{'fraction_non_alphanumeric': 0.04369717676676857, 'fraction_numerical': 0.050350656356770364, 'mean_word_length': 4.774922118380062, 'pattern_counts': {'":': 0, '<': 8, '<?xml version=': 0, '>': 13, 'https://': 1, 'lorem ipsum': 0, 'www.': 1, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
An image-based Pathogen Box screen identifies new compounds with anti-Giardia activity and highlights the importance of assay choice in phenotypic 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 Venture's 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.

Despite growing evidence to suggest that Giardia parasites result in significant morbidity, there is no human vaccine for this pathogen and treatment depends on an arsenal of chemotherapeutics that have limitations including declining efficacy. The most commonly used drugs include the 5-nitroimidazoles and the benzimidazoles, with the 5-nitroimidazole, metronidazole, being widely acknowledged as the "goldstandard" therapy. However, treatment with metronidazole has been associated with clinical failure rates as high as 45-70% (reviewed in Lalle and Hanevik, 2018). Metronidazole is also very distasteful, requires a long treatment regimen (500 mg, 3 times/day for 5-7 days or 2g once/day for 3-5 days (Lalle and Hanevik, 2018)) and can cause side-effects including headache, vomiting, anorexia and nausea (Escobedo and Cimerman, 2007;Lalle, 2010). While the benzimidazoles, such as albendazole, are associated with comparably fewer side effects (Meloni et al., 1990) the efficacy of these agents can be very variable (25-90%) (Gardner and Hill, 2001). To improve these treatment options and to ensure the health and well-being of millions worldwide, particularly young children and those in marginalized communities, new anti-Giardia drugs with different modes of action to current agents need to be identified and developed.
To stimulate the discovery of new anti-infective compounds, the Medicines for Malaria Venture (MMV) developed the Pathogen Box, a collection of 400 molecules, with known activity against one or more key disease-causing pathogens. As all compounds in the Pathogen Box have known biological activity, including cytotoxicity, they represent excellent starting points for drug discovery efforts. To facilitate anti-Giardia drug discovery, the Pathogen Box was recently assessed for compounds displaying activity against Giardia trophozoites (Hennessey et al., 2018). In this study, a transgenic G. duodenalis parasite line expressing red-shifted firefly luciferase under the control of the β-tubulin promoter was used to quantify growth inhibition. Data identified 15 compounds that were able to inhibit growth by at least 95% following 24h exposure at a concentration of 16 μM. The activity of these compounds and an additional three compounds demonstrating > 75% inhibition of G. duodenalis and Cryptosporidium parvum at 16 μM were further assessed in follow-up dose response assays. These data identified ten compounds with 24h IC 50 values < 2 μM (Hennessey et al., 2018). However, the previously published anti-Giardia compounds, mebendazole (IC 50 value of < 1 μM) (Morgan et al., 1993;Katiyar et al., 1994;Chen et al., 2011) and benznidazole (IC 50 values of < 1 μM) (Chen et al., 2011) were not identified as active compounds in this study. In addition, clofazimine and iodoquinol were identified as active compounds (24h IC 50 values of 1.8 μM and 2.5 μM respectively), whereas previous studies had reported these drugs to be inactive (Bonilla-Santiago et al., 2008;Chen et al., 2011). As these discrepancies raised concerns around previously published data, the aim of the current study was to reassess the anti-Giardia activity of the Pathogen Box compounds using wild-type parasites and a previously validated imagebased live cell assay (Hart et al., 2017). The activity of selected compounds was also assessed using 3 H-thymidine incorporation as a comparator.

