Biology subjects

Qahash, T.

Publications and source records attributed to Qahash, T..

4 recordsLinked to original sources

bioRxiv2026

Double Mutations in Plasmodium falciparum Kelch13 drive resistance to next-generation artemisinin derivatives in malaria parasites

New antimalarial compounds are urgently required to overcome artemisinin partial resistance that has emerged in Asia and now Africa. Ozonides are promising next-generation artemisinins that offer the improved pharmacokinetic property of a prolonged in vivo half-life. To assess the potential for parasite resistance to ozonides in an artemisinin-resistant background, we subjected Cambodian Kelch13 (K13) mutant parasites to increasing artefenomel (OZ439) pressure up to in vivo physiological concentrations. Whole-genome sequencing identified a novel non-propeller K13 A212T mutation in OZ439-resistant parasites. Gene editing and drug susceptibility assays revealed that the K13 double mutation R539T+A212T is a determinant of OZ439 resistance. In extended parasite recovery assays, this resistance mechanism was associated with accelerated parasite recrudescence following OZ439 or OZ277 exposure. This phenotype was also observed in K13 C580Y+A212T double mutant parasites. Global metabolomic profiling revealed no changes in the levels of hemoglobin-derived peptides in OZ439-resistant parasites, suggesting that resistance is not associated with drug activation. Instead, double mutant parasites exhibited increased levels of metabolites linked to glutathione, nucleotide, and aspartate-glutamate metabolism, suggesting a higher capacity for redox regulation to tolerate drug-induced oxidative damage. Our findings demonstrate that ozonide resistance can emerge through a novel K13 mutation on the background of existing artemisinin-resistance k13 alleles.

microbiology↗

bioRxiv2026

Functional validation of the Plasmodium falciparum K13 C580Y mutation in recently collected Ethiopian isolates

Recent genomic investigation in Ethiopia identified the first detection of the Plasmodium falciparum kelch13 (K13) C580Y substitution in the Horn of Africa. To assess its functional impact, we introduced C580Y into two recently collected Ethiopian clinical isolates using CRISPR-Cas9 genome editing. Ring-stage survival assays showed significantly elevated in vitro dihydroartemisinin (DHA) survival in edited parasites relative to isogenic controls, demonstrating that C580Y confers artemisinin tolerance in contemporary Ethiopian parasite genetic backgrounds.

microbiology↗

bioRxiv2025

Metabolomics reveals synergistic antimalarial drug pairing effects against Plasmodium falciparum in vitro

BackgroundThe development of new drugs against afflictions that disproportionately impact poorly resourced areas around the globe is an expensive endeavor. As cost-effective alternatives, strategic combinations of approved drugs can be used to enhance the efficacy against Plasmodium falciparum. Understanding the metabolic consequences of such combinations is essential for optimizing treatment strategies and delaying drug resistance. MethodsAn integrated metabolomic and pharmacological analysis was performed on P. falciparum exposed to chloroquine (CQ), pyrimethamine (PY), sulfadoxine (SD), and their combinations (SDPY and SDCQ). Dose{square}response assays were used to quantify drug potency, whereas untargeted metabolomic profiling was used to assess pathway-level perturbations associated with individual and combined treatments. ResultsDose{square}response assays confirmed the nanomolar potency of PY (IC = 12.5 nM) and CQ (IC = 11 nM) compared with the micromolar efficacy of SD (IC = 9.1 {micro}M), which is consistent with its role as a synergistic antifolate partner. Metabolomic profiling revealed that PY strongly disrupted folate-dependent pyrimidine biosynthesis, leading to deoxyuridine and dUMP accumulation, whereas SD caused milder perturbations, which was consistent with DHPS inhibition. CQ produced modest metabolic effects alone but markedly amplified antifolate-induced stress when combined with PY. Drug combinations generated metabolic responses that are distinct from those resulting from individual treatments. Across antifolate combinations, consistent trends included reduced amino acid pools, suppression of thiamine and glutathione metabolism, and enhanced PPP inhibition, leading to broad disruption of nucleotide, redox, and carbon metabolism. ConclusionsPyrimidine suppression has emerged as the central hallmark of antifolate-based therapy in P. falciparum. The distinct synergistic signatures observed with drug combinations support their potential to enhance efficacy and delay resistance. These findings provide a mechanistic foundation for guiding antimalarial combination policies, optimizing therapeutic regimens, and strengthening rational drug-design strategies.

systems biology↗

bioRxiv2024

Measuring Growth, Resistance and Recovery after Artemisinin Treatment of Plasmodium falciparum in a semi-high-throughput Assay

BackgroundArtemisinin partial resistance (ART-R) has spread throughout Southeast Asia and mutations in pfKelch13, the molecular marker of resistance, are widely reported in East Africa. Effective in vitro assays and robust phenotypes are crucial for monitoring populations for the emergence and spread of resistance. The recently developed extended Recovery Ring-stage Survival Assay used a qPCR-based readout to reduce the labor intensiveness for in vitro phenotyping of ART-R and improved correlation with the clinical phenotype of ART-R. Here, we extend and refine this assay to include measurements of parasite growth and recovery after drug exposure. Clinical isolates and progeny from two genetic crosses were used to optimize and validate the reliability of a straight-from-blood, SYBR Green-based qPCR protocol in a 96-well plate format to accurately measure phenotypes for Growth, Resistance, and Recovery. ResultsThe assay determined growth between 6 h and 96 h, resistance at 120 h, and recovery from 120 h and 192 h. Growth can be accurately captured by qPCR and is shown by reproduction of previous growth phenotypes from HB3 x Dd2. Resistance measured at 120 h continually shows the most consistent phenotype for ring stage susceptibility. Recovery identifies an additional response to drug than parasites that are determined sensitive by Fold Change at 120 h. Comparison of progeny phenotypes for Growth vs Resistance showed a minor but significant correlation, whereas Growth vs Recovery and Resistance vs Recovery showed no significant correlation. Additionally, dried blood spot (DBS) samples matched Fold Change measured from liquid samples demonstrating Resistance can be easily quantified using either storage method. ConclusionsThe qPCR-based methodology provides the throughput needed to quickly measure large numbers of parasites for multiple relevant phenotypes. Growth can reveal fitness defects and illuminate relationships between proliferation rates and drug response. Recovery serves as a complementary phenotype to resistance that quantifies the ability of sensitive parasites to tolerate drug exposure. All three phenotypes offer a comprehensive assessment of parasite-drug interaction each with independent genetic determinants of main effect and overlapping secondary effects that should be further. By adapting our method to include DBS, readouts can be easily extended to ex vivo surveillance applications.

microbiology↗