapeutic target of the purine salvage pathway of the malaria parasite Plasmodium falciparum. We identified PfISN1 ligands by in silico screening of a small library of nucleos(t)ide analogues and by thermal shift assays. Starting from a racemic cyclopentyl carbocyclic phosphonate scaffold, we explored the diversity on the nucleobase moiety and also proposed a convenient synthetic pathway to access the pure enantiomers of our initial hit (compound (±)-2). 2,6-Disubstituted purine containing derivatives such as compounds 1, (±)-7e and β-L-(+)-2 showed the most potent inhibition of the parasite in vitro, with low micromolar IC 50 values. These results are remarkable considering the anionic nature of nucleotide analogues, which are known to lack activity in cell culture experiments due to their scarce capacity to cross cell membranes. For the first time, we report the antimalarial activity of a carbocyclic methylphosphonate nucleoside with an L-like configuration., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Nushin Aghajari reports financial support was provided by French National Research Agency., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Diagnosis and treatment of Plasmodium falciparum infections are required for effective malaria control and are pre-requisites for malaria elimination efforts; hence we need to monitor emergence, evolution and spread of drug- and diagnostics-resistant parasites. We deep sequenced key drug-resistance mutations and 1,832 SNPs in the parasite genomes of 609 malaria cases collected during a diagnostic-resistance surveillance study in Ethiopia. We found that 8.0% (95% CI 7.0-9.0) of malaria cases were caused by P. falciparum carrying the candidate artemisinin partial-resistance kelch13 (K13) 622I mutation, which was less common in diagnostic-resistant parasites mediated by histidine-rich proteins 2 and 3 (pfhrp2/3) deletions than in wild-type parasites (P = 0.03). Identity-by-descent analyses showed that K13 622I parasites were significantly more related to each other than to wild type (P < 0.001), consistent with recent expansion and spread of this mutation. Pfhrp2/3-deleted parasites were also highly related, with evidence of clonal transmissions at the district level. Of concern, 8.2% of K13 622I parasites also carried the pfhrp2/3 deletions. Close monitoring of the spread of combined drug- and diagnostic-resistant parasites is needed., (© 2023. The Author(s).)