Primary screen of Pathogen Box compounds
The activity of Pathogen Box compounds was assessed in 96-well flat bottom micro-titre plates (Corning Costar, USA) against G. duodenalis assemblage B parasites BRIS/91/HEPU/1279 at 5 μM in singlicate on two separate occasions. To avoid evaporation in test wells, all outer wells were filled with PBS (200 μL). In addition to test wells (100 μL of 10 μM test compound; 0.5% DMSO) all assay plates included medium only (200 μL medium), negative (100 μL medium), positive (100 μL of 10 μM albendazole; 0.5% DMSO) and vehicle controls (100 μL medium containing 0.5% DMSO) in triplicate wells. All test and control wells, except medium controls, were inoculated with parasites (1.5 × 10 4 BRIS/91/HEPU/1279 trophozoites in 100 μL; final volume 200 μL 0.25% DMSO and final concentration of test compounds 5 μM). Plates were then incubated in sealed chambers filled with 3% O 2 , 5% CO 2 in N 2 for 48h as previously described (Gut et al., 2011;Hart et al., 2017). Plates were imaged using brightfield and digital phase-contrast microscopy using the Operetta (PerkinElmer, USA) and automatically enumerated using Harmony and PhenoLogic™ software as previously described (Hart et al., 2017). Inhibition of parasite growth was calculated as a percentage relative to the vehicle controls minus any background and Z′ values were calculated as previously described (Zhang et al., 1999).

Dose response activity assessments
The dose response activity of selected compounds was assessed in 96-well micro-titre plates by preparing 1:2 serial dilutions of test or control compounds in triplicate (0.2% DMSO; 100 μL). All experiments were performed on at least three occasions, with albendazole control plates included in each assay. Metronidazole control plates were also included when the sensitivity of parasites resistant to this compound was assessed. To avoid evaporation in test wells, all outer wells were filled with PBS (200 μL). In addition to triplicate test wells all assay plates also included medium only (200 μL medium), negative (100 μL medium) and vehicle controls (100 μL medium containing 0.2% DMSO) in triplicate wells. All test and control wells, with the exception of medium only controls, were inoculated with parasites (1.5 × 10 4 trophozoites in 100 μL; final volume 200 μL 0.1% DMSO). Plates were incubated in sealed culture chambers filled with 3% O 2 , 5% CO 2 in N 2 until growth assessment as previously described (Hart et al., 2017). Inhibition of parasite growth was calculated as a percentage relative to the vehicle control minus background and IC 50 values were calculated using log-linear interpolation as previously described (Huber and Koella, 1993). Student's t-tests were used to compare the activity of compounds against the different Giardia parasite lines.

3 H-thymidine incorporation assay
The dose response activity of selected compounds was also assessed using a 3 H-thymidine incorporation assay. Plates were prepared as described for imaging assays (Section 2.4) and all assays were performed in triplicate on at least three occasions. However, following the addition of parasites to assay plates were labelled with 3 H-thymidine (1.0 μCi/well). Plates were then incubated at 37°C in air-tight chambers filled in 3% O 2 , 5% CO 2 , in N 2 for 48 h before being frozen. Parasite growth was indirectly assessed via the assessment of 3 H-thymidine incorporation. In brief, assay plates were thawed, harvested onto glass-fibre filter-mats, air-dried and sealed in sample bags with 5 mL of beta-plate scintillant (PerkinElmer, USA), and counted (Microbeta 2 2450, PerkinElmer, USA) (Bell et al., 1991;Downey et al., 2009). Inhibition of parasite growth was calculated as a percentage relative to the vehicle controls minus any background, and further analysed as previously described (section 2.4).

Minimum lethal concentration assay
Minimum lethal concentration assays were performed using G. duodenalis assemblage B parasites (BRIS/91/HEPU/1279; AB MTZ S ). In brief, duplicate dose response assay plates were prepared in 96-well micro-titre plates as previously described (section 2.4). Following 48 h incubation at 37°C in sealed chambers (3% O 2 , 5% CO 2 in N 2 ), the growth of parasites was assessed by light microscopy. Plates were then incubated on ice for 30 min and the contents of each well transferred to individual 8 mL borosilicate vials filled with culture medium. The minimum lethal concentration or the lowest concentration of each compound that prevented all parasite growth was estimated by light microscopy following a further four days incubation at 37°C. Each assay was performed on three separate occasions with albendazole as the positive control.
