Malaria caused by Plasmodium falciparum remains a major health threat, killing over 600,000 people annually. The spread of resistance to all major antimalarials, including artemisinins, highlights the urgent need for new drugs with distinct mechanisms of action. Here we show that the G-quadruplex ligand RHPS4, an acridine derivative, displays strong antiplasmodial activity against both drug-sensitive and -resistant P. falciparum strains and clinical isolates. RHPS4 primarily targets the trophozoite stage and induces major mitochondrial alterations, including reduction of mitochondrial DNA (mtDNA) and transcriptional dysfunctions. Bioinformatic analyses identified at least eight putative G4-forming sequences within the parasite’s mtDNA. Biophysical studies confirmed G4 folding of at least one sequence and its interaction with RHPS4. These findings indicate that RHPS4 disrupts P. falciparum mitochondrial metabolism through G4 stabilization, leading to parasite death, and establish mtDNA G4 structures as novel therapeutic targets for antimalarial development. ### Competing Interest Statement The authors have declared no competing interest.

Current standard of care, artemisinin-based therapies for malaria, are threatened by emerging drug resistance. Developing antimalarials with novel mechanisms of action and low propensity for resistance is of the highest priority. Here, we explore the target landscape of MMV022224, a promising antimalarial that is active against multiple stages of Plasmodium falciparum and refractory to resistance generation. Using two orthogonal chemical proteomics approaches, chemical pulldown and thermal proteome profiling, we demonstrate that MMV022224 binds selectively and with high affinity to the genetically essential P. falciparum protein kinase 6 (PfPK6), as well as to several additional Plasmodium kinases. Enzymatic studies verify that MMV022224 inhibits PfPK6; however, PfPK6 knockdown does not affect parasite compound susceptibility, confirming that PfPK6 inhibition is not the sole driver of antimalarial activity and that MMV022224 may act through broader, kinase-focused polypharmacology. Employing the same chemical proteomics strategies, we demonstrate that the structurally related azaindole, TCMDC-135051, is a selective inhibitor of the cyclin-dependent kinase PfCLK3. Collectively, these studies demonstrate the value of chemical proteomics for antimalarial drug target deconvolution.

Herein we report the finding and structure determination of a natural product based on the methyldeoxaphomin scaffold family from the fungus Trichocladium asperum that shows promising antiplasmodial activity and selectivity against host cells. In vitro evolution and whole genome analysis in Plasmodium falciparum with the most potent member, NPDG-F (EC 50 of 550 nM in Dd2; 290 nM in 3D7), shows that parasite resistance to methyldeoxaphomins is strongly associated with mutations in PfActin1 (PF3D7_1246200), a critically essential ATPase needed for all stages of parasite development. Molecular docking study with available PfActin1 crystal structure shows NPDG-F occupies the same allosteric binding pocket as the known actin inhibitor cytochalasin D. The direct PfActin1 target engagement in the allosteric site was supported by cross-resistance studies, isobologram analysis with other PfActin1 inhibitors, and the structure–activity relationships for the methyldeoxaphomin family. When added to in vitro culture, NPDG-F induced morphological abnormalities in merozoite cellularization during schizogony in both the Plasmodium blood and liver stages. Our data provide chemical validation that PfActin1 is an attractive, pan-lifecycle target and inform strategies for the design of more selective inhibitors.

This review is focused on hybrid molecules defined as chemical entities with two or more structural domains, as antimalarial drug-candidates, over the past 25 years. Due to their different pharmacophores, such hybrids can interact with a single biological target by different and complementary mechanisms; they can also act simultaneously on several targets having complementary biological functions (dual mode of action), and can theoretically reduce the selection of parasite drug-resistance. This review is not an exhaustive report of all hybrid drugs tested on malaria parasites but a selection of hybrids with pharmacologically relevant antiplasmodial properties and original chemical structures. The choice of pharmacophore synthons and junction arms is obviously decisive. Among the large varieties of hybrid drugs published, emoquine-1 appears at the moment as a promising antimalarial drug candidate, considering 1) its high activities on several multidrug-resistant Plasmodium lab strains and field isolates, 2) its capacity to eliminate the quiescent forms of the artemisinin-resistant parasites, and 3) its curative properties in a malaria mouse model. Such molecules confirm the synergistic effect of hybrid compounds compared to the combination of the pharmacophores leading to novel chemical structures that meet the critical parameters for new antimalarial drugs.

