Novel Type II Fatty Acid Biosynthesis (FAS II) Inhibitors as Multistage Antimalarial Agents

Malaria is a potentially fatal disease caused by Plasmodium parasites and poses a major medical risk in large parts of the world. The development of new, affordable antimalarial drugs is of vital importance as there are increasing reports of resistance to the currently available therapeutics. In addition, most of the current drugs used for chemoprophylaxis merely act on parasites already replicating in the blood. At this point, a patient might already be suffering from the symptoms associated with the disease and could additionally be infectious to an Anopheles mosquito. These insects act as a vector, subsequently spreading the disease to other humans. In order to cure not only malaria but prevent transmission as well, a drug must target both the blood‐ and pre‐erythrocytic liver stages of the parasite. P. falciparum (Pf) enoyl acyl carrier protein (ACP) reductase (ENR) is a key enzyme of plasmodial type II fatty acid biosynthesis (FAS II). It has been shown to be essential for liver‐stage development of Plasmodium berghei and is therefore qualified as a target for true causal chemoprophylaxis. Using virtual screening based on two crystal structures of PfENR, we identified a structurally novel class of FAS inhibitors. Subsequent chemical optimization yielded two compounds that are effective against multiple stages of the malaria parasite. These two most promising derivatives were found to inhibit blood‐stage parasite growth with IC₅₀ values of 1.7 and 3.0 μM and lead to a more prominent developmental attenuation of liver‐stage parasites than the gold‐standard drug, primaquine.     

Artificial Intelligence Applied to the Rapid Identification of New Antimalarial Candidates with Dual-Stage Activity

Increasing reports of multidrug-resistant malaria parasites urge the discovery of new effective drugs with different chemical scaffolds. Protein kinases play a key role in many cellular processes such as signal transduction and cell division, making them interesting targets in many diseases. Protein kinase 7 (PK7) is an orphan kinase from the Plasmodium genus, essential for the sporogonic cycle of these parasites. Here, we applied a robust and integrative artificial intelligence-assisted virtual-screening (VS) approach using shape-based and machine learning models to identify new potential PK7 inhibitors with in vitro antiplasmodial activity. Eight virtual hits were experimentally evaluated, and compound LabMol-167 inhibited ookinete conversion of Plasmodium berghei and blood stages of Plasmodium falciparum at nanomolar concentrations with low cytotoxicity in mammalian cells. As PK7 does not have an essential role in the Plasmodium blood stage and our virtual screening strategy aimed for both PK7 and blood-stage inhibition, we conducted an in silico target fishing approach and propose that this compound might also inhibit P. falciparum PK5, acting as a possible dual-target inhibitor. Finally, docking studies of LabMol-167 with P. falciparum PK7 and PK5 proteins highlighted key interactions for further hit-to lead optimization.     

Probing the Azaaurone Scaffold against the Hepatic and Erythrocytic Stages of Malaria Parasites

The potential of azaaurones as dual‐stage antimalarial agents was investigated by assessing the effect of a small library of azaaurones on the inhibition of liver and intraerythrocytic lifecycle stages of the malaria parasite. The whole series was screened against the blood stage of a chloroquine‐resistant Plasmodium falciparum strain and the liver stage of P. berghei, yielding compounds with dual‐stage activity and sub‐micromolar potency against erythrocytic parasites. Studies with genetically modified parasites, using a phenotypic assay based on the P. falciparum Dd2‐ScDHODH line, which expresses yeast dihydroorotate dehydrogenase (DHODH), showed that one of the azaaurone derivatives has the potential to inhibit the parasite mitochondrial electron‐transport chain. The global urgency in finding new therapies for malaria, especially against the underexplored liver stage, associated with chemical tractability of azaaurones, warrants further development of this chemotype. Overall, these results emphasize the azaaurone chemotype as a promising scaffold for dual‐stage antimalarials.     

