Conference Agenda

Malaria causes significant morbidity and mortality, with 249 million cases and 608,000 deaths in 2022. Progress towards elimination and eradication goals are threatened by parasite drug-resistance. To combat drug-resistance, therapies that possess modes of action different to current antimalarial drugs are needed. The indoloquinolizidine alkaloid natural product alstonine was identified in a screen of compounds from the NatureBank library, Griffith University. Alstonine was shown to have a P. falciparum in vitro IC₅₀ of 0.18 µM, >1,000 selectivity for parasites over human cells, and a slow action phenotype. Alstonine was hypothesized to have a novel mode of action due to a lack of cross-resistance seen by a panel of drug-resistant P. falciparum lines and a different slow action activity to delayed-death compounds. To investigate the antiplasmodial target of alstonine, P. falciparum parasites were generated with 20-fold less sensitivity to alstonine compared to wildtype parasites. Whole genome sequencing of alstonine-resistant subclones identified a point mutation and copy number variation in a gene encoding a putative inner-mitochondrial membrane protein (PfMPV17). CRISPR editing was undertaken to induce the reported point mutation in wildtype P. falciparum parasites. PfMPV17 transgenic parasites displayed a decrease in sensitivity to alstonine, implicating PfMPV17 in the resistance mechanism to alstonine.

The emergence and spread of drug resistance stymies our efforts to control malaria. Clindamycin is a safe and inexpensive antimalarial partner drug candidate. Clindamycin kills malaria parasites by targeting the apicoplast prokaryotic translation machinery, but uncertainties surrounding the clindamycin resistance mechanism have restricted its development in combination therapies. To explore clindamycin resistance, we used two different selection regimes to generate clindamycin resistant lines in human malaria parasites (Plasmodium falciparum). Resistance under both selection regimes resulted from identical point mutations in the apicoplast 23S ribosomal RNA, mirroring the canonical resistance mechanism observed in bacteria.

Notably, the clindamycin resistant parasites exhibited reduced fitness in mosquitoes compared to drug-sensitive parasites. To quantify this transmission deficiency, we developed an amplicon sequencing technique to measure the growth of clindamycin resistant parasites in direct competition with drug-sensitive lines during the mosquito life stages. The mosquito host presents vastly different selection pressures compared to the human blood, and our new assay allows us to quantify parasite fitness during the mosquito stage for the first time. Understanding the impact of Plasmodium clindamycin resistance on transmission will inform decisions about the use of this drug in combination therapy to help curb antimalarial drug resistance.

We developed an axenic amastigote line of Leishmania donovani (MHOM/SD/75/1246/130) that retains macrophage-infecting characteristics and proliferates better than metacyclic promastigotes in vitro and in hamsters. This line was used to induce miltefosine resistance through stepwise increasing drug pressure, achieving stable high-grade resistance (62-fold) both in vitro and in vivo. Unlike previous studies on Leishmania infantum and Leishmania major promastigotes, which showed decreased or no fitness, we assessed the in vitro fitness of our resistant line. We cultured miltefosine-sensitive (Milt-S), miltefosine-resistant (Milt-R), and a 50:50 mix in various conditions, including control, DMSO control, and different concentrations of miltefosine and amphotericin B, for 18 cycles of drug pressure.

The ratio of sensitive to resistant parasites was determined using pyrosequencing of a SNP (D897N) in the miltefosine transporter gene linJ.13.1590. Fitness was defined as the ability of one clone to overtake another over time. Under 3 or 6 μM miltefosine pressure, Milt-R dominated by cycle 5. Without miltefosine or at 1 μM, Milt-S overtook by cycle 8. The fitness advantage is conditional on drug pressure, with Milt-R's advantage manifesting faster under higher miltefosine pressure compared to Milt-S in the absence of drug pressure.

Scabies is a highly contagious dermatological condition caused by Sarcoptes scabiei var. hominis. The therapeutics, ivermectin and permethrin, mainly target parasite nervous system killing only the motile stages. Therefore, they require repeat treatments. Prolonged use of these drugs and patient incompliance to repeat treatments have led to parasite resistance. A single dose treatment which targets all stages of the S. scabiei life cycle is required. Two novel scabicides (FL01 and AB02) were tested as a combination in vitro and pre-clinically. Four groups of eight infected pigs each were treated with a single application of the combination treatment for 4h or 8h or with two-doses of ivermectin or kept untreated. LT100 for the combination was 2h for mites and young eggs, and 8h for late-stage eggs. Only the combination treatment groups were cured and free from parasites from day-1 post-treatment. Clinically, combination treatment groups showed significantly lower pruritus and much improved clinical scores compared the ivermectin and control groups. Maximum absorption of FL01 in the serum and skin was at 8h and 2h and at 2h and 8h for AB02 post-treatment. Efficacy of this combination treatment is significantly higher than available scabicides, indicating that our treatment is a promising novel scabicide.

Cryptosporidium spp., an apicomplexan intestinal parasite, represents a significant global health threat, particularly affecting children. Despite its importance, cryptosporidiosis remains a neglected disease. Research on Cryptosporidium spp. has been hindered by the absence of a tractable, continuous in vitro culture of this parasite. However, our lab has achieved progress in improving several available culture models, including an axenic model. Recent advances in short-term in vitro culture of Cryptosporidium now enable high-throughput sensitivity testing of novel therapeutics targeting this pathogen. In this study, we utilized a newly optimized in vitro drug screening platform to examine, for the first time, the anti-cryptosporidial activity of newly developed synthetic peptoids with potent antimicrobial properties. Utilizing our novel culturing platforms, we have screened a library of 18 peptoids and identified the peptoid TM9 as a promising candidate, demonstrating an anti-cryptosporidial median IC50 of 21 µg/mL. TM9 was further tested for activity against various Cryptosporidium life cycle stages, including invasion and egress, and showed a phenotypic effect against the infectious sporozoite stage. Importantly, toxicity screening revealed that TM9 displayed minimal cytotoxic effects on mammalian host cells. The high efficacy and safety profile of TM9 opens new possibilities for novel treatments and preventative strategies against Cryptosporidium infections.

Antimalarials have historically played a key role in controlling malaria in endemic regions, and for nearly all available antimalarials, cellular targets are unknown. We applied solvent-induced proteome profiling in lysates and live cells with data independent acquisition mass spectrometry (DIA-MS) to identify antimalarial protein targets. We generated the solvent denaturation curve of every single protein identified in the asexual stage of the parasite proteome using increasing percentage of solvent, following which, we choose 4-5 percentages to identify the known targets of current and novel antimalarials in lysate and live infected red blood cells. The experimental workflow involves treatment of P. falciparum infected red blood cells with compounds of interest, solvent exposure, soluble protein isolation, digestion, and DIA-MS using the Orbitrap Astral MS allowing faster throughput with deep coverage and accurate quantification. Further, methodological optimisation in the live cell workflow for the analysis of this intracellular parasite was also necessary, including an enrichment step. We have also developed new approaches for the analysis of the resulting datasets, affording better discrimination of specific compound-induced stabilisation from various experimental artefacts. We successfully identified the known targets of current and novel antimalarials and extended the proteome information of solvent-induced protein precipitation for target identification.