Malaria remains a major global health challenge, with emerging partial resistance to first-line therapies in Africa threatening current control efforts. Drug combinations are essential to improve treatment efficacy and restrain resistance development. However, in vitro assays that quantify parasite viability after drug exposure and characterize pharmacodynamic drug interactions are labor- and resource-intensive, with standard approaches such as the parasite reduction ratio assay limiting systematic, high-resolution evaluation of drug combinations. We present the MUltidimensional Luminescence Test for integration of interactions (MULT-i2), an in vitro assay that enables scalable, high-resolution assessment of parasite viability across multidimensional drug concentration spaces. For dual drug combinations, the MULT-i2 assay characterizes interaction surfaces while requiring ∼50-fold fewer resources and more than two-fold less time than conventional methods, enabling exploration of broader combination scenarios. The assay combines a highly sensitive chemiluminescence readout with inducible reporter expression in Plasmodium falciparum, supporting potential extension to multidimensional combination testing. Using the general pharmacodynamic interaction (GPDI) model, the MULT-i2 assay quantified interaction potency and directionality, confirming and refining the known synergy between atovaquone and proguanil, and revealing detailed interaction patterns for additional drug combinations. Overall, this approach provides an efficient framework for testing and characterizing pharmacodynamic drug interactions and supports the rational development of antimalarial combination therapies.

IntroductionPlasmodium cynomolgi is one of the few malaria parasite species that forms dormant liver stages known as hypnozoites and is therefore a suitable model for Plasmodium vivax, the causative agent of recurrent malaria. Very little is known about the biology and pharmacology of liver stage dormancy, which hampers the search for compounds with anti-hypnozoite activity.MethodsWe present the development of a P. cynomolgi in vitro infection model using stem cell-derived hepatocyte-like cells from Macaca fascicularis. iPS cells were established on feeder free condition and differentiated into hepatocyte-like cells via inducible overexpression of key transcription factors.ResultsThe generated cells were infected with P. cynomolgi sporozoites, and hypnozoite formation as well as schizont development were confirmed by immunofluorescence microscopy.DiscussionThis simian iPS-derived model is a promising alternative to current P. vivax in vitro infection systems to study the mechanisms underlying liver stage dormancy.

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Malaria remains a major global health threat, and the emergence of partial artemisinin resistance challenges current treatment regimens. Reliable antimalarial screening assays are therefore essential for identifying new drug candidates. The parasite reduction ratio (PRR) assay provides valuable pharmacodynamic insights but is limited by its labor-intensive, 14- to 28-day incubation period. We developed an optimized PRR assay protocol using the highly sensitive chemiluminescence-based lacZ/β-galSENSOR readout, reducing assay incubation duration to 7 days while maintaining informative pharmacodynamic parameters, including lag phase, parasite clearance time, parasite reduction ratio, and maximum killing effect. In contrast, the [3H]-hypoxanthine incorporation method failed to detect viable parasites reliably and consistently overestimated drug activity with the shortened protocol. This novel lacZ/β-galSENSOR PRR assay enables laboratories without access to radioactive facilities to evaluate antimalarial compounds efficiently, providing robust time-killing profiles with greater convenience, higher throughput, and lower equipment requirements than existing readout methods.