Given the promising 48 h IC 50 values of MMV676358 and MMV028694 against metronidazole-sensitive G. duodenalis assemblage B parasites (BRIS/91/HEPU/1279; AB MTZ S ) further studies were performed to determine the activity of these compounds against assemblage A (BRIS/87/HEPU/713; AA MTZ S ) and metronidazole resistant parasites ((BRIS/91/HEPU/1279m1; AB MTZ R ; Fig. 3). The minimum lethal concentration of these compounds against BRIS/91/ HEPU/1279; AB MTZ S was also assessed. Data from these studies demonstrated that MMV676358 and MMV028694 were cidal, with minimum lethal concentrations of 0.62 μM and 6.25 μM respectively and that these compounds were active against all parasite lines, with similar 48 h IC 50 values ( Fig. 3A and B). The nitroimidazoles, MMV688262 (delamanid) and metronidazole were used as controls in studies with metronidazole resistant parasites, with data demonstrating both compounds to have reduced activity against these parasites (7-fold higher IC 50 for MMV688262 p < 0.0001 and 4-fold higher IC 50 for metronidazole p = 0.0013 compared to sensitive parasites; Fig. 3C and D). The minimum lethal concentration of albendazole was 0.25 μM (> 0.12 μM), as previously described (Cedillo-Rivera and Munoz, 1992).
An additional four compounds (MMV688844, MMV676395, MMV495543 and MMV687800) that were identified as anti-Giardia hits previously (Hennessey et al., 2018), but did not reach 70% growth inhibition in the current study (< 70% growth inhibition at 5 μM) were also assessed in dose response assays. The IC 50 values of these compounds and representive images obtained during assays (Table 2; Fig. 2) supported initial screening data with all IC 50 values determined to be > 5 μM. As compound was available, and data did not reflect the activity previously reported (Hennessey et al., 2018), the activity of MMV688844 and MMV676395 was also determined using 3 H-thymidine incorporation. Data from these assays further supported these compounds to have IC 50 values of > 5 μM (Table 2). Importantly, mebendazole and albendazole controls were active at concentrations previously reported in the literature in these assays (Table 2).
With demonstrated activity against Plasmodium falciparum and P. berghei (https://www.mmv.org/mmv-open/pathogen-box/aboutpathogen-box), MMV676358 forms part of the malaria disease set within the Pathogen Box. However, the mode of action of this compound against malaria parasites is not known. While our studies investigating the activity of MMV676358 against multiple G. duodenalis assemblages and metronidazole resistant parasites suggests that the mode of action of this compound against Giardia is cidal at sub-μM concentrations ( Fig. 2 and minimum lethal concentration 0.62 μM) and different to the 5-nitroimidazoles, additional studies will be required to determine the target of this compound in these parasites. However, compounds sharing the 3-(1H-imidazo[4,5-b]pyridin-2-yl)pyridin-2amine core of MMV676358 have been shown to modulate the activity of kinases (patent document WO2011006567 and WO2007056155). Further studies investigating the anti-parasitic activity of this compound together with additional analogues appear warranted given that MMV676358 has also demonstrated good selectivity for parasites over human cells (SI > 37; Table 1) and has no obvious structural liabilities (Fig. 1B). In addition, although the previous screen of Pathogen Box compounds against Giardia parasites did not identify MMV676358 as a potent inhibitor of parasites (IC 50 not determined) (Hennessey et al., 2018), likely due to the high screening concentration chosen, this work did demonstrate good inhibition (90 ± 2%) at the screening concentration of 16 μM (Table 1).