Kinase inhibitors are potent therapeutics, but most essential Plasmodium kinases remain unexploited as antimalarial targets. We identified compound 12, a type II kinase inhibitor based on aminopyridine and 2,6-benzimidazole scaffolds, as a lead compound with nanomolar potency, fast action, and in vivo activity in the Plasmodium berghei rodent malaria model. Three-hybrid luciferase fragment complementation, enzymatic studies, and cellular thermal shift assays implicated Plasmodium protein kinase 6 (PfPK6) as the target. However, conditional knockdown of PfPK6 did not alter 12 potency, suggesting complex mechanisms of action. In vitro selection for compound 12 resistance revealed mutations in three transporters: multidrug-resistance protein 1, chloroquine resistance transporter and V-type ATPase, indicating a digestive vacuole site of action. Compound 12 inhibited β-hematin and hemozoin formation while increasing free heme levels, suggesting antimalarial activity via blockade of heme detoxification. Our studies repurpose a safe human kinase inhibitor as a potent, fast-acting antimalarial with established in vivo efficacy.

Elimination of malaria will require new drugs with potent activity against Plasmodium falciparum mature stage V gametocytes, the only stages infective to the mosquito vector. The identification and comprehensive validation of molecules active against these quiescent stages is difficult due to the specific biology of gametocytes, challenges linked to their cultivation in vitro and the lack of animal models suitable for evaluating the transmission-blocking potential of drug candidates in vivo. Here, we present a transmission-blocking drug discovery and development platform that builds on transgenic NF54/iGP1_RE9Hulg8 parasites engineered to conditionally produce large numbers of stage V gametocytes expressing a red-shifted firefly luciferase viability reporter. Besides developing a robust in vitro screening assay for the reliable identification of stage V gametocytocidal compounds, we also establish a preclinical in vivo malaria transmission model based on infecting female humanized NODscidIL2Rγnull mice with pure NF54/iGP1_RE9Hulg8 stage V gametocytes. Using whole animal bioluminescence imaging, we assess the in vivo gametocyte killing and clearance kinetics of antimalarial reference drugs and clinical drug candidates and identify markedly different pharmacodynamic response profiles. Finally, we combine this mouse model with mosquito feeding assays and thus firmly establish a valuable tool for the systematic in vivo evaluation of transmission-blocking drug efficacy.

While progress has been made in the effort to eradicate malaria, the disease remains a significant threat to global health. Acquired resistance to frontline treatments is emerging in Africa, urging a need for the development of novel antimalarial agents. Repurposing human kinase inhibitors provides a potential expedited route given the availability of a diverse array of kinase-targeting drugs that are approved or in clinical trials. Phenotypic screening of a library of type II human kinase inhibitors identified compound 1 as a lead antimalarial, which was initially developed to target human ephrin type A receptor 2 (EphA2). Here, we report a structure-activity relationship study and lead optimization of compound 1, which led to compound 33, with improved antimalarial activity and selectivity.

Our previous work identified a series of 12 xanthoquinodin analogues and 2 emodin-dianthrones with broad-spectrum activities against Trichomonas vaginalis, Mycoplasma genitalium, Cryptosporidium parvum, and Plasmodium falciparum. Analyses conducted in this study revealed that the most active analogue, xanthoquinodin A1, also inhibits Toxoplasma gondii tachyzoites and the liver stage of Plasmodium berghei, with no cross-resistance to the known antimalarial targets PfACS, PfCARL, PfPI4K, or DHODH. In Plasmodium, inhibition occurs prior to multinucleation and induces parasite death following 12 h of compound exposure. This moderately fast activity has impeded resistance line generation, with xanthoquinodin A1 demonstrating an irresistible phenotype in both T. gondii and P. falciparum.

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.

Malaria eradication requires the development of new drugs to combat drug-resistant parasites. We identified bisbenzylisoquinoline alkaloids isolated from Cocculus hirsutus that are active against Plasmodium falciparum blood stages. Synthesis of a library of 94 hemi-synthetic derivatives allowed to identify compound 84 that kills multi-drug resistant clinical isolates in the nanomolar range (median IC50 ranging from 35 to 88 nM). Chemical optimization led to compound 125 with significantly improved preclinical properties. 125 delays the onset of parasitemia in Plasmodium berghei infected mice and inhibits P. falciparum transmission stages in vitro (culture assays), and in vivo using membrane feeding assay in the Anopheles stephensi vector. Compound 125 also impairs P. falciparum development in sporozoite-infected hepatocytes, in the low micromolar range. Finally, by chemical pull-down strategy, we characterized the parasite interactome with trilobine derivatives, identifying protein partners belonging to metabolic pathways that are not targeted by the actual antimalarial drugs or implicated in drug-resistance mechanisms.