Human African Trypanosomiasis (HAT) and Malaria are serious infectious diseases endemic in tropical regions, caused by protozoan parasites, and necessitating an urgent development of new antiprotozoal drugs. As part of our ongoing search for new antiprotozoal steroidal alkaloids from plants, we investigated the methanolic stem bark extract of Holarrhena pubescens (Apocynaceae). H. pubescens is a tropical tree that some Kenyan coastal communities have long used to treat various ailments, including fever and stomach pain. The crude extract, alkaloid fraction, and 16 subfractions acquired through centrifugal partition chromatography (CPC) displayed promising in vitro antiprotozoal activity against Trypanosoma brucei rhodesiense (Tbr) and Plasmodium falciparum (Pf). Partial least squares (PLS) regression modeling of UHPLC/+ESI QqTOF-MS data and the antiprotozoal activity data of the crude extract and its fractions was performed to predict compounds that may be responsible for the observed antiplasmodial activity. Chromatographic separation of the alkaloid fraction afforded one new steroidal alkaloid (5), along with 18 known compounds (1, 2, 4, 6–20), and one artifact (3) that was presumably formed during the acid–base extraction process. The structural characterization of the isolated compounds was accomplished using UHPLC/+ESI-QqTOF-MS/MS and NMR spectroscopy. The isolated compounds were tested for their in vitro antiprotozoal properties against the two aforementioned pathogens, as well as for their cytotoxicity against mammalian cells (L6 cell line). Compounds 2 and 16 (IC50 = 0.2 μmol/L) demonstrated the highest antitrypanosomal activity, with compound 2 showing the highest selectivity (SI = 127). The new compound 5 exhibited the strongest antiplasmodial activity and selectivity against Pf (IC50 = 0.7 μmol/L, SI = 43). Our findings provide further promising antiprotozoal leads for HAT and Malaria.

Neglected tropical diseases (NTDs) remain a major global health challenge, particularly in low- and middle-income countries. Kinetoplastid parasites causing Chagas disease, leishmaniasis, and African trypanosomiasis rely on host purine salvage pathways, making nucleoside analogues attractive therapeutic candidates. However, their clinical utility is limited by poor cellular uptake and rapid metabolism. Herein, we report the application of the ProTide prodrug technology, a clinically validated approach that enhances the intracellular delivery of nucleoside monophosphates for the treatment of kinetoplastids infections. As a proof of concept, a focused library of zidovudine (AZT) and cordycepin ProTide prodrugs was designed, synthesized, and evaluated for antiparasitic activity against T. b. rhodesiense, T. cruzi, and L. donovani, as well as for cytotoxicity in L6 rat myoblasts. Out of these, compound 16 exhibited substantial serum stability and potent activity (IC50 = 5 nM; selectivity index, SI = 2,560) against T. b. rhodesiense with robust activity also observed against T. cruzi and L. donovani. These findings establish the ProTide prodrug technology as a promising strategy for optimizing nucleoside analogues against kinetoplastid parasites and provide a framework for the development of new therapeutics for NTDs.

The protozoan parasite Trypanosoma cruzi causes Chagas disease, which is among the deadliest parasitic infections in Latin America. Current therapies are toxic and lack efficacy against the chronic stage of infection; thus, new drugs are urgently needed. Here, we describe a previously unidentified series of quinazoline compounds with potential against Trypanosoma cruzi and the related trypanosomatid parasites Trypanosoma brucei and Leishmania donovani. We demonstrated partial efficacy of a lead quinazoline compound in a mouse model of acute Chagas disease. Mechanism of action studies using several orthogonal approaches showed that this quinazoline compound series targeted the ATP-binding pocket of T. cruzi lysyl-tRNA synthetase 1 (KRS1). A high-resolution crystal structure of KRS1 bound to the drug indicated binding interactions that led to KRS1 inhibition. Our study identified KRS1 as a druggable target for treating T. cruzi infection in a mouse model. This quinazoline series shows potential for treating Chagas disease but will require further development to become a future treatment for this neglected disease.