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The implementation of artemisinin (ART) combination therapies (ACTs) has greatly decreased deaths caused by Plasmodium falciparum malaria, but increasing ACT resistance in Southeast Asia and Africa could reverse this progress. Parasite population genetic studies have identified numerous genes, single-nucleotide polymorphisms (SNPs), and transcriptional signatures associated with altered artemisinin activity with SNPs in the Kelch13 (K13) gene being the most well-characterized artemisinin resistance marker. However, there is an increasing evidence that resistance to artemisinin in P. falciparum is not related only to K13 SNPs, prompting the need to characterize other novel genes that can alter ART responses in P. falciparum . In our previous analyses of P. falciparum piggyBac mutants, several genes of unknown function exhibited increased sensitivity to artemisinin that was similar to a mutant of K13. Further analysis of these genes and their gene co-expression networks indicated that the ART sensitivity cluster was functionally linked to DNA replication and repair, stress responses, and maintenance of homeostatic nuclear activity. In this study, we have characterized PF3D7&#95;1136600, another member of the ART sensitivity cluster. Previously annotated as a conserved Plasmodium gene of unknown function, we now provide putative annotation of this gene as a Modulator of Ring Stage Translation (MRST). Our findings reveal that the mutagenesis of MRST affects gene expression of multiple translation-associated pathways during the early ring stage of asexual development via putative ribosome assembly and maturation activity, suggesting an essential role of MRST in protein biosynthesis and another novel mechanism of altering the parasite's ART drug response.IMPORTANCE Plasmodium falciparum malaria killed more than 600,000 people in 2021, though ACTs have been critical in reducing malaria mortality as a first-line treatment for infection. However, ACT resistance in Southeast Asia and emerging resistance in Africa are detrimental to this progress. Mutations to Kelch13 (K13) have been identified to confer increased artemisinin tolerance in field isolates, however, genes other than K13 are implicated in altering how the parasite responds to artemisinin prompts additional analysis. Therefore, in this study we have characterized a P. falciparum mutant clone with altered sensitivity to artemisinin and identified a novel gene (PF3D7&#95;1136600) that is associated with alterations to parasite translational metabolism during critical timepoints for artemisinin drug response. Many genes of the P. falciparum genome remain unannotated, posing a challenge for drug-gene characterizations in the parasite. Therefore, through this study, we have putatively annotated PF3D7&#95;1136600 as a novel MRST gene and have identified a potential link between MRST and parasite stress response mechanisms., Competing Interests: The authors declare no conflict of interest.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter (or PfCRT) were shown to be causative of decreased sensitivity to diverse quinoline-based antimalarials. In this report we describe the identification of a post-translational variant of PfCRT using highly characterized antibodies raised against its N- and C-terminal cytoplasmic domains (e.g., 58 and 26 amino acids, respectively). Western blot analyses of P. falciparum protein extracts with anti N-PfCRT antiserum revealed two polypeptides with apparent molecular masses of 52 kDa and 42 kDa, relative to the calculated molecular mass of PfCRT of 48.7 kDa. The 52 kDa polypeptide was detectable with anti C-PfCRT antiserum, only after alkaline phosphatase treatment of P. falciparum extracts. Detailed epitope mapping of anti N- and C-PfCRT antisera revealed epitopes covering two previously identified phosphorylation sites, Ser 411 and Thr 416 , whereby substitution of these residues with Asp amino acid, to mimic phosphorylated residues, dramatically inhibited anti C-PfCRT binding. Consistently, alkaline phosphatase treatment of P. falciparum extract unmasked the binding of anti C-PfCRT to the 52 kDa polypeptide, suggesting that the 52 kDa but not 42 kDa polypeptide is phosphorylated at its C-terminal Ser 411 and Thr 416 . Interestingly, Pfcrt expressed in HEK-293F human kidney cells showed the same reactive polypeptides with anti N- and C-PfCRT antisera, consistent with PfCRT origin of the two polypeptides (e.g., 42 kDa and 52 kDa), but lacking PfCRT phosphorylation at its C-terminal. Immunohistochemical staining of late trophozoite-infected erythrocytes with anti N-or C-PfCRT antisera showed both polypeptides are localized to the parasite's digestive vacuole. Moreover, both polypeptides are detected in chloroquine-susceptible and -resistant strains of P. falciparum. This is the first report describing a post-translationally modified variant of PfCRT. The physiologic role of the 52 kDa phosphorylated PfCRT in P. falciparum remains to be determined., Competing Interests: Declaration of competing interest Both authors agreed to the submission of this manuscript to Biochemical Biophysical Research Communication. Moreover, there are no conflicts of interest from any of the authors., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l-lactate/H + from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target. Small, drug-like FNT inhibitors potently block lactate transport and kill Plasmodium falciparum parasites in culture. The protein structure of Plasmodium falciparum FNT (PfFNT) in complex with the inhibitor has been resolved and confirms its previously predicted binding site and its mode of action as a substrate analog. Here, we investigated the mutational plasticity and essentiality of the PfFNT target on a genetic level, and established its in vivo druggability using mouse malaria models. We found that, besides a previously identified PfFNT G107S resistance mutation, selection of parasites at 3 × IC 50 (50% inhibitory concentration) gave rise to two new point mutations affecting inhibitor binding: G21E and V196L. Conditional knockout and mutation of the PfFNT gene showed essentiality in the blood stage, whereas no phenotypic defects in sexual development were observed. PfFNT inhibitors mainly targeted the trophozoite stage and exhibited high potency in P. berghei- and P. falciparum-infected mice. Their in vivo activity profiles were comparable to that of artesunate, demonstrating strong potential for the further development of PfFNT inhibitors as novel antimalarials., Competing Interests: The authors declare no conflict of interest.

