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The precise mode of action of ganaplacide (KAF156), a phase III antimalarial candidate, remains elusive. Here we employ omics-based methods with the closely related chemical analog, GNF179, to search for potential Plasmodium targets. Ranking potential targets derived from chemical genetics and proteomic affinity chromatography methodologies identifies SEY1 , or Synthetic Enhancement of YOP1, which is predicted to encode an essential dynamin-like GTPase implicated in homotypic fusion of endoplasmic reticulum (ER) membranes. We demonstrate that GNF179 decreases Plasmodium SEY1 melting temperature. We further show that GNF179 binds to recombinant Plasmodium SEY1 and subsequently inhibits its GTPase activity, which is required for maintaining ER architecture. Using ultrastructure expansion microscopy, we find GNF179 treatment changes parasite ER and Golgi morphology. We also confirm that SEY1 is an essential gene in P. falciparum . These data suggest that SEY1 may contribute to the mechanism of action of imidazolopiperazines and is a new and attractive druggable target.

Assessment and proper management of sites contaminated with heavy metals require precise information on the spatial distribution of these metals. This study aimed to predict and map the distribution of Cd, Cu, Ni, Pb, and Zn across the conterminous USA using point observations, environmental variables, and Histogram-based Gradient Boosting (HGB) modeling. Over 9180 surficial soil observations from the Soil Geochemistry Spatial Database (SGSD) (n = 1150), the Geochemical and Mineralogical Survey of Soils (GMSS) (n = 4857), and the Holmgren Dataset (HD) (n = 3400), and 28 covariates (100 m × 100 m grid) representing climate, topography, vegetation, soils, and anthropic activity were compiled. Model performance was evaluated on 20 % of the data not used in calibration using the coefficient of determination (R 2 ), concordance correlation coefficient (ρ c ), and root mean square error (RMSE) indices. Uncertainty of predictions was calculated as the difference between the estimated 95 and 5 % quantiles provided by HGB. The model explained up to 50 % of the variance in the data with RMSE ranging between 0.16 (mg kg -1 ) for Cu and 23.4 (mg kg -1 ) for Zn, respectively. Likewise, ρ c ranged between 0.55 (Cu) and 0.68 (Zn), respectively, and Zn had the highest R 2 (0.50) among all predictions. We observed high Pb concentrations near urban areas. Peak concentrations of all studied metals were found in the Lower Mississippi River Valley. Cu, Ni, and Zn concentrations were higher on the West Coast; Cd concentrations were higher in the central USA. Clay, pH, potential evapotranspiration, temperature, and precipitation were among the model's top five important covariates for spatial predictions of heavy metals. The combined use of point observations and environmental covariates coupled with machine learning provided a reliable prediction of heavy metals distribution in the soils of the conterminous USA. The updated maps could support environmental assessments, monitoring, and decision-making with this methodology applicable to other soil databases, worldwide., 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., (Published by Elsevier B.V.)

Mycotoxin contamination of corn is a pervasive problem that negatively impacts human and animal health and causes economic losses to the agricultural industry worldwide. Historical aflatoxin (AFL) and fumonisin (FUM) mycotoxin contamination data of corn, daily weather data, satellite data, dynamic geospatial soil properties, and land usage parameters were modeled to identify factors significantly contributing to the outbreaks of mycotoxin contamination of corn grown in Illinois (IL), AFL >20 ppb, and FUM >5 ppm. Two methods were used: a gradient boosting machine (GBM) and a neural network (NN). Both the GBM and NN models were dynamic at a state-county geospatial level because they used GPS coordinates of the counties linked to soil properties. GBM identified temperature and precipitation prior to sowing as significant influential factors contributing to high AFL and FUM contamination. AFL-GBM showed that a higher aflatoxin risk index (ARI) in January, March, July, and November led to higher AFL contamination in the southern regions of IL. Higher values of corn-specific normalized difference vegetation index (NDVI) in July led to lower AFL contamination in Central and Southern IL, while higher wheat-specific NDVI values in February led to higher AFL. FUM-GBM showed that temperature in July and October, precipitation in February, and NDVI values in March are positively correlated with high contamination throughout IL. Furthermore, the dynamic geospatial models showed that soil characteristics were correlated with AFL and FUM contamination. Greater calcium carbonate content in soil was negatively correlated with AFL contamination, which was noticeable in Southern IL. Greater soil moisture and available water-holding capacity throughout Southern IL were positively correlated with high FUM contamination. The higher clay percentage in the northeastern areas of IL negatively correlated with FUM contamination. NN models showed high class-specific performance for 1-year predictive validation for AFL (73%) and FUM (85%), highlighting their accuracy for annual mycotoxin prediction. Our models revealed that soil, NDVI, year-specific weekly average precipitation, and temperature were the most important factors that correlated with mycotoxin contamination. These findings serve as reliable guidelines for future modeling efforts to identify novel data inputs for the prediction of AFL and FUM outbreaks and potential farm-level management practices., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Castano-Duque, Winzeler, Blackstock, Liu, Vergopolan, Focker, Barnett, Owens, van der Fels-Klerx, Vaughan and Rajasekaran.)