Diseases caused by trypanosomatid parasites like human African trypanosomiasis (HAT), Chagas disease (CD), leishmaniasis, and malaria are persistent health problems in developing countries that still demand new drug development. The species of the Amaryllidoideae subfamily (Amaryllidaceae) represent a vast source of alkaloids with a wide range of bioactive properties, including antiparasitic effects. The aim of this study was to evaluate the antiparasitic activity of the alkaloids hamayne, 7-hydroxyclivonine, 4-O-methylnangustine, and candimine against Trypanosoma brucei rhodesiense, Trypanosoma cruzi, Leishmania donovani, and Plasmodium falciparum parasites. The alkaloids were isolated from the leaves of Hippeastrum argentinum and Hippeastrum escoipense using several chromatographic techniques and then identified by GC-MS, UPLC-MS/MS, and NMR data. The compounds were assessed against different life cycle stages of these four parasites. Furthermore, the cytotoxic activity of the alkaloids against L6 rat skeletal myoblast cells was tested. P. falciparum was very sensible to 7-hydroxyclivonine. Candimine showed significant antiparasitic activity against all the evaluated parasites, especially T. b. rhodesiense. Candimine merits deeper research regarding its effect against trypanosomatid parasites as a lead compound for the development of alternative treatments for HAT, CD, and malaria.

The naphthoquinone skeleton is known for broad biological applications and, in particular, for antiparasitic efficacy. As part of our ongoing search for new antiprotozoal naphthoquinone derivatives, we incorporated computer-aided optimization models utilizing physicochemical parameters into our approach. Herein, we report on the synthesis of 21 new benzamido–menadione and naphthoquinone derivatives via the Kochi–Anderson reaction. The antiprotozoal activity of all the synthesized compounds was evaluated against Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STIB900. Cytotoxicity towards L6 cells was also determined, and the respective selectivity indices (SI) were calculated. Several ligand efficiency metrics, such as LLE, SILE, and FQ, were calculated, and the results were visualized in scatterplots. Almost all of the synthesized benzamido–menadione derivatives exhibited high activity against NF54 (IC50 < 1 µM), with the strongest activity and excellent selectivity observed in the 2-fluoro-5-trifluoromethylbenzamido derivative 2f (IC50 = 0.021 µM, SI = 10,000). Specific ligand efficiency metrics, such as SILE, LLE or FQ, showed a clear correlation with the corresponding antiplasmodial activities. Toxicity predictions confirmed low acute oral toxicity for most compounds, further supporting their potential as safe drug candidates. Our findings highlight the benzamido–menadione scaffold as a viable option for new antiplasmodial drugs.

Parasites have a severe impact on animal and human health. Parasites like worms, ticks, mites, fleas, biting flies, mosquitoes, and pathogenic protozoa affect humans and their pets as well as their livestock globally, both in terms of severity and numbers. Parasitic infections are a global phenomenon, and they can be associated with severe or mild symptoms but represent a continuous risk of severe diseases for animals and humans. Therefore, effective treatment options and the prevention of infection are key for the wellbeing of pets, livestock, and humans, including the reduction of zoonotic risk of infection. The effective control of parasites in animals can greatly improve their quality of life and is also beneficial for humans; this is threatened by drug-resistant parasite populations. Today’s key areas for improvement of parasite control are as follows: a) convenience of prevention and treatment, b) effectiveness against drug-resistant parasites, c) availability and reduced costs of treatment, and d) control measurements that are environmentally friendly.

We report the synergistic interaction between the complex III inhibitor atovaquone and an ATP synthase inhibitor, venturicidin A, against Plasmodium falciparum . Our results suggest the simultaneous inhibition of the parasite’s primary and alternative pathways for generating the mitochondrial membrane potential to be responsible for this phenomenon, whereby the alternative pathway relies on ATP synthase running in reverse mode. We hypothesize that the synergistic interaction between atovaquone and proguanil could follow a similar mechanism.