Context: Modulation of disease progression is frequently started by identifying biochemical pathway catalyzed by biomolecule that is prone to inhibition by small molecular weight ligands. Such ligands (leads) can be obtained from natural resources or synthetic libraries. However, de novo design based on fragments assembly and optimization is showing increasing success. Plasmodium falciparum parasite depends on glutathione-S-transferase (PfGST) in buffering oxidative heme as an approach to resist some antimalarials. Therefore, PfGST is considered an attractive target for drug development. In this research, fragment-based approaches were used to design molecules that can fit to glutathione (GSH) binding site (G-site) of PfGST., Methods: The involved approaches build molecules from fragments that are either isosteric to GSH sub-moieties (ligand-based) or successfully docked to GSH binding sub-pockets (structure-based). Compared to reference GST inhibitor of S-hexyl GSH, ligands with improved rigidity, synthetic accessibility, and affinity to receptor were successfully designed. The method involves joining fragments to create ligands. The ligands were then explored using molecular docking, Cartesian coordinate's optimization, and simplified free energy determination as well as MD simulation and MMPBSA calculations. Several tools were used which include OPENEYE toolkit, Open Babel, Autodock Vina, Gromacs, and SwissParam server, and molecular mechanics force field of MMFF94 for optimization and CHARMM27 for MD simulation. In addition, in-house scripts written in Matlab were used to control fragments connection and automation of the tools., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs.

The chloroquine resistance transporter, PfCRT, of the human malaria parasite Plasmodium falciparum is sensitive to acidic pH. Consequently, PfCRT operates at 60% of its maximal drug transport activity at the pH of 5.2 of the digestive vacuole, a proteolytic organelle from which PfCRT expels drugs interfering with heme detoxification. Here we show by alanine-scanning mutagenesis that E207 is critical for pH sensing. The E207A mutation abrogates pH-sensitivity, while preserving drug substrate specificity. Substituting E207 with Asp or His, but not other amino acids, restores pH-sensitivity. Molecular dynamics simulations and kinetics analyses suggest an allosteric binding model in which PfCRT can accept both protons and chloroquine in a partial noncompetitive manner, with increased proton concentrations decreasing drug transport. Further simulations reveal that E207 relocates from a peripheral to an engaged location during the transport cycle, forming a salt bridge with residue K80. We propose that the ionized carboxyl group of E207 acts as a hydrogen acceptor, facilitating transport cycle progression, with pH sensing as a by-product., (© 2023. The Author(s).)

Introduction: Pyrimidine nucleotides are essential for the parasite's growth and replication. Parasites have only a de novo pathway for the biosynthesis of pyrimidine nucleotides. Dihydroorotate dehydrogenase (DHODH) enzyme is involved in the rate-limiting step of the pyrimidine biosynthesis pathway. DHODH is a biochemical target for the discovery of new antimalarial agents., Area Covered: This review discussed the development of patented Pf DHODH inhibitors published between 2007 and 2023 along with their chemical structures and activities., Expert Opinion: Pf DHODH enzyme is involved in the rate-limiting fourth step of the pyrimidine biosynthesis pathway. Thus, inhibition of Pf DHODH using species-selective inhibitors has drawn much attention for treating malaria because they inhibit parasite growth without affecting normal human functions. Looking at the current scenario of antimalarial drug resistance with most of the available antimalarial drugs, there is a huge need for targeted newer agents. Newer agents with unique mechanisms of action may be devoid of drug toxicity, adverse effects, and the ability of parasites to quickly gain resistance, and Pf DHODH inhibitors can be those newer agents. Many Pf DHODH inhibitors were patented in the past, and the dependency of Plasmodium on de novo pyrimidine provided a new approach for the development of novel antimalarial agents.

Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure-function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of P f -type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc 1 , and provide guidance for future development of antimalarials targeting Complex III., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Inc.)

Mounting evidence demonstrates that nutritional environment can alter pathogen drug sensitivity. While the rich media used for in vitro culture contains supraphysiological nutrient concentrations, pathogens encounter a relatively restrictive environment in vivo . We assessed the effect of nutrient limitation on the protozoan parasite that causes malaria and demonstrated that short-term growth under physiologically relevant mild nutrient stress (or "metabolic priming") triggers increased tolerance of a potent antimalarial drug. We observed beneficial effects using both short-term survival assays and longer-term proliferation studies, where metabolic priming increases parasite survival to a level previously defined as resistant (>1% survival). We performed these assessments by either decreasing single nutrients that have distinct roles in metabolism or using a media formulation that simulates the human plasma environment. We determined that priming-induced tolerance was restricted to parasites that had newly invaded the host red blood cell, but the effect was not dependent on genetic background. The molecular mechanisms of this intrinsic effect mimic aspects of genetic tolerance, including translational repression and protein export. This finding suggests that regardless of the impact on survival rates, environmental stress could stimulate changes that ultimately directly contribute to drug tolerance. Because metabolic stress is likely to occur more frequently in vivo compared to the stable in vitro environment, priming-induced drug tolerance has ramifications for how in vitro results translate to in vivo studies. Improving our understanding of how pathogens adjust their metabolism to impact survival of current and future drugs is an important avenue of research to slow the evolution of resistance. IMPORTANCE There is a dire need for effective treatments against microbial pathogens. Yet, the continuing emergence of drug resistance necessitates a deeper knowledge of how pathogens respond to treatments. We have long appreciated the contribution of genetic evolution to drug resistance, but transient metabolic changes that arise in response to environmental factors are less recognized. Here, we demonstrate that short-term growth of malaria parasites in a nutrient-limiting environment triggers cellular changes that lead to better survival of drug treatment. We found that these strategies are similar to those employed by drug-tolerant parasites, which suggests that starvation "primes" parasites to survive and potentially evolve resistance. Since the environment of the human host is relatively nutrient restrictive compared to growth conditions in standard laboratory culture, this discovery highlights the important connections among nutrient levels, protective cellular pathways, and resistance evolution., Competing Interests: The authors declare no conflict of interest.

