Metabolomics reveals potentiated metabolic perturbations induced by antimalarial drug combinations against Plasmodium falciparum in vitro

The 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. An integrated metabolomic and pharmacological analysis was performed on P. falciparum parasites exposed to chloroquine (CQ), pyrimethamine (PY), sulfadoxine (SD), and their combinations (SDPY and SDCQ). Dose‒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. Dose‒response assays confirmed the nanomolar potency of PY (IC₅₀ = 12.5 nM) and CQ (IC₅₀ = 11 nM) compared with the micromolar efficacy of SD (IC₅₀ = 9.1 μM), which is consistent with the role of PY as an antifolate partner drug capable of enhancing combinatorial, pathway-level metabolic perturbations. 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. Pyrimidine suppression has emerged as the central hallmark of antifolate-based therapy in P. falciparum. The distinct metabolic perturbations observed with drug combinations suggest enhanced pathway-level responses that may contribute to improved antiplasmodial activity and inform future combination strategies. These findings provide a mechanistic foundation for guiding antimalarial combination policies, optimizing therapeutic regimens, and strengthening rational drug-design strategies.