Chemoproteomics for Plasmodium Parasite Drug Target Discovery

Emerging Plasmodium parasite drug resistance is threatening progress towards malaria control and elimination. While recent efforts in cell-based, high-throughput drug screening have produced first-in-class drugs with promising activities against different Plasmodium life cycle stages, most of these antimalarial agents have elusive mechanisms of action. Though challenging to address, target identification can provide valuable information to facilitate lead optimization and preclinical drug prioritization. Recently, proteome-wide methods for direct assessment of drug-protein interactions have emerged as powerful tools in a number of systems, including Plasmodium. In this review, we will discuss current chemoproteomic strategies that have been adapted to antimalarial drug target discovery, including affinity- and activity-based protein profiling and the energetics-based techniques thermal proteome profiling and stability of proteins from rates of oxidation. The successful application of chemoproteomics to the Plasmodium blood stage highlights the potential of these methods to link inhibitors to their molecular targets in more elusive Plasmodium life stages and intracellular pathogens in the future.     

Characterization of Plasmodium liver stage inhibition by halofuginone

Malaria is a devastating parasitic disease that afflicts one-third of the world's population. Antimalarial drugs in common use address few targets, and their efficacy is being undermined by parasite resistance. Most therapeutics target blood-stage malaria, whereas only few compounds are active against malaria's liver stage, the first stage of the Plasmodium parasite's life cycle within the human host. The identification of inhibitors active against liver-stage malaria would benefit the development of chemical probes to elucidate the poorly understood biology of this phase of the parasite life cycle and could provide agents to prevent and eliminate the disease. Herein we report the development of a live-cell parasite traversal assay in 384-well format amenable to high-throughput screening that exploits the wounding of liver cells by the parasite. This method identifies small molecules that may inhibit the parasite's actin-myosin motor system. The traversal assay, in addition to established methods, was used to evaluate the activity of halofuginone, a synthetic halogenated derivative of the natural alkaloid febrifugine, against liver-stage Plasmodium berghei parasites. Halofuginone was found to inhibit P. berghei sporozoite load in HepG2 cells with an IC(50) value of 17 nM. While the compound does not affect parasite traversal through human liver cells, an inhibition time course assay indicates that it affects essential processes in both early- and late-stage parasite development.     

Pentafluoro-3-hydroxy-pent-2-en-1-ones Potently Inhibit FNT-Type Lactate Transporters from all Five Human-Pathogenic Plasmodium Species

The protozoan parasite Plasmodium falciparum causes the most severe and prevailing form of malaria in sub-Saharan Africa. Previously, we identified the plasmodial lactate transporter, PfFNT, a member of the microbial formate–nitrite transporter family, as a novel antimalarial drug target. With the pentafluoro-3-hydroxy-pent-2-en-1-ones, we discovered PfFNT inhibitors that potently kill P. falciparum parasites in vitro. Four additional human-pathogenic Plasmodium species require attention, that is, P. vivax, most prevalent outside of Africa, and the regional P. malariae, P. ovale and P. knowlesi. Herein, we show that the plasmodial FNT variants are highly similar in terms of protein sequence and functionality. The FNTs from all human-pathogenic plasmodia and the rodent malaria parasite were efficiently inhibited by pentafluoro-3-hydroxy-pent-2-en-1-ones. We further established a phenotypic yeast-based FNT inhibitor screen, and found very low compound cytotoxicity and monocarboxylate transporter 1 off-target activity on human cells, particularly of the most potent FNT inhibitor BH267.meta, allowing these compounds to proceed towards animal model malaria studies.     

Targeting the Erythrocytic and Liver Stages of Malaria Parasites with s‐Triazine‐Based Hybrids

A diversity‐oriented library of s‐triazine‐based hybrids was screened for activity against the chloroquine‐resistant Plasmodium falciparum W2 strain. The most striking result was sub‐micromolar activity against cultured erythrocytic‐stage parasites of hybrid molecules containing one or two 8‐aminoquinoline moieties. These compounds were not clearly toxic to human cells. The most effective blood‐schizontocidal s‐triazine derivatives were then screened for activity against the liver stage of malaria parasites. The s‐triazine hybrid containing two 8‐aminoquinoline moieties and one chlorine atom emerged as the most potent against P. berghei liver‐stage infection, active in the low nanomolar region, combined with good metabolic stability in rat liver microsomes. These results indicate that s‐triazine‐8‐aminoquinoline‐based hybrids are excellent starting points for lead optimization as dual‐stage antimalarials.     

MAEBL Contributes to Plasmodium Sporozoite Adhesiveness

The sole currently approved malaria vaccine targets the circumsporozoite protein—the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.     

