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Sarah Schleiferböck,1,2 Christian Scheurer,1,2 Masataka Ihara,3,4 Isamu Itoh,3,4 Ian Bathurst,5,† Jeremy N Burrows,5 Pascal Fantauzzi,5 Julie Lotharius,5 Susan A Charman,6 Julia Morizzi,6 David M Shackleford,6 Karen L White,6 Reto Brun,1,2 Sergio Wittlin1,21Swiss Tropical and Public Health Institute, Basel, 2University of Basel, Basel, Switzerland; 3Drug Discovery Science Research Center, Hoshi University, Shinagawa, Tokyo, Japan; 4Synstar Japan Co, Ltd, Odawara, Japan; 5Medicines for Malaria Venture, Geneva, Switzerland; 6Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia †Ian Bathurst passed away on 26 June 2011Abstract: The objective of this work was to characterize the in vitro (Plasmodium falciparum) and in vivo (Plasmodium berghei) activity profile of the recently discovered lead compound SSJ-183. The molecule showed in vitro a fast and strong inhibitory effect on growth of all P. falciparum blood stages, with a tendency to a more pronounced stage-specific action on ring forms at low concentrations. Furthermore, the compound appeared to be equally efficacious on drug-resistant and drug-sensitive parasite strains. In vivo, SSJ-183 showed a rapid onset of action, comparable to that seen for the antimalarial drug artesunate. SSJ-183 exhibited a half-life of about 10 hours and no significant differences in absorption or exposure between noninfected and infected mice. SSJ-183 appears to be a promising new lead compound with an attractive antimalarial profile.Keywords: antimalarial studies, cross-resistance, stage-specificity, Plasmodium falciparum

Abstract Background Semi-synthetic artemisinin derivatives are powerful peroxidic drugs in artemisinin-based combination therapy (ACT) recommended as first-line treatment of Plasmodium falciparum malaria in disease-endemic countries. Studies by Eckstein-Ludwig and co-workers showed both thapsigargin and artemisinin specifically inhibit the sarcoplasmic reticulum Ca2+−ATPase of Plasmodium falciparum (PfATP6). In the present study the type of interaction between thapsigargin and artemisinin derivatives as well as the ozonide OZ277 (RBx11160 or arterolane) was evaluated in parasite cultures. The latter compound is an adamantane-based peroxide and the first fully synthetic clinical candidate recently registered in India by Ranbaxy Laboratories Ltd. for anti-malarial combination therapy. Methods Drug interaction studies were performed using a previously described fixed ratio method and anti-malarial activity measured using the [3H] hypoxanthine incorporation assay. Results The sum 50% and 90% fractional inhibitory concentration (∑FIC50, 90) of the interaction of thapsigargin with OZ277, artemether or artesunate, against NF54 and K1 strains of P. falciparum ranged from 0.9 to 1.4. Conclusion The interaction of thapsigargin with OZ277, artesunate or artemether was additive, data consistent with previous observations indicating that activity of anti-malarial peroxides does not derive from reversible interactions with parasite targets.

Abstract Background The standard in vitro test to assess anti-malarial activity of chemical compounds is the [3H]hypoxanthine incorporation assay. It is a radioactivity-based method to measure DNA replication of Plasmodium in red blood cells. The method is highly reproducible, however, the handling of radioactive material is costly, hazardous and requires the availability of appropriate technology and trained staff. Several other ways to evaluate in vitro anti-malarial activity do exist, all with their own assets and limitations. Methods The newly developed double-antibody sandwich ELISA described here is based on the properties of a non-overlapping pair of monoclonal antibodies directed against Plasmodium falciparum aldolase. This glycolytic enzyme possesses some unique nucleotide sequences compared to the human isoenzymes and has been highly conserved through evolution. Out of twenty possibilities, the most sensitive antibody pair was selected and used to quantitatively detect parasite aldolase in infected blood lysates. Results A total of 34 compounds with anti-malarial activity were tested side-by-side by ELISA and the [3H]hypoxanthine incorporation assay. The novel ELISA provided IC50s closely paralleling those from the radioactivity-based assay (R = 0.99, p < 0.001). At the investigated assay conditions (72 h incubation time, parasitaemia = 0.3%), the assay was found to be reproducible and easy to perform. Conclusion The newly developed ELISA presents several advantages over the comparative method, the [3H]hypoxanthine incorporation assay. The assay is highly reproducible, less hazardous (involves no radioactivity) and requires little and cheap technical equipment. Relatively unskilled personnel can conduct this user-friendly assay. All this makes it attractive to be employed in resource-poor laboratories.