Introduction: Aflatoxin (AFL), a secondary metabolite produced from filamentous fungi, contaminates corn, posing significant health and safety hazards for humans and livestock through toxigenic and carcinogenic effects. Corn is widely used as an essential commodity for food, feed, fuel, and export markets; therefore, AFL mitigation is necessary to ensure food and feed safety within the United States (US) and elsewhere in the world. In this case study, an Iowa-centric model was developed to predict AFL contamination using historical corn contamination, meteorological, satellite, and soil property data in the largest corn-producing state in the US., Methods: We evaluated the performance of AFL prediction with gradient boosting machine (GBM) learning and feature engineering in Iowa corn for two AFL risk thresholds for high contamination events: 20-ppb and 5-ppb. A 90%-10% training-to-testing ratio was utilized in 2010, 2011, 2012, and 2021 ( n  = 630), with independent validation using the year 2020 ( n  = 376)., Results: The GBM model had an overall accuracy of 96.77% for AFL with a balanced accuracy of 50.00% for a 20-ppb risk threshold, whereas GBM had an overall accuracy of 90.32% with a balanced accuracy of 64.88% for a 5-ppb threshold. The GBM model had a low power to detect high AFL contamination events, resulting in a low sensitivity rate. Analyses for AFL showed satellite-acquired vegetative index during August significantly improved the prediction of corn contamination at the end of the growing season for both risk thresholds. Prediction of high AFL contamination levels was linked to aflatoxin risk indices (ARI) in May. However, ARI in July was an influential factor for the 5-ppb threshold but not for the 20-ppb threshold. Similarly, latitude was an influential factor for the 20-ppb threshold but not the 5-ppb threshold. Furthermore, soil-saturated hydraulic conductivity (Ksat) influenced both risk thresholds., Discussion: Developing these AFL prediction models is practical and implementable in commodity grain handling environments to achieve the goal of preventative rather than reactive mitigations. Finding predictors that influence AFL risk annually is an important cost-effective risk tool and, therefore, is a high priority to ensure hazard management and optimal grain utilization to maximize the utility of the nation's corn crop., 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 © 2023 Branstad-Spates, Castano-Duque, Mosher, Hurburgh, Owens, Winzeler, Rajasekaran and Bowers.)

New antimalarial treatments with novel mechanism of action are needed to tackle Plasmodium falciparum infections that are resistant to first-line therapeutics. Here we report the exploration of MMV692140 (2) from the Pathogen Box, a collection of 400 compounds that was made available by Medicines for Malaria Venture (MMV) in 2015. Compound 2 was profiled in in vitro models of malaria and was found to be active against multiple life-cycle stages of Plasmodium parasites. The mode of resistance, and putatively its mode of action, was identified as Plasmodium falciparum translation elongation factor 2 (PfeEF2), which is responsible for the GTP-dependent translocation of the ribosome along mRNA. The compound maintains activity against a series of drug-resistant parasite strains. The structural motif of the tetrahydroquinoline (2) was explored in a chemistry program with its structure-activity relationships examined, resulting in the identification of an analog with 30-fold improvement of antimalarial asexual blood stage potency., (© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Malaria control programs continue to be threatened by drug resistance. To identify new antimalarials, we conducted a phenotypic screen and identified a novel tetrazole-based series that shows fast-kill kinetics and a relatively low propensity to develop high-level resistance. Preliminary structure-activity relationships were established including identification of a subseries of related amides with antiplasmodial activity. Assaying parasites with resistance to antimalarials led us to test whether the series had a similar mechanism of action to chloroquine (CQ). Treatment of synchronized Plasmodium falciparum parasites with active analogues revealed a pattern of intracellular inhibition of hemozoin (Hz) formation reminiscent of CQ's action. Drug selections yielded only modest resistance that was associated with amplification of the multidrug resistance gene 1 ( pfmdr1 ). Thus, we have identified a novel chemical series that targets the historically druggable heme polymerization pathway and that can form the basis of future optimization efforts to develop a new malaria treatment.

