Pascal Mäser

As part of our search for new natural chemicals with bioactivity against protozoan pathogens, we have previously reported on the isolation, identification and antiprotozoal activity of over 100 alkaloids with an aminosteroid or related scaffold. We have now investigated the hypothesis that these chemically related compounds also share common mechanisms of action regarding their activity against Trypanosoma brucei rhodesiense (human East African trypanosomiasis) and Plasmodium falciparum (tropical malaria). Furthermore, their cytotoxic activity against mammalian cells (L6 rat skeletal myoblasts) was also included in the investigation. We found that mammalian cytotoxicity is generally lower and there is only a low degree of mutual correlation between the susceptibility of the two parasites and mammalian cells to the compounds’ toxic effects. With the molecular targets being unknown, the 3D-QSAR study was conducted using the Open3Dqsar software. The resulting models show the main structural features influencing the different biological effects. They indicate that indeed related molecular targets underly the cytotoxic, antitrypanosomal and antiplasmodial activity based on structural determinants sufficiently different enough to warrant a significant level of parasite-selectivity. The results of this study offer structural explanations for the observed differences in aminosteroids’ action against the mentioned pathogens and provide a starting point for optimization and simplification of the natural structures towards leads for antiprotozoal drug development.

Human African trypanosomiasis (HAT) is caused by two subspecies of Trypanosoma brucei , T. b. gambiense and T. b. rhodesiense , with the former accounting for over 95% of all cases. HAT is an elusive disease, which is fatal unless treated, with a focal prevalence in the rural areas of tropical Africa where the tsetse fly vectors are endemic. At the same time, HAT is one of the very few infectious diseases where global elimination seems possible. The incidence is at a historic low, and new orally bioavailable drugs have been developed that will expand the coverage of treatment in endemic regions. In particular, acoziborole has passed the clinical development as a single-dose oral cure of T. b. gambiense infection. However, to win the endgame against HAT, acoziborole also needs to be extended to T. b. rhodesiense . In order to strengthen the preclinical dossier of acoziborole, we conducted an in-depth evaluation of acoziborole against the bloodstream forms of all three T. brucei subspecies with a focus on T. b. rhodesiense . Acoziborole had consistent and high activity against different T. b. rhodesiense strains, including a multidrug-resistant field isolate, with IC ₅₀ values around 600 nM. Acoziborole is faster acting than fexinidazole, and it exhibits irreversible cidality against T. b. rhodesiense at concentrations of ≥4.3 μM (1.6 μg/ml) after 15 h of incubation. At very short exposure times (≤8 h), T. b. gambiense appeared to be more sensitive than T. b. rhodesiense . However, given the extremely long elimination half-life of acoziborole in the human body, this difference will be irrelevant for its trypanocidal action in patients. We therefore conclude that acoziborole is expected to be as effective against r -HAT as against g -HAT in patient populations.

Introduction: Plasmodium 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. Methods: We 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. Results: The generated cells were infected with P. cynomolgi sporozoites, and hypnozoite formation as well as schizont development were confirmed by immunofluorescence microscopy. Discussion: This simian iPS-derived model is a promising alternative to current P. vivax in vitro infection systems to study the mechanisms underlying liver stage dormancy.

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 MU ltidimensional L uminescence T est for i ntegration of i nteractions (MULT-i ² ), an in vitro assay that enables scalable, high-resolution assessment of parasite viability across multidimensional drug concentration spaces. For dual drug combinations, the MULT-i ² 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-i ² 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.

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.

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 modelling of UHPLC/+ESI QqTOF-MS data and 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.

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-gal^SENSOR 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-gal^SENSOR 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.

In this study, we describe the synthesis of isobenzofuranones and isoindolinones using the Claisen–Schmidt condensation via the formation of a chalcone intermediate, followed by a 5-exo-trig cyclization facilitated by the carboxylate group under basic conditions. The desired products were obtained in good yields. Subsequently, Michael-lactamization reaction of the isobenzofuranones was employed to synthesize isoindolinone derivatives. The three-component Mannich-lactamization reaction was also explored to synthesize isoindolinones from 2–hydroxyacetophenone, phthalaldehydic acid, and amine derivatives. Furthermore, the synthesized compound 5c exhibited low EC₅₀ (μM) values against Leishmania donovani (L. donovani), with no significant cytotoxicity observed against L-6 cells.

In continuation of our recent report on the antileishmanial activity of an ethanolic extract from leaves of Ptilostemon chamaepeuce subsp. cyprius (Pcc, Asteraceae), we have now isolated the main sesquiterpene lactone, deacylcynaropicrin, along with a minor derivative, 13-hydroxy-11β,13-dihydro-deacylcynaropicrin. The main constituent was tested for antileishmanial activity against promastigotes and amastigotes of Leishmania infantum (Lin), the causative agent of visceral leishmaniasis. Both STLs were tested against additional protozoan pathogens, including L. donovani, Trypanosoma brucei rhodesiense (Tbr), and Plasmodium falciparum. The STL cynaropicrin from Artichoke (Cynara cardunculus), a congener of deacylcynaropicrin with a hydroxymethacrylate ester group, previously known to possess antiprotozoal activity, was retested against Lin for direct comparison. Cynaropicrin was found to be much more potent than either its deacyl congener or the hydroxylated derivative from Pcc against all tested parasites and also against an isolated parasite enzyme, Tbr pteridine reductase (TbPTR1). The ester moiety of cynaropicrin significantly enhances the antiprotozoal activity of this STL. Since cynaropicrin also displayed significant cytotoxicity against mammalian cells (L6 and J774A.1 cell lines), its utility as candidate for further development appears limited. However, this study provides valuable new insight into the structure–activity relationships of these compounds.