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.

Emerging resistance to current antimalarials is reducing their effectiveness and therefore there is a need to develop new antimalarial therapies. Toward this goal, high throughput screens against the P. falciparum asexual parasite identified the pyrazolopyridine 4-carboxamide scaffold. Structure-activity relationship analysis of this chemotype defined that the N1-tert-butyl group and aliphatic foliage in the 3- and 6-positions were necessary for activity, while the inclusion of a 7'-aza-benzomorpholine on the 4-carboxamide motif resulted in potent anti-parasitic activity and increased aqueous solubility. A previous report that resistance to the pyrazolopyridine class is associated with the ABCI3 transporter was confirmed, with pyrazolopyridine 4-carboxamides showing an increase in potency against parasites when the ABCI3 transporter was knocked down. The low metabolic stability intrinsic to the pyrazolopyridine scaffold and the slow rate by which the compounds kill asexual parasites resulted in poor performance in a P. berghei asexual blood stage mouse model. Lowering the risk of resistance and mitigating the metabolic stability and cytochrome P450 inhibition will be challenges in the future development of the pyrazolopyrimidine antimalarial class., 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 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A total synthesis of the enantiopure syn,syn-tosyl-samroiyotmycin A, a C 2 -symmetric 20-membered antimalarial macrodiolide with syn,syn-configuration of the 8,24-dihydroxy-9,25-dimethyl units and it's enantiopure anti,anti-derivative is described. The synthesis was accomplished utilizing a linear approach in 7 steps and 3 % overall yield via a sequence of diastereoselective methylation of SuperQuat oxazolidinone auxiliary, cross metathesis and Yamaguchi macrolactonization of fully functionalized seco-acids. By a similar approach we gained access to several samroiyotmycin analogues and precursors. Antimalarial activity was tested on multi-resistant (K1) and sensitive (Nf54) P. falciparum strains providing insight into structure activity relationships. Both tosyl-oxazol unit as well as the syn-configuration of the two contiguous stereogenic centers turned out to be beneficial for antiplasmodial activity. For instance, syn,syn-tosyl-samroiyotmycin A showed 3.4 times higher activities than the "tosyl-free" natural product., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Malaria remains a global health concern as drug resistance threatens treatment programs. We identified a piperidine carboxamide (SW042) with anti-malarial activity by phenotypic screening. Selection of SW042-resistant Plasmodium falciparum (Pf) parasites revealed point mutations in the Pf&#95;proteasome β5 active-site (Pfβ5). A potent analog (SW584) showed efficacy in a mouse model of human malaria after oral dosing. SW584 had a low propensity to generate resistance (minimum inoculum for resistance [MIR] >10 9 ) and was synergistic with dihydroartemisinin. Pf&#95;proteasome purification was facilitated by His 8 -tag introduction onto β7. Inhibition of Pfβ5 correlated with parasite killing, without inhibiting human proteasome isoforms or showing cytotoxicity. The Pf&#95;proteasome&#95;SW584 cryoelectron microscopy (cryo-EM) structure showed that SW584 bound non-covalently distal from the catalytic threonine, in an unexplored pocket at the β5/β6/β3 subunit interface that has species differences between Pf and human proteasomes. Identification of a reversible, species selective, orally active