Cladosporin, a unique natural product from the fungus Cladosporium cladosporioides, exhibits nanomolar inhibitory activity against Plasmodium falciparum by targeting its cytosolic lysyl-tRNA synthetase (PfKRS) to inhibit protein biosynthesis. Due to its exquisite selectivity towards pathogenic parasites, cladosporin has become a very promising lead compound for developing antiparasitic drugs to treat drug-resistant malaria and cryptosporidiosis infections. Here we review the recent research progress of cladosporin covering aspects of the chemical synthesis, biosynthesis, bioactivity, cellular target and structure-activity relationship., (© 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound 70 ) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP). The eukaryotic translation initiation factor 3 (EIF3i) subunit I was identified as the main target protein stabilized by compound 70 in Plasmodium falciparum . This protein has never been characterized in malaria parasites. P. falciparum parasite lines were generated expressing either a HA tag or an inducible knockdown of the PfEIF3i gene to further characterize the target protein. PfEIF3i was stabilized in the presence of compound 70 in a cellular thermal shift Western blot assay, pointing that PfEIF3i indeed interacts with quinoline-quinazoline-based inhibitors. In addition, PfEIF3i-inducible knockdown blocks intra-erythrocytic development in the trophozoite stage, indicating that it has a vital function. We show that PfEIF3i is mostly expressed in late intra-erythrocytic stages and localizes in the cytoplasm. Previous mass spectrometry reports show that PfEIF3i is expressed in all parasite life cycle stages. Further studies will explore the potential of PfEIF3i as a target for the design of new antimalarial drugs active all along the life cycle of the parasite.

The chloroquine resistance transporter (PfCRT) confers resistance to a wide range of quinoline and quinoline-like antimalarial drugs in Plasmodium falciparum, with local drug histories driving its evolution and, hence, the drug transport specificities. For example, the change in prescription practice from chloroquine (CQ) to piperaquine (PPQ) in Southeast Asia has resulted in PfCRT variants that carry an additional mutation, leading to PPQ resistance and, concomitantly, to CQ re-sensitization. How this additional amino acid substitution guides such opposing changes in drug susceptibility is largely unclear. Here, we show by detailed kinetic analyses that both the CQ- and the PPQ-resistance conferring PfCRT variants can bind and transport both drugs. Surprisingly, the kinetic profiles revealed subtle yet significant differences, defining a threshold for in vivo CQ and PPQ resistance. Competition kinetics, together with docking and molecular dynamics simulations, show that the PfCRT variant from the Southeast Asian P. falciparum strain Dd2 can accept simultaneously both CQ and PPQ at distinct but allosterically interacting sites. Furthermore, combining existing mutations associated with PPQ resistance created a PfCRT isoform with unprecedented non-Michaelis-Menten kinetics and superior transport efficiency for both CQ and PPQ. Our study provides additional insights into the organization of the substrate binding cavity of PfCRT and, in addition, reveals perspectives for PfCRT variants with equal transport efficiencies for both PPQ and CQ., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gomez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In vitro evolution of drug resistance is a powerful approach for identifying antimalarial targets, however, key obstacles to eliciting resistance are the parasite inoculum size and mutation rate. Here we sought to increase parasite genetic diversity to potentiate resistance selections by editing catalytic residues of Plasmodium falciparum DNA polymerase δ. Mutation accumulation assays reveal a ~5-8 fold elevation in the mutation rate, with an increase of 13-28 fold in drug-pressured lines. Upon challenge with the spiroindolone PfATP4-inhibitor KAE609, high-level resistance is obtained more rapidly and at lower inocula than wild-type parasites. Selections also yield mutants with resistance to an "irresistible" compound, MMV665794 that failed to yield resistance with other strains. We validate mutations in a previously uncharacterised gene, PF3D7&#95;1359900, which we term quinoxaline resistance protein (QRP1), as causal for resistance to MMV665794 and a panel of quinoxaline analogues. The increased genetic repertoire available to this "mutator" parasite can be leveraged to drive P. falciparum resistome discovery., (© 2023. The Author(s).)