Selective Inhibition of an Apicoplastic Aminoacyl‐tRNA Synthetase from Plasmodium falciparum

The resistance of malaria parasites to available drugs continues to grow, and this makes the need for new antimalarial therapies pressing. Aminoacyl‐tRNA synthetases (ARSs) are essential enzymes and well‐established antibacterial targets and so constitute a promising set of targets for the development of new antimalarials. Despite their potential as drug targets, apicoplastic ARSs remain unexplored. We have characterized the lysylation system of Plasmodium falciparum, and designed, synthesized, and tested a set of inhibitors based on the structure of the natural substrate intermediate: lysyl‐adenylate. Here we demonstrate that selective inhibition of apicoplastic ARSs is feasible and describe new compounds that that specifically inhibit Plasmodium apicoplastic lysyl‐tRNA synthetase and show antimalarial activities in the micromolar range.     

N‐Cinnamoylation of Antimalarial Classics: Quinacrine Analogues with Decreased Toxicity and Dual‐Stage Activity

Plasmodium falciparum, the causative agent of the most lethal form of malaria, is becoming increasingly resistant to most available drugs. A convenient approach to combat parasite resistance is the development of analogues of classical antimalarial agents, appropriately modified in order to restore their relevance in antimalarial chemotherapy. Following this line of thought, the design, synthesis and in vitro evaluation of N‐cinnamoylated quinacrine surrogates, 9‐(N‐cinnamoylaminobutyl)‐amino‐6‐chloro‐2‐methoxyacridines, is reported. The compounds were found to be highly potent against both blood‐stage P. falciparum, chloroquine‐sensitive 3D7 (IC₅₀=17.0–39.0 nM ) and chloroquine‐resistant W2 and Dd2 strains (IC₅₀=3.2–41.2 and 27.1–131.0 nM , respectively), and liver‐stage P. berghei (IC₅₀=1.6–4.9 μM ) parasites. These findings bring new hope for the possible future “rise of a fallen angel” in antimalarial chemotherapy, with a potential resurgence of quinacrine‐related compounds as dual‐stage antimalarial leads.     

N‐Cinnamoylation of Antimalarial Classics: Effects of Using Acyl Groups Other than Cinnamoyl toward Dual‐Stage Antimalarials

In a follow‐up study to our reports of N‐cinnamoylated chloroquine and quinacrine analogues as promising dual‐stage antimalarial leads with high in vitro potency against both blood‐stage Plasmodium falciparum and liver‐stage Plasmodium berghei, we decided to investigate the effect of replacing the cinnamoyl moiety with other acyl groups. Thus, a series of N‐acylated analogues were synthesized, and their activities against blood‐ and liver‐stage Plasmodium spp. were assessed along with their in vitro cytotoxicities. Although the new N‐acylated analogues were found to be somewhat less active and more cytotoxic than their N‐cinnamoylated counterparts, they equally displayed nanomolar activities in vitro against blood‐stage drug‐sensitive and drug‐resistant P. falciparum, and significant in vitro liver‐stage activity against P. berghei. Therefore, it is demonstrated that simple N‐acylated surrogates of classical antimalarial drugs are promising dual‐stage antimalarial leads.     

Inhibition of Eimeria tenella CDK‐Related Kinase 2: From Target Identification to Lead Compounds

Apicomplexan parasites encompass several human‐ and animal‐pathogenic protozoans such as Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella causes coccidiosis, a disease that afflicts chickens, leading to tremendous economic losses to the global poultry industry. The considerable increase in drug resistance makes it necessary to develop new therapeutic strategies against this parasite. Cyclin‐dependent kinases (CDKs) are key molecules in cell‐cycle regulation and are therefore prominent target proteins in parasitic diseases. Bioinformatics analysis revealed four potential CDK‐like proteins, of which one—E. tenella CDK‐related kinase 2 (EtCRK2)—has already been characterized by gene cloning and expression. 1 By using the CDK‐specific inhibitor flavopiridol in EtCRK2 enzyme assays and schizont maturation assays (SMA), we could chemically validate CDK‐like proteins as potential drug targets. An X‐ray crystal structure of human CDK2 (HsCDK2) served as a template to build protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP binding site between EtCRK2 and HsCDK2, as well as chicken CDK3, were addressed for the optimization of selective ATP‐competitive inhibitors. Virtual screening and “wet‐bench” high‐throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized, leading to a set of four promising lead compounds for development as EtCRK2 inhibitors.     

