article: There is need for a more efficient cell-based assay amenable to high-throughput drug screening against Giardia lamblia. Here, we report the development of a screening method utilizing G. lamblia engineered to express red-shifted firefly luciferase. Parasite growth and replication were quantified using D-luciferin as a substrate in a bioluminescent read-out plateform. This assay was validated for reproducibility and reliability against the Medicines for Malaria Venture (MMV) Pathogen Box compounds. For G. lamblia, forty-three compounds showed ≥ 75% inhibition of parasite growth in the initial screen (16 μM), with fifteen showing ≥ 95% inhibition. The Pathogen Box was also screened against Nanoluciferase expressing (Nluc) C. parvum, yielding 85 compounds with ≥ 75% parasite growth inhibition at 10 μM, with six showing ≥ 95% inhibition. A representative set of seven compounds with activity against both parasites were further analyzed to determine the effective concentration that causes 50% growth inhibition (EC50) and cytotoxicity against mammalian HepG2 cells. Four of the seven compounds were previously known to be effective in treating either Giardia or Cryptosporidium. The remaining three shared no obvious chemical similarity with any previously characterized anti-parasite diarrheal drugs and offer new medicinal chemistry opportunities for therapeutic development. These results suggest that the bioluminescent assays are suitable for large-scale screening of chemical libraries against both C. parvum and G. lamblia.
  Cryptosporidium and Giardia are widely acknowledged as significant waterborne pathogens, as both are major contributors to the global health burden of diarrheal diseases in children under the age of five [1,2]. Since Giardia and Cryptosporidium infections are among the most common cause of clinical and asymptomatic parasitic diseases in children in resource-limited environments, a new “dual use” therapeutic would be a very valuable treatment option. Giardia replicates through binary fission and colonizes the small intestine of vertebrate hosts by attachment through a ventral disk [3,4]. This can affect the host’s ability to effectively absorb necessary nutrients [5]. Young children can suffer physical stunting and wasting, cognitive impairment, and fine motor movement problems [5,6]. Food and Drug Administration (FDA) approved treatments for giardiasis include metronidazole, chemically related nitroimidazole drugs, and albendazole. However, a substantial number of clinical presentations are resistant to these treatments [7,8]. A combined treatment regimen of metronidazole and albendazole or quinacrine can be highly effective for patients with metronidazole-resistant giardiasis [9,10] but toxicity often limits therapy. Cryptosporidium infections are chronic and, in some cases, fatal in immune compromised patients [11–14]. Infection is caused by ingestion of environmentally resistant infective stages, called cysts (G. lamblia) or oocysts (C. parvum), upon passage in the feces [15]. Ingestion of as few as 10 cysts or oocysts can lead to clinical episodes [16–18]. Nitazoxanide is the only FDA-approved therapy for cryptosporidiosis. However, nitazoxanide has shown little efficacy in immunocompromised patients [19] and is not approved for use in children under the age of one [20]. Consequently, there is an increasing need to develop alternative drugs for both cryptosporidiosis and giardiasis [21]. Methods to accelerate drug discovery by high-throughput screening of large chemical libraries will require development of assay platforms that provide reliability, sensitivity, have easily observable readouts, and are both cost and time efficient. A bioluminescent assay that fits these criteria for high-throughput drug screening of inhibitors against C. parvum was recently described [22,23]. Screening of molecules against G. lamblia parasites traditionally involved microscopic counting of parasites [24], or utilizing a MOXI cell coulter counter [25], both of which require manual counting of each well in an assay plate. Semi high-throughput assays using resazurin to measure cell viability [26] and another based on automated image analysis by cell stained-DAPI signal read-out have also been explored [27]. Most recently, a digital phase-contrast microscopy morphology-based assay method with enumeration by software was developed, which does not require cell staining [28]. The morphology-based assay is comparable to the previously reported DAPI stain method, but it relies solely on expensive software to identify and count parasites based on size and morphology [27,28]. Stable expression of Escherichia coli β-glucuronidase A (GusA) as a reporter gene for G. lamblia growth measurement was also described as suitable for high-throughput drug screening [29]. We now describe the development of an efficient bioluminescent assay for measuring growth inhibition of trophozoites using an engineered G. lamblia strain expressing a red-shifted firefly luciferase PpyRE9h gene [30]. The Pathogen Box (www. pathogenbox. org; Medicines for Malaria Venture (MMV), Geneva, Switzerland) is a set of 400 molecules directed to a variety of “neglected disease” pathogens, including Plasmodium, Mycobacterium tuberculosis, helminths, kinetoplastids, Cryptosporidium, Toxoplasma, and dengue virus. Among the 400 molecules are 26 reference compounds [31] that are known bioactives against particular pathogens (https: //www. pathogenbox. org/about-pathogen-box/composition). The Pathogen Box was screened for activity against G. lamblia and C. parvum cells, in an attempt to find potential dual pathogen-inhibiting molecules.