series with low resistance propensity provides a path for drugging this essential target., Competing Interests: Declaration of interests B.L. is a Medicines for Malaria Venture (MMV) employee. J.M.R., S.F.C., B.L., S.G., S.A.C., and M.A.P. are inventors on a patent application covering the described compounds., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Insurance benefit design influences whether individuals with diabetes who require a continuous glucose monitor (CGM) to provide real-time feedback on their blood glucose levels can obtain the CGM device from either a pharmacy or a durable medical equipment supplier. The impact of the acquisition channel on device adherence and health care costs has not been systematically evaluated., Objective: This study aims to compare the adherence rates for patients new to CGM therapy and the costs of care for individuals who obtained CGM devices from a pharmacy versus acquisition through a durable medical equipment supplier using retrospective claims analysis., Methods: Using the Mariner commercial claims database, individuals aged >18 years with documented diabetes and an initial CGM claim during the first quarter of 2021 (2021 Q1, index date) were identified. Patients had to maintain uninterrupted enrollment for a duration of 15 months but file no CGM claim during the 6 months preceding the index date. We used direct matching to establish comparable pharmacy and durable medical equipment cohorts. Outcomes included quarterly adherence, reinitiation, and costs for the period from 2021 Q1 to the third quarter of 2022 (2022 Q3). Between-cohort differences in adherence rates and reinitiation rates were analyzed using z tests, and cost differences were analyzed using 2-tailed t tests., Results: Direct matching was used to establish comparable pharmacy and durable medical equipment cohorts. A total of 2356 patients were identified, with 1178 in the pharmacy cohort and 1178 in the durable medical equipment cohorts. Although adherence declined over time in both cohorts, the durable medical equipment cohort exhibited significantly superior adherence compared to the pharmacy cohort at 6 months (pharmacy n=615, 52% and durable medical equipment n=761, 65%; P<.001), 9 months (pharmacy n=579, 49% and durable medical equipment cohorts n=714, 61%; P<.001), and 12 months (pharmacy 48% and durable medical equipment n=714, 59%; P<.001). Mean annual total medical costs for adherent patients in the pharmacy cohort were 53% higher than the durable medical equipment cohort (pharmacy US $10,635 and durable medical equipment US $6967; P<.001). In nonadherent patients, the durable medical equipment cohort exhibited a significantly higher rate of therapy reinitiation during the period compared to the pharmacy cohort (pharmacy 61/613, 10% and durable medical equipment 108/485, 22%; P<.001)., Conclusions: The results from this real-world claims analysis demonstrate that, in a matched set, individuals who received their CGM through a durable medical equipment supplier were more adherent to their device. For individuals who experienced a lapse in therapy, those whose supplies were provided through the durable medical equipment channel were more likely to resume use after an interruption than those who received their supplies from a pharmacy. In the matched cohort analysis, those who received their CGM equipment through a durable medical equipment supplier demonstrated a lower total cost of care., (©Jason C Allaire, Consuela Dennis, Arti Masturzo, Steven Wittlin. Originally published in JMIR Diabetes (https://diabetes.jmir.org), 22.07.2024.)