Melatonin Signaling and Its Modulation of PfNF-YB Transcription Factor Expression in Plasmodium falciparum

Malaria is one of the most severe tropical infectious diseases. More than 220 million people around the world have a clinical malaria infection and about one million die because of Plasmodium annually. This parasitic pathogen replicates efficiently in its human host making it difficult to eradicate. It is transmitted by mosquito vectors and so far mosquito control programs have not effectively eliminated this transmission. Because of malaria’s enormous health and economic impact and the need to develop new control and eventual elimination strategies, a big research effort has been made to better understand the biology of this parasite and its interactions with its vertebrate host. Determination of the genome sequence and organization, the elucidation of the role of key proteins, and cell signaling studies have helped to develop an understanding of the molecular mechanisms that provide the parasite’s versatility. The parasite can sense its environment and adapt to benefit its survival, indeed this is essential for it to complete its life cycle. For many years we have studied how the Plasmodium parasite is able to sense melatonin. In this review we discuss the melatonin signaling pathway and its role in the control of Plasmodium replication and development.     

Repurposing Anticancer Drugs To Tackle Malaria

Despite considerable efforts, malaria remains one of the most devastating infectious disease worldwide. In the absence of an effective vaccine, the prophylaxis and management of Plasmodium infections still rely on the therapeutic use of antimalarial agents. However, the emergence of resistant parasites has jeopardized the efficiency of virtually all antimalarial drugs, including artemisinin combination therapies (ACTs). Thus, there is an urgent need for innovative treatments with novel targets to avoid or overcome drug resistance. In this context, Huang & colleagues prioritized compounds that can block the activity of epigenetic enzymes, and described the discovery of a selective P. falciparum histone deacetylase (HDAC) inhibitor with high activity against various stages of the parasite. These findings may pave the way toward developing new lead compounds with broad-spectrum activity, thus facilitating malaria treatment and elimination.     

A Sweet Galactose Transfer: Metabolic Oligosaccharide Engineering as a Tool To Study Glycans in Plasmodium Infection

The introduction of chemical reporter groups into glycan structures through metabolic oligosaccharide engineering (MOE) followed by bio-orthogonal ligation is an important tool to study glycosylation. We show the incorporation of synthetic galactose derivatives that bear terminal alkene groups in hepatic cells, with and without infection by Plasmodium berghei parasites, the causative agent of malaria. Additionally, we demonstrated the contribution of GLUT1 to the transport of these galactose derivatives, and observed a consistent increase in the uptake of these compounds going from naïve to P. berghei-infected cells. Finally, we used MOE to study the interplay between Plasmodium parasites and their mosquito hosts, to reveal a possible transfer of galactose building blocks from the latter to the former. This strategy has the potential to provide new insights into Plasmodium glycobiology as well as for the identification and characterization of key glycan structures for further vaccine development.     

PV1 Protein from Plasmodium falciparum Exhibits Chaperone-Like Functions and Cooperates with Hsp100s

Plasmodium falciparum parasitophorous vacuolar protein 1 (PfPV1), a protein unique to malaria parasites, is localized in the parasitophorous vacuolar (PV) and is essential for parasite growth. Previous studies suggested that PfPV1 cooperates with the Plasmodium translocon of exported proteins (PTEX) complex to export various proteins from the PV. However, the structure and function of PfPV1 have not been determined in detail. In this study, we undertook the expression, purification, and characterization of PfPV1. The tetramer appears to be the structural unit of PfPV1. The activity of PfPV1 appears to be similar to that of molecular chaperones, and it may interact with various proteins. PfPV1 could substitute CtHsp40 in the CtHsp104, CtHsp70, and CtHsp40 protein disaggregation systems. Based on these results, we propose a model in which PfPV1 captures various PV proteins and delivers them to PTEX through a specific interaction with HSP101.     

The Impact of Activity-Based Protein Profiling in Malaria Drug Discovery

Activity-based protein profiling (ABPP) is an approach used at the interface of chemical biology and proteomics that uses small molecular probes to provide dynamic fingerprints of enzymatic activity in complex proteomes. Malaria is a disease caused by Plasmodium parasites with a significant death burden and for which new therapies are actively being sought. Here, we compile the main achievements from ABPP studies in malaria and highlight the probes used and the different downstream platforms for data analysis. ABPP has excelled at studying Plasmodium cysteine proteases and serine hydrolase families, the targeting of the proteasome and metabolic pathways, and in the deconvolution of targets and mechanisms of known antimalarials. Despite the major impact in the field, many antimalarials and enzymatic families in Plasmodium remain to be studied, which suggests ABPP will be an evergreen technique in the field.     