  The Pathogen Box (MMV) molecules were obtained as 10 mM DMSO stocks and stored at -20 °C. Metronidazole (Sigma, St. Louis, MO), a commercially available drug, was included in the study as a preliminary control of the assay, while quinacrine was used in cytotoxicity screening. Giardia lamblia (WBC6, ATCC 50803) trophozoites were grown in TYI-S-33 medium supplemented with 10% bovine serum and 0. 05 mg/mL bovine bile [32]. Transgenic C. parvum strain UGA1 expressing Nanoluciferase (Nluc) [22] used in this study was grown in HCT-8 cells. Cultures were incubated at 37°C. Oligonucleotide pairs used to generate the Giardia transfection construct are the RE9 primers (5' ACCATGGAATCTAGAATGGAGGACGCCAAGAACAT 3' and 5' TGGATCCTCTTAATTAATCAGATCTTGCCGCCCTTCTTGGCC 3' ); pBetaTub-Gib-forward (5' TCTGCAAGTTAATTTTTGGCCCCTAGGTCGGATCAAGACTTCAAATTAGAAA 3' ) and BetaTubUTR-Gib-reverse (5' TTATTTGACCATCGTACTTGCAACTAGTGAGCTCGGTACCAGCTGATCGGCGC 3' ). The expression plasmid (pBetaTubulinPro: : PpyRE9h: : BetaTubulin3' UTR/pPAC-integ) was constructed by cloning the PpyRE9h luciferase gene downstream of a β-Tubulin promoter. First, the entire open reading frame of the RE9hum (thermostable red-shifted firefly luciferase PpyRE9h) was digested from the parent plasmid vector pTRIX2-RE9h using XbaI and BamHI. The RE9hum fragment, representing the 2. 8 kb region, was amplified and subcloned into mNeonGreen-C18-β-tubulin [33] after excising the tubulin gene and mNeonGreen with NcoI and BamHI and digesting the amplicon with the same enzymes forming pβTub: : PpyRE9h: : βTub3’UTR. The pβTub: : PpyRE9h: : βTub3’UTR fragment was amplified using Phusion High-fidelity DNA polymerase (Thermo Fisher Scientific, Rockford, IL) and cloned into pPacV-integ digested with EcoRI and PmlI, using Gibson cloning. The vector pβTub: : PpyRE9h: : βTub3’UTR/pPacV-integ was used to integrate into chromosome 5 by homologous recombination [34]. For integration, 30 μg of pβTub: : PpyRE9h: : βTub3’UTR/pPacV-integ was linearized with SwaI overnight, precipitated with ethanol and incubated with 300 μL of chilled G. lamblia cells (~13x106/mL) for 30 minutes before electroporation (Bio-Rad GenePulser X at 375 V, 1000 uF, 750 ohms). After electroporation, cells were incubated on ice for 10 minutes before being transferred to fresh media at 37 °C. Transfectants were selected with puromycin after overnight recovery, as previously described [35]. To determine optimal D-luciferin (Gold BioTechnology Inc. , St. Louis, MO) concentration, substrate was serially diluted 1: 2 in PBS from 100 mg/mL to 0. 052 mg/mL. Parasite numbers were kept constant at 50,000 cells/mL in a volume of 250 μL per well. Plates were incubated for 30 minutes at 37 °C on a plate shaker. Wild-type G. lamblia (non-luciferase expressing) was used as a control. To determine the optimal parasite concentration, an initial cell concentration of ~1x106 cells/mL was serially diluted 1: 2, to a minimum concentration of ~500 cells/mL. Luminescence was measured at 20,30 and 40 minutes in white polystyrene, flat bottom 96-well plates (Corning Incorporated, Kennebunk, ME) on an EnVision plate reader (PerkinElmer, Waltham, MA). Preliminary effective concentration that causes 50% growth inhibition (EC50) assays were performed on the luciferase expressing and wild-type (control) G. lamblia cells to establish a correlation between manually counted cells and the bioluminescent signal read out. Trophozoites were harvested by chilling cultures on ice for 30 minutes and plated by adding 150 μL per well at a concentration of 250,000 cells/mL. Metronidazole (Sigma, St. Louis, MO) was used as a control and was serially diluted 1: 2 from 40 μM to 0. 04 μM and 150 μL was added to the 150 μL of the G. lamblia trophozoites. Plates were covered in plastic low-evaporation lids, sealed in individual anaerobic BD GasPak Bio-Bags (Becton Dickinson, San Jose, CA) and incubated at 37 °C for 24 hours. Plates were then iced for 30 minutes and 250 μL of resuspended cells were transferred to a solid white 96-well plate and 50 μL of 10 mg/mL D-luciferin was added to a final reaction concentration of 1. 