Conference Agenda

Platelet Factor 4 Derived Internalisation Peptide (PDIP), based on the antimicrobial peptide-like domain of human PF4, exhibits activity against Plasmodium. PDIP rapidly kills parasites by penetrating Plasmodium-infected erythrocytes and destroying the digestive vacuole. Why PDIP penetrates only infected but not healthy erythrocytes and destroys only the digestive vacuole is unclear. We hypothesised that differences in lipid composition between infected erythrocyte and parasite membranes alter membrane polarity and fluidity, thereby facilitating PDIP’s differential interactions.

To test this, we used the membrane dyes Nile Red and Laurdan, which report polarity and fluidity through emission‑wavelength shifts quantified as ratiometric indices. These ratios were measured for uninfected and Plasmodium-infected erythrocyte membranes, and the intracellular parasite, with and without PDIP treatment. We found comparable fluidity between infected and uninfected erythrocyte membranes. Polarity differed significantly, ranking from most to least: intracellular parasite > uninfected > and infected erythrocyte membranes. These polarity differences may contribute to PDIP’s ability to selectively enter infected cells and suggested that parasites contain more polar lipids that may facilitate PDIP’s activity. PDIP treatment significantly increased polarity and reduced fluidity for all membrane types. This indicates a non-specific interaction of the peptide independent of translocation that alters membrane properties non-destructively.

Widespread resistance to all current antimalarials threatens the control and eradication of malaria. Second-generation bis-triazines display low nanomolar potency and fast-killing asexual P. falciparum. However, the novel mechanism of action (MOA) remains unknown.

In vitro combination drug-pulse assays using various inhibitors were performed to identify potential modulators of bis-triazine activity. We also included two antimalarial candidates currently under development with the Medicines for Malaria Venture (MMV) with some structural similarity to the bis-triazines. E64d, a cysteine protease inhibitor, bafilomycin A1, a V-ATPase inhibitor and chloroquine all caused antagonism of trophozoite-stage activity across the bis-triazine analogues and MMV candidates (between 2 and 10-fold increases in IC₅₀). All three activity modulators are known to localise to the digestive vacuole and indicates potential involvement of the haemoglobin digestion pathway in the MOA of these series.

3-hour ring-stage survival assays with an artemisinin-resistant clinical isolate and a Pf3D7 line genetically modified to induce knockdown of essential falcipain-3 resulted in decreased activity (up to 20-fold increases in IC₅₀) for both MMV candidates and one bis-triazine analogue. The current lead bis-triazine analogue, however, observed no change or slight hypersensitisation. Uninterrupted haemoglobin digestion appears to be vital for these compounds to maintain their fast-killing activity.

Malaria remains one of the most devastating infectious diseases globally. Resistance to frontline antimalarials continues to compromise control efforts, highlighting the urgent need for novel therapeutic strategies. We previously identified the strobilurin compound MMV1794211 as a highly potent antiplasmodial agent with low-picomolar activity against blood-stage Plasmodium falciparum. Biochemical and enzymatic analyses demonstrated that MMV1794211 targets Complex III of the parasite mitochondrial electron transport chain (mtETC), a clinically validated antimalarial target. Our recent structure-activity relationship studies have identified key chemical elements underlying its potency and enabled the synthesis of derivative compounds with favourable antiplasmodial activity and enhanced selectivity for parasites over human cells. In vitro evolution experiments generated parasites carrying a single mutation in cytochrome b conferring high-level resistance to MMV1794211. Drug sensitivity profiling of the mutant revealed pan-strobilurin resistance; however, no cross-resistance was observed with established antimalarials or other mtETC inhibitors currently under investigation. Ongoing work includes assessing the fitness costs associated with strobilurin-resistance mutations and developing derivative compounds that remain active in resistant parasites. Collectively, our study provides a comprehensive evaluation of strobilurins as a chemically distinct class of antiplasmodial agents and highlights their promise for future therapeutic development.