Plasmodione is a potent early antiplasmodial compound. A metabolic study on mice treated with plasmodione revealed that 6-hydroxy-plasmodione was the main metabolite eliminated in the urine of treated mice. To block the metabolic pathway in the host, the introduction of fluorine at C-6 of the 3-benzylmenadione core was applied and showed potent antiplasmodial activity similar to that of the plasmodione analogue in vitro. In this work, a library of 38 6-fluoro-3-benzylmenadione analogues (a series) was constructed by incorporating structurally diverse groups in place of the 4-(trifluoromethyl) substituent present in the antiplasmodial plasmodione, via three synthetic routes. All new compounds were tested against the P. falciparum NF54 strain and for cytotoxicity with the rat L6 line. With a fluorine atom at C-6, A-a-21 was revealed to be the only compound from the a series, superior to the 6-H- analogue from the b series, with an IC50 value of 70 nM versus 200 nM. Then, five other fluorine-based 3-benzylmenadiones, in which the fluorine was introduced in various positions of the 3-benzylmenadione core, were synthetized to assist our understanding of the impact of fluorine on antiplasmodial potencies in vitro; in particular, the aim here was to compare the effects of human serum and P. berghei species in these drug screens. This was also conducted in vivo with the P. berghei-infected mouse model. In the P. berghei species assay, PD and the 4'-fluoro-3'-trifluoromethyl-benzylmenadione A-b-9 exhibited a similar antiplasmodial behavior toward P. falciparum versus P. berghei. In the human serum versus Albumax assays, only the 6-fluoro-plasmodione showed a lower shift factor between Albumax assays and human serum conditions, suggesting a lower protein binding for the 6-F-PD compared to plasmodione or A-b-9. In vivo, 6-fluoro-plasmodione proved to be the most potent 3-benzylmenadione, reducing parasitemia by 50% after oral administration at 50 mg/kg.

Malaria remains a major global health problem that has been exacerbated by the impact of the COVID-19 pandemic on health systems. To combat this, the World Health Organization (WHO) has set a target of driving forward research into innovative treatment methods such as new drugs and vaccines. Quinones, particularly 1,4-naphthoquinones, have been identified as promising candidates for the development of antiprotozoal drugs. Herein, we report several methods for the preparation of 2-benzyl-1,4-naphthoquinones. In particular, the silver-catalyzed Kochi-Anderson radical decarboxylation is well suited for the preparation of these compounds. The antiprotozoal activity of all synthesized compounds was evaluated against Plasmodium falciparum NF54 and Trypanosoma brucei rhodesiense STIB900. Cytotoxicity towards L6 cells was also determined, and the respective selectivity indices (SI) were calculated. The synthesized compounds exhibited good antiplasmodial activity against the P. falciparum (NF54) strain, particularly (2-fluoro-5-trifluoromethylbenzyl)-menadione 2e, which showed strong efficacy and high selectivity (IC50 = 0.006 µM, SI = 7495). In addition, these compounds also displayed favorable physicochemical properties, suggesting that the benzylnaphthoquinone scaffold may be a viable option for new antiplasmodial drugs.

Lefleuganan, a clinical stage drug candidate for the treatment of cutaneous leishmaniasis, is a synthetic nonapeptide inspired by the natural antimicrobial peptide leucinostatin A, exhibiting excellent antiprotozoal activity in the low nM range. Lefleuganan shows strongly reduced acute toxicity, making it amenable for clinical use. Here, using a broad set of in vivo and in vitro measurements using purified enzymes, we find that leucinostatin A, but not lefleuganan, specifically targets the mitochondrial ATP synthase, inhibiting ATP synthesis by the human, bovine, and yeast enzyme in the nanomolar range. In a structure-activity relationship study covering the chemical space between the two compounds, hydroxyleucine at position 7 in leucinostatin A is identified as the key responsible moiety for specific ATP synthase inhibition and systemic toxicity. Our data suggest that efficient antiprotozoal activity of these class of compounds is mediated by efficient energetic uncoupling of negatively charged membranes.