67 mg/mL. Plates were incubated on a plate shaker at 37 °C for 30 minutes and read on an EnVision Plate Reader. A duplicate plate was concurrently set up and assayed using a MOXI Z Mini Automated Cell Counter Kit (Orflo, Ketchum, ID). Results obtained from the bioluminescence assay and the MOXI counter data were compared to evaluate their correlation to each another. Albendazole (Sigma, St. Louis, MO), an alternative giardiasis therapeutic, was assayed in concentrations from 4 μM to 0. 004 μM with only the luciferase expressing G. lamblia cells. Growth inhibition and EC50 determination assays were performed using UGA1 Nluc expressing C. parvum strain in HCT-8 cells (ATCC©, Manassas, VA) as previously described [23]. HCT-8 cells were inoculated onto 96-well plates and allowed to grow for 48 hours to 90–100% confluence and then treated with single concentration (10 μM) or serially diluted compounds prior to oocyst infection. C. parvum oocysts were incubated for 10 minutes in 10% bleach at room temperature and then washed with DPBS. One thousand oocysts per well were applied to the 96 well plates with RPMI-1640 medium supplemented with 10% horse serum and 1% penicillin/streptomycin at the same time as compound addition and incubated for 48 hours. The cell monolayers were lysed for 1 hour before adding Nano-Glo® luciferase reagent (Promega, Madison, WI), and measuring relative luminescence units (RLU) in an EnVision Plate Reader (Perkin Elmer, Waltham, MA). Several parameters, including reproducibility and reliability, were previously evaluated to optimize this assay for low and high-throughput (HTP) screening of small molecule compounds against C. parvum [23]. EC50 values were calculated using Prism 7 software (GraphPad Software, San Diego, CA). The 400 compounds in the Pathogen Box were initially screened against the bioluminescent strain of G. lamblia in duplicate at 16 μM as described above. Further experiments were carried out to determine the EC50 values of molecules that were found to have ≥ 95% inhibition of G. lamblia or C. parvum cell growth after the initial screen. Compounds were serially diluted 1: 2 starting at an initial concentration of 8 μM to a final 0. 015 μM for G. lamblia. C. parvum screening against the Pathogen Box compounds was performed as previously described starting at a concentration of 10 μM [23]. Assays to determine EC50 values were also performed for a subset of compounds found to have ≥ 75% growth inhibition of both G. lamblia and C. parvum in the initial screens as potential dual inhibiting molecules. Mammalian cell cytotoxicity assay data performed on HepG2 (human hepatocellular carcinoma) cells and described as CC20 or CC50 values are provided in supporting documentation that accompanied the Pathogen Box for approximately three quarters of the 400 compounds. However, no details of the assay conditions or parameters were provided [31]. In this study, selected compounds were further tested for cytotoxicity against HepG2 cells as previously described [36]. Briefly, cells were exposed to two-fold serial dilutions of compounds for 48 hours and cytotoxicity was assessed using the AlamarBlue® viability assay (Thermo Fisher Scientific, Waltham, MA). Each dilution was assayed in quadruplicate and concentrations causing 50% growth inhibition (CC50) were calculated by non-linear regression using GraphPad Prism 7 software (GraphPad Software, San Diego, CA). The data was analysed in Microsoft Excel and Prism 7 software (GraphPad Software, San Diego, CA). A nonlinear regression sigmoidal dose-response curve fit was applied to dose-response data for both half maximal effective concentration and half maximal cytotoxic concentrations.