The neglected tropical disease, schistosomiasis, is caused by trematode blood flukes of the Schistosoma genus and infects approximately 200 million people worldwide. With just one partially effective drug available for disease treatment, new drugs are urgently needed. Herein, a series of 47 phthalimide (Pht) analogues possessing high-value bioactive scaffolds (i.e., benzimidazole and 1,2,3,-triazoles) was synthesized by click-chemistry. Compounds were evaluated for anti-schistosomal activity in culture against somules (post-infective larvae) and adults of Schistosoma mansoni, their predicted ADME (absorption, distribution, metabolism, and excretion) properties, and toxicity vs. HepG2 cells. The majority showed favorable parameters for surface area, lipophilicity, bioavailability and Lipinski score. Thirteen compounds were active at 10 µM against both somules and adults ( 6d , 6f , 6i - 6l , 6n - 6p , 6s , 6r' , 6t' and 6w ). Against somules, the majority caused degeneracy and/or death after 72 h; whereas against adult parasites, five compounds ( 6l , 6d , 6f , 6r' and 6s ) elicited degeneracy, tegumental (surface) damage and/or death. Strongest potency against both developmental stages was recorded for compounds possessing n-butyl or isobutyl as a linker, and a pentafluorophenyl group on triazole. Apart from five compounds for which anti-parasite activity tracked with toxicity to HepG2 cells, there was apparently no toxicity to HepG2 cells (EC 50 values ≥50 µM). The data overall suggest that phthaloyl-triazole compounds are favorable synthons for additional studies as anti-schistosomals., Competing Interests: The authors declare no competing financial interest.

Plasmodium parasites possess a protein with homology to Niemann-Pick Type C1 proteins (Niemann-Pick Type C1-Related protein, NCR1). We isolated parasites with resistance-conferring mutations in Plasmodium falciparum NCR1 (PfNCR1) during selections with three diverse small-molecule antimalarial compounds and show that the mutations are causative for compound resistance. PfNCR1 protein knockdown results in severely attenuated growth and confers hypersensitivity to the compounds. Compound treatment or protein knockdown leads to increased sensitivity of the parasite plasma membrane (PPM) to the amphipathic glycoside saponin and engenders digestive vacuoles (DVs) that are small and malformed. Immuno-electron microscopy and split-GFP experiments localize PfNCR1 to the PPM. Our experiments show that PfNCR1 activity is critically important for the composition of the PPM and is required for DV biogenesis, suggesting PfNCR1 as a novel antimalarial drug target., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: EI, SD, SB, JB, EO, ML, SG, JN, EW, AV, DG No competing interests declared, (© 2019, Istvan et al.)

Plasmodium parasites, the causative agent of malaria infections, rapidly evolve drug resistance and escape detection by the human immune response via the incredible mutability of its genome. Understanding the genetic mechanisms by which Plasmodium parasites develop antimalarial resistance is essential to understanding why most drugs fail in the clinic and designing the next generation of therapies. A systematic genomic analysis of 262 Plasmodium falciparum clones with stable in vitro resistance to 37 diverse compounds with potent antimalarial activity was undertaken with the main goal of identifying new drug targets. Despite several challenges inherent to this method of in vitro drug resistance generation followed by whole genome sequencing, the study was able to identify a likely drug target or resistance gene for every compound for which resistant parasites could be generated. Known and novel P falciparum resistance mediators were discovered along with several new promising antimalarial drug targets. Surprisingly, gene amplification events contributed to one-third of the drug resistance acquisition events. The study can serve as a model for drug discovery and resistance analyses in other similar microbial pathogens amenable to in vitro culture., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