Natural products (NP) have proven to be a rich source of potentially bioactive compounds, and metabolomics is the current method of choice for characterizing natural extracts. To integrate the vast amount of data and information produced by modern metabolomics workflows, we recently developed a sample-centric approach for the semantic enrichment and alignment of metabolomics datasets. The resulting Experimental Natural Products Knowledge Graph (ENPKG) is queryable and integrates both newly acquired digitalized experimental data and information, and previously reported knowledge. It allows the highlighting of putative bioactive compounds at the extract level by comparing, for example, the occurrence of compounds of a given chemical class with bioactivity results. Using this approach, we recently described potent anti-Trypanosoma cruzi activity of two rotenoids, deguelin and rotenone. These compounds were identified in six active extracts from four plant species: Cnestis palala (Connaraceae), Chadsia grevei, Pachyrhizus erosus, and Desmodium heterophylum (Fabaceae). In this work, we present the results of the phytochemical investigation of four of these extracts and the establishment of a library of structural analogs for in vitro bioactivity testing. This work led to the isolation, characterization, and biological evaluation of the anti-T. cruzi potential of 41 compounds, including 11 rotenoids and seven compounds reported for the first time. Thanks to modern metabolite annotation and single-step isolation procedures, this work also demonstrates the possibility of considering natural extract libraries as a reservoir of rapidly accessible pure NPs. This perspective could increase the options for NP research and help accelerate NP drug discovery efforts.

This study explores the synthesis and evaluation of novel 1,2,3-triazole-methyl-1,4-naphthoquinone hybrids, focusing on their electrochemical properties and antiparasitic efficacies against two human blood-dwelling parasites Plasmodium falciparum and Schistosoma mansoni. Using copper-catalyzed azide-alkyne cycloaddition (CuAAC), a well-established tool in click chemistry, two synthetic routes were assessed to develop alpha- and beta-[triazole-methyl]-menadione derivatives. By optimizing the CuAAC reaction conditions, yields were significantly improved, reaching up to 94 % for key intermediates and resulting in the formation of a library of approximately 30 compounds. Biological evaluation of the compounds in antiparasitic drug assays demonstrated notable antischistosomal potencies, while no significant activity was observed for the same series against P. falciparum parasites. Electrochemical and 'benzylic' oxidation studies confirmed that the active 'benzoyl' metabolite responsible for the antiplasmodial activity of plasmodione cannot be generated. These findings highlight the potential of triazole-linked menadione hybrids as promising early candidates for antischistosomal drug development, and provides insights into structure-activity relationships crucial for future therapeutic strategies.

The emergence of drug resistance to nearly all antimalarials following their rollout underscores the need for novel chemotypes with novel modes of action to replenish the antimalarial drug-development pipeline. We identified a novel class of compounds in the antimalarial armory. Compound 31, characterized by a 2-hydroxyphenyl benzamide scaffold, displays potent activity against blood-stage and mature sexual stages of Plasmodium falciparum and no toxicity in human cells. Resistance selection studies with 31 identified a previously unknown point mutation in the P. falciparum multidrug-resistance protein 1 (pfmdr1) gene, for which we confirmed causality by CRISPR/Cas9-based gene editing as the primary mediator of resistance. No cross-resistance toward first-line antimalarials was identified in compound 31-resistant parasites. Proteomics studies indicated that the primary mode of action of 31 is through direct binding to cytosolic ribosomal subunits, thereby inhibiting protein synthesis in the parasite. Taken together, compound 31 is a promising starting point for the development of a next-generation antimalarial.

New chemical entities are consistently being investigated in antimalarial drug discovery and they require animal models for toxicity and efficacy testing. Murine models in searching for novel antimalarial drugs are inevitable because they show unique similarities to human physiology during malaria pathogenesis. Therefore, they provide a preclinical basis (following in vitro assessments of newly identified lead compounds) for further assessment in the drug development pipeline. Specific mouse strains, non-humanized and humanized, have successfully been infected with rodent Plasmodium species and the human Plasmodium falciparum respectively. Infected mice provide a platform for the assessment of treatment options being sought. In vivo pharmacokinetic evaluations are necessary when determining the fate of new lead compounds in addition to the efficacy assessment of these chemical entities. This review highlights specific murine models important for antimalarial drug discovery and their resultant critical in vivo pharmacokinetic, safety, and efficacy assessments necessary for making appropriate choices of lead compounds.