  Wild-type G. lamblia cells were transfected with pβTub: : PpyRE9h: : βTub3’UTR/pPacV-integ (Fig 1), selected clones express red-shifted firefly luciferase PpyRE9h as validated by detectable bioluminescence signals when incubated with serial concentrations of D-luciferin in parallel to the wild-type strain (Fig 2a). Optimum luminescence was achieved after 30 minutes of incubation with D-luciferin (Fig 2b). The optimal concentration of D-luciferin was determined to be 1. 67 mg/mL, as bioluminescence signals plateaued above this concentration within 30 minutes (Fig 2b) demonstrating no reasonable need for higher concentrations. The amount of red-shifted firefly luciferase-driven bioluminescence activity was proportional to the number of viable transfected G. lamblia parasites per well, as determined by a comparative analysis with direct cell count of both the wild type and transfectant strains (Fig 2c). Considering G. lamblia’s doubling time of 8 hours [33], the optimal starting concentration of cells to achieve the highest bioluminescent signal was determined to be ~125,000 cells/mL, as this would result in an ending concentration of ≥106 cells/mL after 24 hours of growth (Fig 2c). A preliminary experiment to determine the reliability of the bioluminescence-based G. lamblia assay for drug inhibitory activities was performed using metronidazole and albendazole. The metronidazole EC50 values obtained from 125,000 cells/mL by MOXI coulter counter (EC50 = 1. 06 μM) and the luciferase-based assay (EC50 = 2. 24 μM) are within the acceptable range for experimental errors. The luciferase-based assay EC50 value of 0. 41 μM was obtained for albendazole (Fig 3). These EC50 values confirmed the reliability of the luminescence signal-based assay as compared to previous cell counting methods. Furthermore, the experimentally derived EC50 value of standard clinical giardiasis therapies using this new assay track with previously documented literature values of ~5 μM [37] or 8. 65 μM [38] for metronidazole and 0. 23 μM for albendazole (reference strain—WB) [38]. The screening of the Pathogen Box against G. lamblia at 16 μM led to the identification of one hundred and four compounds with ≥ 50% inhibition, forty-three compounds with ≥ 75% inhibition of cell growth, among which twenty-two showed ≥ 90% and fifteen compounds showed ≥ 95% inhibition (S1 Table and Table 1). EC50 values determined for thirteen of the molecules with ≥ 95% inhibition ranged from 0. 5 to >10 μM (Table 1). Two (MMV153413 and MMV688283) of the fifteen hits with ≥ 95% inhibition at 16 μM initial screening were deemed to be false positives since they were not potent in the follow up serial titration of the drugs. Screening against C. parvum at 10 μM yielded one hundred and eighty-five compounds with >50% inhibition, eighty-six compounds with ≥75% inhibition, twenty-five compounds with ≥ 90% inhibition and six compounds with ≥ 95% inhibition (S1 Table). The six compounds with cell growth inhibition ≥95% were tested to determine their EC50 values against C. parvum. The compounds MMV676477, MMV688853, MMV002817, MMV085499, MMV019189 and MMV102872 have C. parvum EC50 values of 0. 22, >10,0. 48,1. 93,2. 52, and 0. 14 μM respectively. Of the 400 Pathogen Box molecules, there were sixteen molecules with activity against both parasites at ≥75% inhibition. EC50 analysis was performed for a representative subset of seven of these dual hit molecules. All seven molecules had EC50 values of ≤ 5 μM on both parasites within the selected cytotoxicity criteria (HepG2 ≥5-fold of the observed EC50 value). Previously described anti-giardia and/or anti-cryptosporidiosis drugs auranofin [39,40], nitazoxanide [41], nifurtimox [38], and clofazimine [41] were found as dual hits in this screen. Furthermore, EC50s obtained using the new assay platform are very similar to those obtained for the wild-type G. lamblia strain and available literature-based comparisons (Table 2, S2 Table). Auranofin is highlighted as a potent hit against G. lamblia and C. parvum (Table 2), in agreement with literature results (G. lamblia EC50 = 4–6 μM in [40]; C. parvum EC50 = 2 μM in [39]). Clofazimine (G. lamblia EC50 = 1. 