MMV028694, the second compound with sub-μM activity against Giardia parasites, also forms part of the malaria disease set, but has demonstrated activity against multiple parasites including Trypanosoma (Duffy et al., 2017). While the mode of action of MMV028694 has not been reported, structurally similar compounds have been identified as inhibitors of human MAP kinase enzymes (Palmer et al., 2013), so the anti-parasitic activity of MMV028694 may be associated with kinase inhibition (reviewed in (Veale, 2019). MAP kinase enzymes have been identified in G. duodenalis and could represent promising new therapeutic targets (Manning et al., 2011). Importantly studies with metronidazole resistant parasites suggest that the activity of MMV028694 is different to this currently used 5-nitroimidazole (Fig. 3). This observation taken together with the cidal (minimum lethal concentrations 6.25 μM) anti-Giardia activity and selectivity of this compound (IC 50 < 1 μM; SI > 10; Table 1), supports further mode of action and structure activity relationship studies. In addition, MMV028694 has no obvious structural liabilities and its anti-Giardia activity, while lower than reported in the current study, was supported by Hennessey et al.
Giardia protein kinases) (Manning et al., 2011). While less research has been performed with MMV022478, this compound has also demonstrated promising activity against multiple parasites including Toxoplasma (Spalenka et al., 2018) and Schistosoma species (Pasche et al., 2019). However, the activity of this compound against human protein kinase C (PKCβII) suggests that structure activity relationship activity optimisation studies would be required to further develop the activity of this compound (SI~6 to > 7; Table 1).
The remaining two Pathogen Box compounds with anti-Giardia IC 50 values of ≤2 μM, MMV021660 and MMV687812, belong to the tuberculosis compound set. While MMV687812 is part of a group of compounds designed to inhibit Mycobacterium DNA gyrase (Shirude et al., 2013), the mode of action of MMV021660 against M. tuberculosis is less clear (reviewed in (Veale, 2019). While further studies would be required to define and optimise the activity of these compounds for Giardia, this work is feasible given the availability of analogues and is highly likely to result in the development of compounds with a different mode of action to those currently used in the clinic. Additional studies against drug resistant parasites should also be pursued given limited compound availability precluded this work in the current study. Importantly, although the dose response activity of MMV021660 was not assessed as part of the previously reported anti-Giardia Pathogen Box screen, both MMV021660 and MMV687812 were reported to inhibit the growth of parasites (90 ± 4% growth inhibition at 16 μM and 24 h IC 50 1.2 μM respectively) further supporting additional studies with these compounds.
A comparison of the data generated in the current study with that 95.4 ± 8.0 99 ± 0 9.1 × 10 −7 ± 2.3 × 10 −7 1.9 × 10 −6 ± 4.0 × 10 −8 3.9 × 10 −6 ± 1.3 × 10 −6 MMV688844 24.6 ± 9.7 98 ± 0 > 10 × 10 −6 > 10 × 10 −6 2.3 × 10 −6 ± 4.3 × 10 −7 MMV676395 13.8 ± 19.5 98 ± 0 > 10 × 10 −6 > 10 × 10 −6 1.6 × 10 −6 ± 2.0 × 10 −7 MMV495543 0.0 ± 0.0 97 ± 2 > 10 × 10 −6 Not assessed 2.8 × 10 −6 ± 2.1 × 10 −7 MMV687800 clofazimine 59.1 ± 34.5 97 ± 0 6.6 × 10 −6 ± 7.0 × 10 −7 Not assessed 1.8 × 10 −6 ± 2.3 × 10 −7 mebendazole 94.9 ± 5.8 26 ± 0 5.0 × 10 −8 ± 7.0 × 10 −9 1.6 × 10 −7 ± 9.0 × 10 −8 Not assessed; > 1.6 × 10 −5 albendazole 92.3 ± 8.1 Not assessed 9.0 × 10 −8 ± 2.0 × 10 −8 1.7 × 10 −7 ± 4.0 × 10 −8 4.1 × 10 −7 ± Fig. 2. The effect of selected compounds on Giardia trophozoite growth. Giardia trophozoites were treated with vehicle control or test compounds to a final concentration of 5 μM for 48 h. Brightfield images were then taken using a PerkinElmer Operetta® using a 20× Olympus objective. Scale bar indicates 50 μm. reported by Hennessey et al. (2018), highlighted the importance of assay choice in phenotypic drug discovery. While both studies identified most known anti-Giardia