Conference Agenda

On an annual basis >300 million people develop giardiasis, a disease that impacts child development and the long-term health of many adults. However, there is no vaccine for this disease and treatment options are failing due to multiple factors including drug resistant parasites. Moreover, current treatment strategies are monotherapies that do little to combat the development of drug resistance. To improve this position combination therapies that include new compounds with unique mechanisms of action are needed. However, little has been done to identify best practice combination therapies for giardiasis.

As a mechanism to pave the way towards the development of highly effective therapies for giardiasis we have been investigating the activity of novel compounds and compound combinations against Giardia parasites in vitro. These studies have identified multiple synergistic compound combinations that may be useful in the in vivo including combinations that have Interaction (I) values >3.0 (p<0.05). While in vivo studies are now needed to determine how these combination therapies work in clinical settings, taken together with pharmacokinetic data, these results are exciting and suggest that novel antigiardial combination therapies can be developed to help combat treatment refractory giardiasis.

Toxoplasma gondii causes ocular toxoplasmosis, a vision-threatening retinal infection. Current therapeutics are not curative. ELQs are promising drugs not yet evaluated for ocular toxoplasmosis.

Human retinal pigment epithelial cells (ARPE-19 line) were infected with T. gondii GT-1 or GPHT tachyzoites and treated with ELQ-316 or ELQ-685. Tachyzoite growth 50% inhibitory concentrations (IC_50s) were calculated. Tachyzoite invasion, replication, and egress were assayed. Cell viability was evaluated by XTT assay. Cell response transcripts were measured by RT-qPCR.

ELQ IC_50s for GT-1 and GPHT were nanomolar-range and approximately equivalent (ELQ-316: 22.96±4.33nM vs. 21.23±3.92nM; ELQ-685: 1.08±0.34nM vs. 0.88±0.20nM; unpaired t-test, p>0.05). Both treatments significantly reduced replication (tachyzoites/rosette: ELQs ≤1.77±0.07 vs. untreated ≥6.85±0.66, p<0.0001; one-way ANOVA). Tachyzoite invasion and egress were not impacted by either drug. ELQs ≤12µM did not reduce cell viability (mitochondrial respiration (arbitrary units): ELQ 12µM 2.37±0.13 vs. untreated 2.19±0.07; p>0.05; one-way ANOVA). In GT-1-infected cells, ELQ treatment reduced 7 transcripts (CCL2, CXCL8, ICAM1, IL1B, IL6, NFKB1, PDCD1LG2; p≤0.0042) and increased 3 transcripts (CXCL10, TGFB2, VCAM1; p≤0.0048), versus untreated controls (one-way ANOVA). In GPHT-infected cells, ELQ treatment reduced 5 transcripts (CXCL8, ICAM1, IL6, NFKB1, REL; p≤0.043).

ELQs were non-toxic in ARPE-19 cells, reduced T. gondii replication, and altered host inflammatory mediator expression.

Plasmodium falciparum, the deadliest human malaria parasite, has developed resistance to all clinically used antimalarials, highlighting the need for new compounds with novel modes of action. Pantothenate analogues kill P. falciparum by targeting the biosynthesis or utilisation of coenzyme A (CoA), an essential enzyme cofactor. Pantothenamides (PanAms), pantothenate analogues in which the carboxyl group is replaced by an amide group, exhibit potent in vitro activity. Unfortunately, PanAms are degraded in vivo by human pantetheinase. Modification of the labile amide bond has given rise to pantetheinase-resistant PanAm mimics. AH-2-45, a ring-substituted PanAm mimic, exhibits nanomolar antiplasmodial activity and is metabolised by CoA biosynthesis enzymes into a CoA antimetabolite, proposed to inhibit downstream CoA-dependent pathways. However, its precise target remains unknown. Whole-genome sequencing of in vitro-generated AH-2-45-resistant P. falciparum revealed a missense mutation in the gene encoding the endoplasmic reticulum-resident glycerol-3-phosphate 1-O-acyltransferase (PfGPAT), an essential CoA-dependent enzyme involved in phospholipid biosynthesis. We are currently genetically validating PfGPAT as the AH-2-45 resistance determinant and characterising the functional impact of the resistance-associated mutation using PfGPAT activity assays. Elucidating this mechanism may establish PfGPAT as a novel antimalarial drug target and guide structural optimisations of the AH-2-45 antimetabolite and related PanAm mimics.

WM382 is an inhibitor for Plasmepsin IX and X in Plasmodium spp blood and liver stages. These proteases are essential and ubiquitous in Plasmodium spp., where they process diverse proteins involved in egress and invasion of host cells. This makes them ideal targets for drug intervention. Plasmepsin IX and X are also expressed in sporozoites. To decipher their role in this stage, WM382 was administered to mosquitoes after Plasmodium falciparum infection. The number of developing oocysts was the same irrespective of WM382 treatment, though a modest increase in size was detected following drug administration, suggesting differential parasite development. Furthermore, the number of salivary gland sporozoites was dramatically reduced when mosquitoes were treated with WM382. This phenotype points to a defect in egress of P. falciparum sporozoites from the oocyst. We identified accumulation of unprocessed precursors of the essential protein AMA1 in haemolymph and salivary gland sporozoites when treating with WM382, while processing of CSP remained unaffected. Some WM382-treated sporozoites could still invade salivary glands but they displayed significant loss-of-function phenotypes during cell traversal and infection of human HC04 hepatocytes. These results show a role of plasmepsin IX/X during the mosquito stages, raising the possibility of control interventions during transmission.

Cattle ticks are a burden on global agriculture, with chemical treatments the primary method of control. However, ticks are being selected for resistance to these chemicals. Current tests for chemical resistance are slow and require laboratory facilities, inhibiting testing. In this project, we aim to undertake deep sequencing to define polymorphisms controlling resistance and build a database for monitoring of resistance changes. In preliminary studies, a survey and sampling system has been established to capture cattle ticks from across the infested range. Based on initial collections, resistance has been assessed across ten properties, defining resistance to synthetic pyrethroids (SP), amitraz, fluazuron and macrocyclic lactones. A newly developed rapid test for chemical resistance (RaTexT^TM) was used to define resistance to a synthetic pyrethroid, deltamethrin. Resistance to deltamethrin has been found at 100% of properties regardless of whether this chemical is currently in use. In contrast, fluazuron resistance was found at 40% of properties and only at properties where it is currently in use. Future work will assess the allele frequencies of the known polymorphism driving SP resistance, kdr, to determine the accuracy of rapid testing methods and deep sequencing will be undertaken on resistant and susceptible ticks for each chemical class.