Systematic Analysis of Clemastine,a Candidate Apicomplexan Parasite-Selective Tubulin-Targeting Agent

Apicomplexan parasites, such as Toxoplasma gondii, Plasmodium spp., Babesia spp., and Cryptosporidium spp., cause significant morbidity and mortality. Existing treatments are problematic due to toxicity and the emergence of drug-resistant parasites. Because protozoan tubulin can be selectively disrupted by small molecules to inhibit parasite growth, we assembled an in vitro testing cascade to fully delineate effects of candidate tubulin-targeting drugs on Toxoplasma gondii and vertebrate host cells. Using this analysis, we evaluated clemastine, an antihistamine that has been previously shown to inhibit Plasmodium growth by competitively binding to the CCT/TRiC tubulin chaperone as a proof-of-concept. We concurrently analyzed astemizole, a distinct antihistamine that blocks heme detoxification in Plasmodium. Both drugs have EC₅₀ values of ~2 µM and do not demonstrate cytotoxicity or vertebrate microtubule disruption at this concentration. Parasite subpellicular microtubules are shortened by treatment with either clemastine or astemizole but not after treatment with pyrimethamine, indicating that this effect is not a general response to antiparasitic drugs. Immunoblot quantification indicates that the total α-tubulin concentration of 0.02 pg/tachyzoite does not change with clemastine treatment. In conclusion, the testing cascade allows profiling of small-molecule effects on both parasite and vertebrate cell viability and microtubule integrity.     

An Endoperoxide‐Based Hybrid Approach to Deliver Falcipain Inhibitors Inside Malaria Parasites

The emergence of artemisinin‐resistant Plasmodium falciparum malaria in Southeast Asia has reinforced the urgent need to discover novel chemotherapeutic strategies to treat and control malaria. To address this problem, we prepared a set of dual‐acting tetraoxane‐based hybrid molecules designed to deliver a falcipain‐2 (FP‐2) inhibitor upon activation by iron(II) in the parasite digestive vacuole. These hybrids are active in the low nanomolar range against chloroquine‐sensitive and chloroquine‐resistant P. falciparum strains. We also demonstrate that in the presence of FeBr₂ or within infected red blood cells, these molecules fragment to release falcipain inhibitors with nanomolar protease inhibitory activity. Molecular docking studies were performed to better understand the molecular interactions established between the tetraoxane‐based hybrids and the cysteine protease binding pocket residues. Our results further indicate that the intrinsic activity of the tetraoxane partner compound can be masked, suggesting that a tetraoxane‐based delivery system offers the potential to attenuate the off‐target effects of known drugs.     

Synthesis,Inhibition Potency,Binding Mode,and Antiprotozoal Activities of Fluorescent Inhibitors of Trypanothione Reductase Based on Mepacrine‐Conjugated Diaryl Sulfide Scaffolds

Trypanothione reductase (TR) is a flavoenzyme unique to trypanosomatid parasites and a target for lead discovery programs. Various inhibitor scaffolds have emerged in the past, exhibiting moderate affinity for the parasite enzyme. Herein we show that the combination of two structural motifs of known TR inhibitors — diaryl sulfides and mepacrine — enables the simultaneous addressing of two hydrophobic patches in the active site. The binding efficacy of these conjugates is enhanced over that of the respective parent inhibitors. They show K_ic values for the parasite enzyme down to 0.9±0.1 μm and exhibit high selectivity for TR over human glutathione reductase (GR). Despite their considerable molecular mass and in some cases permanent positive charges, in vitro studies revealed IC₅₀ values in the low micromolar to sub‐micromolar range against Trypanosoma brucei rhodesiense and Trypanosoma cruzi, as well as the malaria parasite Plasmodium falciparum, which lack trypanothione metabolism. The inhibitors exhibit strong fluorescence due to their aminoacridine moiety. This feature allows visualization of the drugs in the parasite where high accumulation was observed by fluorescence microscopy even after short exposure times.