Background: Malaria remains one of the most serious public health problems in sub-Saharan Africa and Mozambique is the world's fourth largest contributor, with 4.7% of disease cases and 3.6% of total deaths due to malaria. Its control relies on the fight against the vector and treatment of confirmed cases with anti-malarial drugs. Molecular surveillance is an important tool for monitoring the spread of anti-malarial drug resistance., Methods: A cross-sectional study recruited 450 participants with malaria infection detected by Rapid Diagnostic Tests, from three different study sites (Niassa, Manica and Maputo) between April and August 2021. Correspondent blood samples were collected on filter paper (Whatman® FTA® cards), parasite DNA extracted and pfk13 gene sequenced using Sanger method. SIFT software (Sorting Intolerant From Tolerant) was used, predict whether an amino acid substitution affects protein function., Results: No pfkelch13-mediated artemisinin resistance gene mutation was detected in this study settings. However, non-synonymous mutations were detected at prevalence of 10.2%, 6% and 5% in Niassa, Manica and Maputo, respectively. Most (56.3%) of the reported non-synonymous mutations were due to substitution at the first base of the codon, 25% at the second base and 18.8% at the third base. Additionally, 50% of non-synonymous mutations showed a SIFTscore bellow cut off value of 0.05, therefore, they were predicted to be deleterious., Conclusion: These results do not show an emergence of artemisinin resistance cases in Mozambique. However, the increased number of novel non-synonymous mutations highlights the relevance of increasing the number of studies focused on the molecular surveillance of artemisinin resistance markers, for its early detection., (© 2023. The Author(s).)

Cell surface-bound human Hsp70 (hHsp70) sensitises tumour cells to the cytolytic attack of natural killer (NK) cells through the mediation of apoptosis-inducing serine protease, granzyme B (GrB). hHsp70 is thought to recruit NK cells to the immunological synapse via the extracellularly exposed 14 amino acid sequence, TKDNNLLGRFELSG, known as the TKD motif of Hsp70. Plasmodium falciparum-infected red blood cells (RBCs) habour both hHsp70 and an exported parasite Hsp70 termed PfHsp70-x. Both PfHsp70-x and hHsp70 share conserved TKD motifs. The role of PfHsp70-x in facilitating GrB uptake in malaria parasite-infected RBCs remains unknown, but hHsp70 enables a perforin-independent uptake of GrB into tumour cells. In the current study, we comparatively investigated the direct binding of GrB to either PfHsp70-x or hHsp70 in vitro. Using ELISA, slot blot assay and surface plasmon resonance (SPR) analysis, we demonstrated a direct interaction of GrB with hHsp70 and PfHsp70-x. SPR analysis revealed a higher affinity of GrB for PfHsp70-x than hHsp70. In addition, we established that the TKD motif of PfHsp70-x directly interacts with GrB. The data further suggest that the C-terminal EEVN motif of PfHsp70-x augments the affinity of PfHsp70-x for GrB but is not a prerequisite for the binding. A potent antiplasmodial activity (IC 50 of 0.5 µM) of GrB could be demonstrated. These findings suggest that the uptake of GrB by parasite-infected RBCs might be mediated by both hHsp70 and PfHsp70-x. The combined activity of both proteins could account for the antiplasmodial activity of GrB at the blood stage., (© 2023. The Author(s).)