We adapted the RNA FISH Stellaris method to specifically detect the expression of Plasmodium genes by flow cytometry and ImageStream (Flow-FISH). This new method accurately quantified the erythrocytic forms of (1) Plasmodium falciparum and Plasmodium vivax and (2) the sexual stages of P vivax from patient isolates. ImageStream analysis of liver stage sporozoites using a combination of surface circumsporozoite protein (CSP), deoxyribonucleic acid, and 18S RNA labeling proved that the new Flow-FISH is suitable for gene expression studies of transmission stages. This powerful multiparametric single-cell method offers a platform of choice for both applied and fundamental research on the biology of malaria parasites.

Malaria eradication requires the development of new drugs to combat drug-resistant parasites. The search for new chemical scaffolds that target novel pathways of the human malaria parasite Plasmodium falciparum is of highest priority. We identified bisbenzylisoquinoline alkaloids isolated from Cocculus hirsutus . (trilobine derivatives) as active in the nanomolar range against P. falciparum blood stages. Synthesis of a library of 94 hemi-synthetic derivatives allowed us to identify compound 84 that kills multi-drug resistant clinical isolates in the nanomolar range (median IC 50 ranging from 35-88nM). Efforts were made to obtain compounds with significantly improved preclinical properties. Out of those, compound 125 delays the onset of parasitemia in P. berghei infected mice and inhibits P. falciparum transmission stages in vitro (culture assays) and in vivo using membrane feeding assay in the Anopheles stephensi vector. Compound 125 also impairs P. falciparum development in sporozoite-infected hepatocytes, in the low micromolar range. Finally, we used a chemical pull-down strategy to identify potential protein targets of this chemical family. Mass spectrometry analysis identified the parasite interactome with trilobine derivatives, identifying protein partners belonging to metabolic pathways that have not been previously targeted by antimalarial drugs or implicated in drug-resistance mechanisms.

Plasmodium vivax is the most widespread human malaria parasite. Due to the presence of extravascular reservoirs and relapsing infections from dormant liver stages, P. vivax is particularly difficult to control and eliminate. Experimental research is hampered by the inability to maintain P. vivax cultures in vitro, due to its tropism for immature red blood cells (RBCs). Here, we describe a new humanized mice model that can support efficient human erythropoiesis and maintain long-lasting multiplication of inoculated cryopreserved P. vivax parasites and their sexual differentiation, including in bone marrow. Mature gametocytes were transmitted to Anopheles mosquitoes, which led to the formation of salivary gland sporozoites. Importantly, blood-stage P. vivax parasites were maintained after the secondary transfer of fresh or frozen infected bone marrow cells to naïve chimeras. This model provides a unique tool for investigating, in vivo, the biology of intraerythrocytic P. vivax .

The Apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that include Toxoplasma gondii , Plasmodium , and Cryptosporidium spp., which infect humans and animals and cause severe parasitic diseases. Available therapeutics against these diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. We use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index when used against T. gondii . We have identified Tg PRP4K of T. gondii as the primary target of altiratinib using genetic target deconvolution, which highlighted key residues within the kinase catalytic site that conferred drug resistance when mutated. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for Tg PRP4K and for its Plasmodium falciparum counterpart, Pf CLK3. Our data identified structural features critical for binding of the other Pf CLK3 inhibitor, TCMDC-135051. Consistent with the splicing control activity of this kinase family, we have shown that altiratinib can cause global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum . Thus, our data establish parasitic PRP4K/CLK3 as a potential pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

A commonly observed survival strategy in protozoan parasites is the sequential expression of clonally variant-surface antigens to avoid elimination by the host's immune response. In malaria-causing P. falciparum, the immunovariant erythrocyte-membrane protein-1 (PfEMP1) adhesin family, encoded by var genes, is responsible for both antigenic variation and cytoadherence of infected erythrocytes to the microvasculature. Until recently, the biological function of these variant genes was believed to be restricted to intraerythrocytic developmental stages. With the advent of new technologies, var gene expression has been confirmed in transmission and pre-erythrocytic stages. Here, we discuss how repurposing of var gene expression beyond chronic blood-stage infection may be critical for successful transmission.

