Novel Antiplasmodial Natural Products Identified Through a Modified Bioluminescence-Based Rate-of-Kill Assay

<b>Background/Objectives</b>: The discovery of antimalarial compounds with novel mechanisms of action and distinct rates of kill (RoK) is essential to address emerging drug resistance in <i>Plasmodium falciparum</i>. Natural product libraries represent a valuable and chemically diverse source of potential new antiplasmodial scaffolds. This study aimed (i) to evaluate a modified bioluminescence relative rate-of-kill (mBRRoK) assay as a rapid triage platform for screening large compound libraries with previously unknown antiplasmodial activity, enabling simultaneous assessment of potency and RoK, and (ii) to identify novel compounds with potent and selective in vitro erythrocytic activity. <b>Methods:</b> A fixed two-concentration mBRRoK screen was applied to 1165 compounds from the PhytoQuest natural product library. Antiplasmodial activity and RoK profiles were assessed over 48 h using two genetically distinct luciferase-expressing <i>P. falciparum</i> strains (Dd2^luc and NF54^luc) with distinct drug resistance backgrounds. Reproducibility was evaluated across biological replicates. Selected hits underwent secondary profiling, including EC₅₀ determination and HepG2 cytotoxicity assessment to establish potency and selectivity. <b>Results</b>: The primary screen identified 36 lead compounds demonstrating potent activity within 48 h, encompassing both fast- and slow-acting phenotypes. Activity was reproducible and largely strain-independent across both parasite lines. Secondary profiling prioritised four compounds (<b>100657</b>, <b>101158</b>, <b>101160</b>, and <b>101173</b>) with nanomolar-to-micromolar antiplasmodial potency and favourable selectivity indices relative to mammalian cell cytotoxicity. <b>Conclusions</b>: The mBRRoK assay provides a robust and scalable framework for integrating potency and pharmacodynamic assessment in early antimalarial discovery. This strategy enabled efficient prioritisation of selective natural product-derived leads with distinct killing profiles, supporting their progression toward further optimisation and preclinical development.