79 μM and C. parvum EC50 = 3. 3 μM) is a promising dual treatment option to further explore. The remaining tested hits (Table 2) share no obvious chemistry with these previously characterized anti-parasitic drugs. One of the best examples of this novel dual-inhibiting compound is MMV010576 which had 3. 0 and 1. 9 μM EC50 against C. parvum and G. lamblia in cell proliferation assays, respectively (Table 2). The other is MMV676501 with detectable cytostatic effects on mammalian cells at high concentrations of 50. 1 μM (Table 2). These have reasonable safety indices (EC50 of both parasites/CC50 against mammalian cells) for further exploration. There were no specific data on the biological activity of inhibitors against G. lamblia as documented for C. parvum in the MMV Pathogen_Box_Activity_Biological_Data_Smiles (https: //www. pathogenbox. org/about-pathogen-box/composition). This makes direct comparison of biological activity for G. lamblia obtained in our study against the Pathogen Box data sheet challenging. However, 11 compounds in the Pathogen Box series were designated as anti-cryptosporidiosis agents. Pathogen Box datasheet reported EC50s between 0. 05 μM and >5 μM for compounds MMV675968, MMV675993, MMV675994, MMV676050, MMV676053, MMV676599, MMV688853 and MMV688854. Initial screening data in our study found C. parvum cell growth inhibition at 10 μM to be between 79 and 96% with substantially lower effects on G. lamblia trophozoites (S1 Table). Hence, they were not progressed to the next stage of EC50 determination based on our dual hit selection criteria. Compound MMV675968 was however, a dual hit in the initial screen with 88 and 90% inhibition of C. parvum and G. lamblia growth, respectively. In the follow up serial dilution assays, MMV675968 had EC50s of 2. 1 μM and >10 μM for C. parvum and G. lamblia, respectively, thus ruling it out as a dual hit. MMV676191 had a 36% inhibitory activity on C. parvum cells in our initial screen at 10 μM, in agreement with the Pathogen Box datasheet reported EC50 of >10 μM. Dual hit compounds MMV676604 (C. parvum EC50 of 2. 9 μM) and MMV676602 (C. parvum EC50 of 0. 31 μM) were also not effective in the follow up EC50 assay against G. lamblia. The most obvious conclusion from the comparative analysis of anti-Cryptosporidiosis activity as provided by the MMV Pathogen Box datasheet with those obtained in this study is that there is great overlap in the results against C. parvum cells. Compounds with parasite EC50s ≤5 μM and within the selected cytotoxicity criteria (on mammalian HepG2 cells) were deemed potent hits. Sixteen compounds are dual hits with ≥75% initial growth inhibition against both parasites, three molecules were dropped from further consideration due to HepG2 cytotoxicity data from MMV and reduced efficacy in the follow up assay to determine their EC50s for G. lamblia. These include compounds MMV676602 and MMV676604. Recent interest in compound MMV687807 as a potential compound that may be further developed as an agent against Candida albicans [42] and Toxoplasma gondii [43] despite undesirable activity at 0. 7 μM against HepG2 (Table 2) argues for its inclusion in our analysis against G. lamblia. However, MMV687807 was too toxic to the HCT-8 host cells in the C. parvum assay that a reliable EC50 value could not be determined. Although auranofin showed high cytotoxicity levels of 0. 50 μM against HepG2 cells (Tables 1 and 2), it has already entered clinical trials as a possible anti-giardial [44]. Similarly, nitazoxanide showed activity against C. parvum and was considered a legitimate hit being already approved for clinical use. Altogether, seven molecules with EC50s ≤5 μM and within the selected cytotoxicity barrier (in HepG2 ≥5-fold of the observed EC50) were deemed potent dual hits.