Structure-activity relationship studies of 2,8-disubstituted-1,5-naphthyridines, previously reported as potent inhibitors of Plasmodium falciparum ( Pf ) phosphatidylinositol-4-kinase β (PI4K), identified 1,5-naphthyridines with basic groups at 8-position, which retained Plasmodium PI4K inhibitory activity but switched primary mode of action to the host hemoglobin degradation pathway through inhibition of hemozoin formation. These compounds showed minimal off-target inhibitory activity against the human phosphoinositide kinases and MINK1 and MAP4K kinases, which were associated with the teratogenicity and testicular toxicity observed in rats for the Pf PI4K inhibitor clinical candidate MMV390048. A representative compound from the series retained activity against field isolates and lab-raised drug-resistant strains of Pf . It was efficacious in the humanized NSG mouse malaria infection model at a single oral dose of 32 mg/kg. This compound was nonteratogenic in the zebrafish embryo model of teratogenicity and has a low predicted human dose, indicating that this series has the potential to deliver a preclinical candidate for malaria.

New antimalarial drug candidates that act via novel mechanisms are urgently needed to combat malaria drug resistance. Here, we describe the multi-omic chemical validation of Plasmodium M1 alanyl metalloaminopeptidase as an attractive drug target using the selective inhibitor, MIPS2673. MIPS2673 demonstrated potent inhibition of recombinant Plasmodium falciparum ( Pf A-M1) and Plasmodium vivax ( Pv A-M1) M1 metalloaminopeptidases, with selectivity over other Plasmodium and human aminopeptidases, and displayed excellent in vitro antimalarial activity with no significant host cytotoxicity. Orthogonal label-free chemoproteomic methods based on thermal stability and limited proteolysis of whole parasite lysates revealed that MIPS2673 solely targets Pf A-M1 in parasites, with limited proteolysis also enabling estimation of the binding site on Pf A-M1 to within ~5 Å of that determined by X-ray crystallography. Finally, functional investigation by untargeted metabolomics demonstrated that MIPS2673 inhibits the key role of Pf A-M1 in haemoglobin digestion. Combined, our unbiased multi-omic target deconvolution methods confirmed the on-target activity of MIPS2673, and validated selective inhibition of M1 alanyl metalloaminopeptidase as a promising antimalarial strategy., Competing Interests: CG, MC, GS, RE, TM, CW, ND, NV, CM, NC, SD, SW, SD, VA, TD, PS, SM, DC No competing interests declared, (© 2024, Giannangelo, Challis et al.)

Background: A global death toll of 608,000 in 2022 and emerging parasite resistance to artemisinin, the mainstay of antimalarial chemotherapy derived from the Chinese herb Artemisia annua, urge the development of novel antimalarials. A clinical trial has found high antimalarial potency for aqueous extracts of A. annua as well as its African counterpart Artemisia afra, which contains only trace amounts of artemisinin. The artemisinin-independent antimalarial activity of A. afra points to the existence of other antimalarials present in the plant. However, the publication was retracted due to ethical and methodological concerns in the trial, so the only evidence for antimalarial activity of A. afra is built on in vitro studies reporting efficacy only in the microgram per milliliter range., Hypothesis: Our study aims to shed more light on the controversy around the antimalarial activity of A. afra by assessing its efficacy in mice. In particular, we are testing the hypothesis that A. afra contains a pro-drug that is inactive in vitro but active in vivo after metabolization by the mammalian host., Methods: Plasmodium berghei-infected mice were treated once or thrice (on three consecutive days) with various doses of A. afra, A. annua, or pure artemisinin., Results: Aqueous powder suspensions of A. annua but not A. afra showed antimalarial activity in mice., Conclusion: Our experiments conducted in mice do not support the pro-drug hypothesis., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier GmbH.)

To combat the global burden of malaria, development of new drugs to replace or complement current therapies is urgently required. Here, we show that the compound MMV1557817 is a selective, nanomolar inhibitor of both Plasmodium falciparum and Plasmodium vivax aminopeptidases M1 and M17, leading to inhibition of end-stage hemoglobin digestion in asexual parasites. MMV1557817 can kill sexual-stage P. falciparum , is active against murine malaria, and does not show any shift in activity against a panel of parasites resistant to other antimalarials. MMV1557817 -resistant P. falciparum exhibited a slow growth rate that was quickly outcompeted by wild-type parasites and were sensitized to the current clinical drug, artemisinin. Overall, these results confirm MMV1557817 as a lead compound for further drug development and highlights the potential of dual inhibition of M1 and M17 as an effective multi-species drug-targeting strategy.IMPORTANCEEach year, malaria infects approximately 240 million people and causes over 600,000 deaths, mostly in children under 5 years of age. For the past decade, artemisinin-based combination therapies have been recommended by the World Health Organization as the standard malaria treatment worldwide. Their widespread use has led to the development of artemisinin resistance in the form of delayed parasite clearance, alongside the rise of partner drug resistance. There is an urgent need to develop and deploy new antimalarial agents with novel targets and mechanisms of action. Here, we report a new and potent antimalarial compound, known as MMV1557817 , and show that it targets multiple stages of the malaria parasite lifecycle, is active in a preliminary mouse malaria model, and has a novel mechanism of action. Excitingly, resistance to MMV15578117 appears to be self-limiting, suggesting that development of the compound may provide a new class of antimalarial., Competing Interests: The authors declare no conflict of interest.

