Conference Agenda

Trichomonas vaginalis (Tv) is a human parasite, causing urogenital tract infections and is commonly treated with metronidazole. Rising resistance rates have become an issue in patient treatment.

The mechanisms of resistance are not yet fully understood, but it was shown previously that strain T_V C1 displayed a similar pattern of up- and downregulated proteins when depleted of iron as its highly metronidazole-resistant cell line Tv C1 res. Proteomic analysis was performed to fully determine the differences in protein expression between wildtype, iron depleted, and resistant Tv C1, to thereby identify proteins specifically up- or downregulated in the context of metronidazole resistance. To this end, C1 cells were cultured with 2,2-bipyridine for iron depletion, alongside metronidazole resistant and wildtype C1 cell lines. Protein extraction was performed with TCA/acetone, followed by enzymatic digestion using Trypsin/ LysC and 3 kDa FASP filter units. Peptides were separated using a nano-HPLC Ultimate 3000 RSLC system directly coupled to a high-resolution Q-Exactive HF Orbitrap mass spectrometer via ESI interface. Using a quantitative proteomics approach, we were able to highlight proteomic differences between resistant, non-resistant, and iron depleted parasitic cells, to gain further understanding in the formation of metronidazole resistance in Tv.

Human trichomoniasis, a globally prevalent sexually transmitted infection caused by Trichomonas vaginalis, affects approximately 278 million people each year. It presents a challenge due to resistance to the current treatment, Metronidazole (MTZ), which is also associated with side effects. Cannabis sativa, with more than 100 phytocannabinoids and numerous studies for therapeutic applications, including parasitic infections, has undergone a significant shift in acceptance worldwide, highlighted by legalizations and substantial revenue projections. In this context, the present study delves into the effects of cannabinoids, specifically WIN 55,212-2, Cannabivarin (CBV), CBD-rich oil, and THC-rich oil, showcasing their anti-parasitic actions that influence the growth and morphology of T. vaginalis. The analysis extends to encompass the pharmacokinetic properties of these cannabinoids. Among the analyzed cannabinoids, CBV stands out for adhering to Lipinski's rules, indicating its potential suitability for oral drug delivery. They also demonstrated inhibitory effects on the growth of T. vaginalis trophozoites and a reduction in the parasite's adhesion to host cells. Several morphological alterations were observed, such as membrane projections, blebbing, autophagosomes and damaged hydrogenosomes. These results highlight the need for further research to explore the therapeutic potential of cannabinoids and understand their mechanisms of action in T. vaginalis.

Giardia duodenalis is a gastrointestinal parasite causing ~200 million symptomatic infections annually, disproportionately in lower socioeconomic tiers and children. Chemotherapeutic interventions are limited to nitroheterocyclic antibiotics such as metronidazole. However, high doses are toxic and drug-resistant treatment failures occur in up to 20% of cases, highlighting the urgency of novel and safer chemotherapeutics.

We previously identified a drug-like kinase inhibitor (IC₅₀=114nM). To identify the kinase target(s) in Giardia, we immobilise this compound to azide-agarose and -magnetic supports through “Click” chemistry, to “pulldown” its high affinity target(s). Pilot pulldown experiments showed different enrichment and non-specific binding profiles for either sets of beads. To minimise non-specific binding, we use “blank” beads and competitive binding as negative controls, with “baited” beads for the samples.

Out of the 4900 proteins in the annotated G. duodenalis proteome, DIA library-free searches identified 2065 and 931 proteins from these respective pulldowns. Relative to “blank” agarose and magnetic beads, we pulled down 819 and 132 significant proteins respectively. When further cross-referenced to secondary controls, we identify eight significantly enriched proteins from the baited agarose, and ten significantly enriched proteins from the baited magnetic beads; two kinases were identified from the baited agarose and were selected for downstream validation.

Screening for drug efficacy and safety for disease agents has historically been a costly and labour-intensive process, particularly for parasites which can have complex in vitro needs, despite efforts to improve the process.

We developed a custom robotics platform with the potential to concurrently screen for compound efficacy for parasite targets, off target effects and toxicity. A Tecan Fluent robotics platform was integrated with a gas-controlled incubator (Cytomat) and SparkCyto, capable of automated tissue culture maintenance and the capacity to perform whole cell assays using fluorescence, absorbance, bioluminescent and microscopy, followed by dose-titration on selected hits.

The platform was validated for Giardia duodenalis inhibition and compound specificity testing. From a small library of compounds 7% had potent selective inhibition of G. duodenalis, 12 of which had IC₅₀’s between 0.1 – 4.9 µM.

In conclusion, we describe a unique, high throughput platform for anti-parasite drug discovery, with the potential to initiate 18,000 assays per day. The platform could be adapted to accommodate a vast range of assays, from helminth motility assays to intracellular anti-parasite efficacy or enzyme target assays, greatly expanding the scope and improving the efficiency of anti-parasite drug discovery programs.

Drug resistance hampers the treatment of giardiasis, one of the world’s most common gastro-intestinal parasitic diseases. On an annual basis ~200 million people develop giardiasis, a disease that impacts child development and the long-term health of many adults. However, there is no vaccine for giardiasis 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. Our team has identified new compounds with potent and selective activity against Giardia parasites. Our lead drug candidates are well-tolerated, have in vivo activity, do not impact the microbiome of mice, and have in vitro synergistic activity with currently used benzimidazole treatments. Importantly, one of the most promising candidates identified has cleared infections in mice. Preliminary data suggest that our new compounds have a different mode of action to currently used drugs that is linked to the unique cytoskeleton of Giardia parasites. Further studies with our lead candidates are now under way and will be discussed.

Giardia duodenalis is a common gastrointestinal parasite that causes ~280 million cases of giardiasis each year. While giardiasis is a ubiquitous disease that causes high rates of morbidity, Giardia is a neglected parasite with limited treatment options that are failing at an increasing rate. To improve this position, new treatment strategies and drugs with novel modes of action are desperately needed. This includes the identification and recommendation of drug combination strategies to combat treatment refractory cases. Recent work in our laboratories have identified two novel compound series with potent activity against G. duodenalis that have demonstrated tolerability and activity in mice. To further understand the clinical potential of these new anti-Giardia compounds, I have been further characterizing their in vitro interactions and biological activities. These data will be presented and discussed.