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Dans et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC 50 s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC 50 s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside (1) with IC 50 s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid (2), ursolic acid (3), quafrinoic acid (4), quinovic acid (5), quinovic acid 3-O-β-D-fucopyranoside (6), oleanolic acid (7), oleanolic acid 3-acetate (8), friedelin (9), β-sitosterol (10a), stigmasterol (10b) and stigmasterol 3-O-β-D-glucopyranoside (11). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside (1) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria., (© 2023 Wiley-VHCA AG, Zurich, Switzerland.)

The appearance in 2008 in western Cambodia of Plasmodium falciparum tolerant to artemisinin, defined by longer parasite clearance time following drug administration and in vitro by a slightly higher survival rate of the ring stage after a 3-h treatment with 700 nM artemisinin (or analogues, collectively termed ART), has raised concerns of the possible loss of this frontline antimalarial [used in the form of an artemisinin combination therapy (ACT)], with its low IC 50 value against the ring stage and pleiotropic pro-drug/poison property. The key genetic marker of ART tolerance phenotype is a number of non-synonymous mutations in Pfkelch13 propeller domain. This results in defective assembly at the ring stage of a cytostome structure located at cytoplasmic side of the parasite membrane required for invagination of a double-membrane endosome carrying host cytosol haemoglobin to the digestive vacuole. The consequential deprivation of amino acids initiates ring stage parasites bearing the causal mutations in PfK13 (or other key cytostome components) entry into a dormant state ("Sleeping Beauty"), which, after a duration longer than that the short-lived ART, "Sleeping Beauty" ring parasite resumes its normal, but accelerated, development to maintain the 48-h intra-erythrocytic life-cycle. We posit that when ART-tolerant P. falciparum has acquired under ART stress the causative PfK13 mutation (not obligatory if mutations occur in other critical cytostome components), together with other necessary mutations to adjust to the new normalcy and to provide survival competitiveness, ART-tolerant parasite has now evolved into a genetically programmed "Sleeping Beauty". The onus of preventing the spread of ART-tolerant P. falciparum lies with the efficacy of ACT partner drug, hence the recommendation of a triple ACT (TACT). Nevertheless, attention should also be focussed on understanding the mechanisms of dormancy, such as induction, maintenance and recovery, to enable discovery and development of novel antimalarials targeting this unique parasite stage., Competing Interests: Declaration of competing interest The authors declare no competing interest., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Identifying how small molecules act to kill malaria parasites can lead to new "chemically validated" targets. By pressuring Plasmodium falciparum asexual blood stage parasites with three novel structurally-unrelated antimalarial compounds (MMV665924, MMV019719 and MMV897615), and performing whole-genome sequence analysis on resistant parasite lines, we identify multiple mutations in the P. falciparum acyl-CoA synthetase (ACS) genes PfACS10 (PF3D7&#95;0525100, M300I, A268D/V, F427L) and PfACS11 (PF3D7&#95;1238800, F387V, D648Y, and E668K). Allelic replacement and thermal proteome profiling validates PfACS10 as a target of these compounds. We demonstrate that this protein is essential for parasite growth by conditional knockdown and observe increased compound susceptibility upon reduced expression. Inhibition of PfACS10 leads to a reduction in triacylglycerols and a buildup of its lipid precursors, providing key insights into its function. Analysis of the PfACS11 gene and its mutations point to a role in mediating resistance via decreased protein stability., (© 2023. The Author(s).)

Current malaria treatments are threatened by drug resistance, and new drugs are urgently needed. In a phenotypic screen for new antimalarials, we identified ( S )-SW228703 (( S )-SW703), a tyrosine amide with asexual blood and liver stage activity and a fast-killing profile. Resistance to ( S )-SW703 is associated with mutations in the Plasmodium falciparum cyclic amine resistance locus ( Pf CARL) and P. falciparum acetyl CoA transporter ( Pf ACT), similarly to several other compounds that share features such as fast activity and liver-stage activity. Compounds with these resistance mechanisms are thought to act in the ER, though their targets are unknown. The tyramine of ( S )-SW703 is shared with some reported Pf CARL-associated compounds; however, we observed that strict S-stereochemistry was required for the activity of ( S )-SW703, suggesting differences in the mechanism of action or binding mode. ( S )-SW703 provides a new chemical series with broad activity for multiple life-cycle stages and a fast-killing mechanism of action, available for lead optimization to generate new treatments for malaria.