Giardia duodenalis is an intestinal parasite that causes giardiasis, a widespread human gastrointestinal disease. Treatment of giardiasis relies on a small arsenal of compounds that can suffer from limitations including side-effects, variable treatment efficacy and parasite drug resistance. Thus new anti-Giardia drug leads are required. The search for new compounds with anti-Giardia activity currently depends on assays that can be labour-intensive, expensive and restricted to measuring activity at a single time-point. Here we describe a new in vitro assay to assess anti-Giardia activity. This image-based assay utilizes the Perkin-Elmer Operetta® and permits automated assessment of parasite growth at multiple time points without cell-staining. Using this new approach, we assessed the Malaria Box compound set for anti-Giardia activity. Three compounds with sub-μM activity (IC50 0.6-0.9 μM) were identified as potential starting points for giardiasis drug discovery.

Giardia duodenalis, the most prevalent human intestinal parasite causes the disease, giardiasis. On an annual basis G. duodenalis infects ~1 billion people, of which ~280 million develop symptomatic disease. Giardiasis can be severe and chronic, causing malnutrition, stunted growth and poor cognitive development in children. Current treatment options rely on drugs with declining efficacy and side-effects. To improve the health and well-being of millions of people world-wide, new anti-Giardia drugs with different modes of action to currently used drugs are required. The Medicines for Malaria Ventures Pathogen Box, a collection of bio-active compounds specifically chosen to stimulate infectious disease drug discovery, represents an opportunity for the discovery of new anti-Giardia agents. While the anti-Giardia activity of Pathogen Box compounds has been reported, this work failed to identify known anti-Giardia controls within the compound set. It also reported the activity of compounds previously screened and shown to be inactive by others, suggesting data may be inaccurate. Given these concerns the anti-Giardia activity of Pathogen Box compounds was re-assessed in the current study. Data from this work identified thirteen compounds with anti-Giardia IC50 values ≤2 μM. Five of these compounds were reference compounds (marketed drugs with known anti-microbial activity), or analogues of compounds with previously described anti-Giardia activity. However, eight, including MMV676358 and MMV028694, which demonstrated potent sub-μM IC50s against assemblage A, B and metronidazole resistant parasites (0.3 μM and 0.9 μM respectively), may represent new leads for future drug development. Interestingly, only four of these compounds were identified in the previously reported Pathogen Box screen highlighting the importance of assay selection and design when assessing compounds for activity against infectious agents.

On an annual basis the flagellate protozoan, Giardia duodenalis, is responsible for an estimated one billion human infections of which approximately two hundred million cause disease. However, the treatment of Giardia infections is reliant on a small group of chemotherapeutic classes that have a broad spectrum of antimicrobial activity and increasing treatment failure rates. To improve this situation, we need new drugs. In this study we screened the Compounds Australia Scaffolds Library for compounds with potent and selective activity against these parasites. Unlike previous drug discovery efforts that have focused on drug repurposing, this library is comprised of commercially available synthetic compounds arranged into lead-like scaffolds to facilitate structure activity relationship assessments and de novo drug discovery. A screen of 2451 compounds in this library identified 40 hits (>50% inhibitory activity at 10 µM, over 48 h). Secondary testing identified three compounds with IC50 values <1 μM and >50-fold selectivity for parasites over mammalian cells and a hit series, CL9406, comprising compounds with potent (lowest IC50 180 nM) and selective activity for Giardia parasites. The most promising compound in this series, SN00797640, displayed selective activity against assemblage A, B, and metronidazole resistant parasites which was parasiticidal (minimum lethal concentration 625 nM) and synergistic with albendazole. SN00797640 was well-tolerated when administered to mice at doses of 50 mg/kg daily for three days paving the way for pre-clinical in vivo activity assessment.

Giardia duodenalis is the causative agent of the neglected diarrhoeal disease giardiasis. While often self-limiting, giardiasis is ubiquitous and impacts hundreds of millions of people annually. It is also a common gastro-intestinal disease of domestic pets, wildlife, and livestock animals. However, despite this impact, there is no vaccine for Giardia currently available. In addition, treatment relies on chemotherapies that are associated with increasing failure rates. To identify new treatment options for giardiasis we recently screened the Compounds Australia Scaffold Library for new chemotypes with selective anti-Giardia activity, identifying three compounds with sub-μM activity and promising selectivity. Here we extended these studies by examining the anti-Giardia activity of series CL9569 compounds. This compound series was of interest given the promising activity (IC50 1.2 μM) and selectivity demonstrated by representative compound, SN00798525 (1). Data from this work has identified an additional three thieno [3,2-b]pyrrole 5-carboxamides with anti-Giardia activity, including 2 which displayed potent cytocidal (IC50 ≤ 10 nM) and selective activity against multiple Giardia strains, including representatives from both human-infecting assemblages and metronidazole resistant parasites. Preclinical studies in mice also demonstrated that 2 is well-tolerated, does not impact the normal gut microbiota and can reduce Giardia parasite burden in these animals.

A screen for compounds with antigiardial activity in the Compounds Australia Scaffolds library identified SN00797640 (supplied structure being 8-acylaminodibenzoxazepinone 1) as a hit compound with potent anti-parasitic activity (concentration for 50% growth inhibition of Giardia duodenalis, IC50 0.18 μM). To further explore the structure–activity relationships in this series, compound 1 and analogues, including its 7-acylaminodibenzoxazepinone regioisomer (2), were synthesized and assessed for anti-Giardia activity. While regioisomer 2 demonstrated antigiardial activity, resynthesized 1 and other 8-acylaminodibenzoxazepinone analogues were inactive. Comparison of spectroscopic and physical properties demonstrated the correct structure of SN00797640 to be 7-acylamino regioisomer 2. These results highlight the importance of independent synthesis in verifying the structure and activity of screening hits.