Antimalarial resistance to the first-line partner drug piperaquine (PPQ) threatens the effectiveness of artemisinin-based combination therapy. In vitro piperaquine resistance is characterized by incomplete growth inhibition, i.e. increased parasite growth at higher drug concentrations. However, the 50% inhibitory concentrations (IC 50 ) remain relatively stable across parasite lines. Measuring parasite viability of a drug-resistant Cambodian Plasmodium falciparum isolate in a parasite reduction ratio (PRR) assay helped to better understand the resistance phenotype towards PPQ. In this parasite isolate, incomplete growth inhibition translated to only a 2.5-fold increase in IC 50 but a dramatic decrease of parasite killing in the PRR assay. Hence, this pilot study reveals the potential of in vitro parasite viability assays as an important, additional tool when it comes to guiding decision-making in preclinical drug development and post approval. To the best of our knowledge, this is the first time that a compound was tested against a drug-resistant parasite in the in vitro PRR assay., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Walz, Sax, Scheurer, Tamasi, Mäser and Wittlin.)

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Brad Sleebs reports financial support was provided by National Health and Medical Research Council. Susan Charman reports financial support was provided by National Health and Medical Research Council. Paul Gilson reports financial support was provided by National Health and Medical Research Council. Alan Cowman reports financial support was provided by National Health and Medical Research Council. William Nguyen reports financial support was provided by National Health and Medical Research Council. Tania de Koning Ward reports financial support was provided by National Health and Medical Research Council. If there are other authors, they 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 Masson SAS.. All rights reserved.)

Malaria is caused by parasites of the Plasmodium genus and remains one of the most pressing human health problems. The spread of parasites resistant to or partially resistant to single or multiple drugs, including frontline antimalarial artemisinin and its derivatives, poses a serious threat to current and future malaria control efforts. In vitro drug assays are important for identifying new antimalarial compounds and monitoring drug resistance. Due to its robustness and ease of use, the [ 3 H]-hypoxanthine incorporation assay is still considered a gold standard and is widely applied, despite limited sensitivity and the dependence on radioactive material. Here, we present a first-of-its-kind chemiluminescence-based antimalarial drug screening assay. The effect of compounds on P. falciparum is monitored by using a dioxetane-based substrate (AquaSpark β-D-galactoside) that emits high-intensity luminescence upon removal of a protective group (β-D-galactoside) by a transgenic β-galactosidase reporter enzyme. This biosensor enables highly sensitive, robust, and cost-effective detection of asexual, intraerythrocytic P. falciparum parasites without the need for parasite enrichment, washing, or purification steps. We are convinced that the ultralow detection limit of less than 100 parasites of the presented biosensor system will become instrumental in malaria research, including but not limited to drug screening.