In this work we aimed to develop parasite-selective histone deacetylase inhibitors (HDAC) inhibitors with activity against the disease-causing asexual blood stages of Plasmodium as well as causal prophylactic and/or transmission blocking properties. We report the design, synthesis, and biological testing of a series of 13 terephthalic acid-based HDAC inhibitors. All compounds showed low cytotoxicity against human embryonic kidney (HEK293) cells (IC 50 : 8->51 μm), with 11 also having sub-micromolar in vitro activity against drug-sensitive (3D7) and multidrug-resistant (Dd2) asexual blood-stage P. falciparum parasites (IC 50 ≈0.1-0.5 μm). A subset of compounds were examined for activity against early- and late-stage P. falciparum gametocytes and P. berghei exo-erythrocytic-stage parasites. While only moderate activity was observed against gametocytes (IC 50 >2 μm), the most active compound (N 1 -((3,5-dimethylbenzyl)oxy)-N 4 -hydroxyterephthalamide, 1 f) showed sub-micromolar activity against P. berghei exo-erythrocytic stages (IC 50 0.18 μm) and >270-fold better activity for exo-erythrocytic forms than for HepG2 cells. This, together with asexual-stage in vitro potency (IC 50 ≈0.1 μm) and selectivity of this compound versus human cells (SI>450), suggests that 1 f may be a valuable starting point for the development of novel antimalarial drug leads with low host cell toxicity and multi-stage anti-plasmodial activity., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Current efforts to reduce the global burden of malaria are threatened by the rapid spread throughout Asia of Plasmodium falciparum resistance to artemisinin-based combination therapies, which includes increasing rates of clinical failure with dihydroartemisinin plus piperaquine (PPQ) in Cambodia. Using zinc finger nuclease-based gene editing, we report that addition of the C101F mutation to the chloroquine (CQ) resistance-conferring PfCRT Dd2 isoform common to Asia can confer PPQ resistance to cultured parasites. Resistance was demonstrated as significantly higher PPQ concentrations causing 90% inhibition of parasite growth (IC 90 ) or 50% parasite killing (50% lethal dose [LD 50 ]). This mutation also reversed Dd2-mediated CQ resistance, sensitized parasites to amodiaquine, quinine, and artemisinin, and conferred amantadine and blasticidin resistance. Using heme fractionation assays, we demonstrate that PPQ causes a buildup of reactive free heme and inhibits the formation of chemically inert hemozoin crystals. Our data evoke inhibition of heme detoxification in the parasite's acidic digestive vacuole as the primary mode of both the bis -aminoquinoline PPQ and the related 4-aminoquinoline CQ. Both drugs also inhibit hemoglobin proteolysis at elevated concentrations, suggesting an additional mode of action. Isogenic lines differing in their pfmdr1 copy number showed equivalent PPQ susceptibilities. We propose that mutations in PfCRT could contribute to a multifactorial basis of PPQ resistance in field isolates. IMPORTANCE The global agenda to eliminate malaria depends on the continued success of artemisinin-based combination therapies (ACTs), which target the asexual blood stages of the intracellular parasite Plasmodium Partial resistance to artemisinin, however, is now established in Southeast Asia, exposing the partner drugs to increased selective pressure. Plasmodium falciparum resistance to the first-line partner piperaquine (PPQ) is now spreading rapidly in Cambodia, resulting in clinical treatment failures. Here, we report that a variant form of the Plasmodium falciparum chloroquine resistance transporter, harboring a C101F mutation edited into the chloroquine (CQ)-resistant Dd2 isoform prevalent in Asia, can confer PPQ resistance in cultured parasites. This was accompanied by a loss of CQ resistance. Biochemical assays showed that PPQ, like CQ, inhibits the detoxification of reactive heme that is formed by parasite-mediated catabolism of host hemoglobin. We propose that novel PfCRT variants emerging in the field could contribute to a multigenic basis of PPQ resistance., (Copyright © 2017 Dhingra et al.)

MMV007564 is a novel antimalarial benzimidazolyl piperidine chemotype identified in cellular screens. To identify the genetic determinant of MMV007564 resistance, parasites were cultured in the presence of the compound to generate resistant lines. Whole genome sequencing revealed distinct mutations in the gene named Plasmodium falciparum cyclic amine resistance locus (pfcarl), encoding a conserved protein of unknown function. Mutations in pfcarl are strongly associated with resistance to a structurally unrelated class of compounds, the imidazolopiperazines, including KAF156, currently in clinical trials. Our data demonstrate that pfcarl mutations confer resistance to two distinct compound classes, benzimidazolyl piperidines and imidazolopiperazines. However, MMV007564 and the imidazolopiperazines, KAF156 and GNF179, have different timings of action in the asexual blood stage and different potencies against the liver and sexual blood stages. These data suggest that pfcarl is a multidrug-resistance gene rather than a common target for benzimidazolyl piperidines and imidazolopiperazines.