compounds and the compounds discussed above as active agents, there were several obvious differences in the data generated. One clear difference was the inability of the Hennessey et al. (2018) screen to demonstrate the anti-Giardia activity of mebendazole (26 ± 0% inhibition after 24h exposure to 16 μM compared to a 24 h IC 50 0.59 μM in the current study; Table 1). While the authors indicate that this discrepancy may be due to slow-action, we saw no indication of this in the current study (Table 1) and hypothesize that the reduced activity of mebendazole may have been associated with the genetic modification made to the parasites used during this research. The selection of transgenic parasites with puromycin has been shown to alter their gene expression (Su et al., 2007). In addition, the transgenic G. duodenalis parasite line used in this work expressed a red-shifted firefly luciferase under the control of the β-tubulin promoter and the activity of benzimidazoles, including mebendazole, in Giardia has been linked to β-tubulin and the inhibition of cytoskeleton polymerization (Holberton and Ward, 1981;Chavez et al., 1992;MacDonald et al., 2004). This idea is further supported by the relatively high IC 50 reported for albendazole in this study (Table 1) and may indicate a limitation of the assay to detect the activity of compounds with this defined mode of action. The Pathogen Box screen performed by Hennessey et al. (2018), also failed to detect the previously reported anti-Giardia activity of benznidazole (IC 50 values of < 1 μM) (Chen et al., 2011) (57 ± 10% inhibition at 16 μM). While the reason for this observation is unknown, particularly given that the work was performed with high compound concentrations in an anaerobic environment, it may be also be associated with the altered gene expression of transgenic parasites (Su et al., 2007). Although we have observed the activity of selected nitroimidazole compounds to be time dependent (e.g. Table 1 metronidazole 24 h IC 50 4.8 μM vs 48 h IC 50 2.0 μM), this idea does not adequately explain the poor activity of benznidazole reported by Hennessey et al. (2018) in their 24 h assay. The current study detected the activity of benznidazole at 24 and 48 h in a microaerophilic environment (3% O 2 , 5% CO 2 in N 2 ), which is known to inhibit the activation of benznidazole and other nitroheterocyclic compounds (Edwards and Shanson, 1980). Indeed, the reduced activity of nitazoxanide (67.8% inhibition at 5 μM) reported in the current study, is likely to be associated with assay environment (3% O 2 , 5% CO 2 in N 2 ). Previous work has shown nitazoxanide activation to be more sensitive to oxygen than metronidazole (Muller et al., 2015). The microaerophilic conditions used in the current study were chosen to better mimic the oxygen available in the mammalian intestine (He et al., 1999;Zheng et al., 2015).
The current study was unable to confirm the anti-Giardia activities of clofazimine (MMV687800; IC 50 < 5 μM; Table 2; Fig. 2) and iodoquinol (MMV002817) (Supplemental Table S1). However, the activity of clofazimine was highly variable in our hands, an observation that may be associated with the activity reported by Hennessey et al. (2018). We were also unable to confirm the activity of MMV495543, MMV676395, MMV676501 and MMV688844 (Supplemental Table S1) with additional imaging and 3 H-thymidine assays for selected compounds also supporting these findings ( Fig. 2; Table 2).
In an era of declining anti-Giardia drug efficacy, the current study has identified several new compounds that warrant further investigation. These studies may be particularly beneficial given that the modes of action of identified compounds appear novel and that many of these compounds have already been studied by multiple groups, making analogues and resources readily available. The current study has also highlighted the impact of phenotypic assay design on data generation, directly demonstrating the potential impact of transgenic parasites and assay screening concentration on study outcomes.

Declaration of competing interest
None.