Toward addressing the cardiotoxicity liability associated with the antimalarial drug astemizole (AST, hERG IC 50 = 0.0042 μM) and its derivatives, we designed and synthesized analogues based on compound 1 ( Pf NF54 IC 50 = 0.012 μM; hERG IC 50 = 0.63 μM), our previously identified 3-trifluoromethyl-1,2,4-oxadiazole AST analogue. Compound 11 retained in vitro multistage antiplasmodium activity (ABS Pf NF54 IC 50 = 0.017 μM; gametocytes Pf iGc/ Pf LGc IC 50 = 1.24/1.39 μM, and liver-stage Pb HepG2 IC 50 = 2.30 μM), good microsomal metabolic stability (MLM CL int < 11 μL·min -1 ·mg -1 , E H < 0.33), and solubility (150 μM). It shows a ∼6-fold and >6000-fold higher selectivity against human ether-á-go-go-related gene higher selectively potential over hERG relative to 1 and AST, respectively. Despite the excellent in vitro antiplasmodium activity profile, in vivo efficacy in the Plasmodium berghei mouse infection model was diminished, attributable to suboptimal oral bioavailability ( F = 14.9%) at 10 mg·kg -1 resulting from poor permeability (log  D 7.4 = -0.82). No cross-resistance was observed against 44 common Pf mutant lines, suggesting activity via a novel mechanism of action., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

We discovered dibenzannulated medium-ring keto lactams (11,12-dihydro-5 H -dibenzo[ b , g ]azonine-6,13-diones) as a new antimalarial chemotype. Most of these had chromatographic LogD 7.4 values ranging from <0 to 3 and good kinetic solubilities (12.5 to >100 μg/mL at pH 6.5). The more polar compounds in the series (LogD 7.4 values of <2) had the best metabolic stability (CL int values of <50 μL/min/mg protein in human liver microsomes). Most of the compounds had relatively low cytotoxicity, with IC 50 values >30 μM, and there was no correlation between antiplasmodial activity and cytotoxicity. The four most potent compounds had Plasmodium falciparum IC 50 values of 4.2 to 9.4 nM and in vitro selectivity indices of 670 to >12,000. They were more than 4 orders-of-magnitude less potent against three other protozoal pathogens ( Trypanosoma brucei rhodesiense , Trypanosoma cruzi , and Leishmania donovani ) but did have relatively high potency against Toxoplasma gondii , with IC 50 values ranging from 80 to 200 nM. These keto lactams are converted into their poorly soluble 4(1 H )-quinolone transannular condensation products in vitro in culture medium and in vivo in mouse blood. The similar antiplasmodial potencies of three keto lactam-quinolone pairs suggest that the quinolones likely contribute to the antimalarial activity of the lactams.

Thanks to its expertise in clinical research, epidemiology, infectious diseases, microbiology, parasitology, public health, translational research and tropical medicine, coupled with deeply rooted partnerships with institutions in low- and middle-income countries (LMICs), the Swiss Tropical and Public Health Institute (Swiss TPH) has been a key contributor in many drug research and development consortia involving academia, pharma and product development partnerships. Our know-how of the maintenance of parasites and their life-cycles in the laboratory, plus our strong ties to research centres and disease control programme managers in LMICs with access to field sites and laboratories, have enabled systems for drug efficacy testing in vitro and in vivo, clinical research, and modelling to support the experimental approaches. Thus, Swiss TPH has made fundamental contributions towards the development of new drugs - and the better use of old drugs - for neglected tropical diseases and infectious diseases of poverty, such as Buruli ulcer, Chagas disease, food-borne trematodiasis (e.g. clonorchiasis, fascioliasis and opisthorchiasis), human African trypanosomiasis, leishmaniasis, malaria, schistosomiasis, soil-transmitted helminthiasis and tuberculosis. In this article, we show case the success stories of molecules to which Swiss TPH has made a substantial contribution regarding their use as anti-infective compounds with the ultimate aim to improve people's health and well-being., (Copyright 2023 Pascal Mäser, Sonja Bernhard, Reto Brun, Christian Burri, Sébastien Gagneux, Manuel W. Hetzel, Marcel Kaiser, Christian Lengeler, Gerd Pluschke, Elisabeth Reus, Matthias Rottmann, Jürg Utzinger, Louisa Warryn, Sergio Wittlin, Jennifer Keiser. License: This work is licensed under a Creative Commons Attribution 4.0 International License.)

With the resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany, of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250 was prioritized for further optimization. The potency of the series lead compound ( WEHI-518 ) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound, MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting that this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasite's life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB that clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies that showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

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.