Malaria parasites need to cope with changing environmental conditions that require strong countermeasures to ensure pathogen survival in the human and mosquito hosts. The molecular mechanisms that protect Plasmodium falciparum homeostasis during the complex life cycle remain unknown. Here, we identify cytosine methylation of tRNAAsp (GTC) as being critical to maintain stable protein synthesis. Using conditional knockout (KO) of a member of the DNA methyltransferase family, called Pf-DNMT2, RNA bisulfite sequencing demonstrated the selective cytosine methylation of this enzyme of tRNAAsp (GTC) at position C38. Although no growth defect on parasite proliferation was observed, Pf-DNMT2KO parasites showed a selective downregulation of proteins with a GAC codon bias. This resulted in a significant shift in parasite metabolism, priming KO parasites for being more sensitive to various types of stress. Importantly, nutritional stress made tRNAAsp (GTC) sensitive to cleavage by an unknown nuclease and increased gametocyte production (>6-fold). Our study uncovers an epitranscriptomic mechanism that safeguards protein translation and homeostasis of sexual commitment in malaria parasites. IMPORTANCE P. falciparum is the most virulent malaria parasite species, accounting for the majority of the disease mortality and morbidity. Understanding how this pathogen is able to adapt to different cellular and environmental stressors during its complex life cycle is crucial in order to develop new strategies to tackle the disease. In this study, we identified the writer of a specific tRNA cytosine methylation site as a new layer of epitranscriptomic regulation in malaria parasites that regulates the translation of a subset of parasite proteins (>400) involved in different metabolic pathways. Our findings give insight into a novel molecular mechanism that regulates P. falciparum response to drug treatment and sexual commitment.

Introductory paragraph The apicomplexa comprise a large phylum of single-celled, obligate intracellular protozoa that infect humans and animals and cause severe parasitic diseases. Available therapeutics against these devastating diseases are limited by suboptimal efficacy and frequent side effects, as well as the emergence and spread of resistance. Here, we use a drug repurposing strategy and identify altiratinib, a compound originally developed to treat glioblastoma, as a promising drug candidate with broad spectrum activity against apicomplexans. Altiratinib is parasiticidal and blocks the development of intracellular zoites in the nanomolar range and with a high selectivity index. We have identified Tg PRP4K of T. gondii as the primary target of altiratinib by genetic target deconvolution, highlighting key residues within the kinase catalytic site that, when mutated, confer resistance to the drug. We have further elucidated the molecular basis of the inhibitory mechanism and species selectivity of altiratinib for Tg PRP4K as well as for its P. falciparum counterpart Pf CLK3. Our data also point to structural features critical for binding of the other Pf CLK3 inhibitor, TCMDC-135051. Consistent with the role of this kinase family in splicing in a broad spectrum of eukaryotes, we have shown that altiratinib causes global disruption of splicing, primarily through intron retention in both T. gondii and P. falciparum . Thus, our data establish parasitic PRP4K/CLK3 as a promising pan-apicomplexan target whose repertoire of inhibitors can be expanded by the addition of altiratinib.

Background: Expanding resistance to multiple antimalarials, including chloroquine, in South-East Asia (SEA) urges the development of new therapies. AQ-13, a chloroquine derivative, is a new drug candidate for treating malaria caused by Plasmodium falciparum. Objectives: Possible cross-resistance between the 4-aminoquinolines amodiaquine, piperaquine and AQ-13 has not been assessed. In vitro parasite growth assays were used to characterize the susceptibility of multidrug-resistant and susceptible P. falciparum patient isolates to AQ-13. Methods: A [3H]hypoxanthine uptake assay and a 384-well high content imaging assay were used to assess efficacy of AQ-13 and desethyl-amodiaquine against 38 P. falciparum isolates. Results: We observed a strong cross-resistance between the chloroquine derivative amodiaquine and AQ-13 in Cambodian P. falciparum isolates (Pearson correlation coefficient of 0.8621, P < 0.0001). Conclusions: In light of the poor efficacy of amodiaquine that we described recently in Cambodia, and its cross resistance with AQ-13, there is a significant risk that similar clinical efficacy of AQ-13-based combinations should be anticipated in areas of amodiaquine resistance.