  The bioluminescent assay for Giardia growth described here was optimized and found to be comparable to assays performed with automated cell counters. Reproducibility was validated by comparing known literature EC50s of metronidazole, auranofin, nitazoxanide and nifurtimox, finding all to be within a two-fold difference of our results. This suggests that the described bioluminescent assay can efficiently be used for semi high-throughput drug screening. Furthermore, the found dual-inhibiting compounds are of great public health importance since these could be suitable for co-infections or in situations where the precise diarrheal-causing pathogens cannot be determined. The luciferase-expressing G. lamblia strain could also be adapted for in vivo experimentation in infected animal models, advancing drug development and screening for G. lamblia similar to the way that Nluc expressing C. parvum has for Cryptosporidium [22,23].
summary: Development of effective single agent therapy that could be used to treat either cryptosporidiosis or giardiasis could significantly benefit those in developing regions of the world where diagnosis may be delayed or uncertain. However, asymptomatic presentations of these diseases and overlapping symptomatology with other parasitic diarrheal diseases are significant public health challenges for which there are limited available remedies. An effective drug developed based on phenotypic properties against G. lamblia and C. parvum would likely have some impact on other diarrheal parasites and be useful as a prophylactic indication against asymptomatic diseases. An important step towards this goal is the use of fast and inexpensive methods to identify new potential drug candidates. Among the goals of this study is the design of an efficient bioluminescent assay that can be used to measure growth inhibition of G. lamblia trophozoites in an efficient, high-throughput screen of large compound libraries. Stable expression of the luciferase gene introduced into Giardia was observed over a 24-hour incubation period. The assay was validated for reproducibility and reliability against known G. lamblia drugs with the obtained EC50 values within a statistically acceptable range, compared to literature values. A test of the high-throughput drug screening capability of the assay was verified in a parallel screen of G. lamblia and C. parvum with the aim of finding potential dual pathogen-inhibiting molecules. Seven compounds inhibited both parasites at low micromolar levels as confirmed by follow up assays. This series of inhibitors could be further developed as therapeutics and potentially become an important new tool for giardiasis and cryptosporidiosis treatments.
keywords: luciferase assay
  luciferase
  medicine and health sciences
  pathology and laboratory medicine
  giardiasis
  giardia
  enzymes
  tropical diseases
  enzymology
  cryptosporidium
  parasitic diseases
  parasitic protozoans
  toxicology
  biochemical analysis
  enzyme assays
  protozoans
  neglected tropical diseases
  bioassays and physiological analysis
  cryptosporidiosis
  research and analysis methods
  giardia lamblia
  parasitic intestinal diseases
  proteins
  oxidoreductases
  cryptosporidium parvum
  protozoan infections
  cytotoxicity
  biochemistry
  eukaryota
  biology and life sciences
  organisms
title: Screening of the Pathogen Box for inhibitors with dual efficacy against Giardia lamblia and Cryptosporidium parvum