Development of a Cytopathic Effect-Based Phenotypic Screening

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Development of a novel cytopathic effect-based phenotypic screening assay against Cryptosporidium Alexander T. Chao, Boon Heng Lee, Kah Fei Wan, Jeremy Selva, Bin Zou, Peter Gedeck, David Beer, Thierry T. Diagana, Ghislain Bonamy, and Ujjini H. Manjunatha ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00247 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 20, 2018

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ACS Infectious Diseases

Development of a novel cytopathic effect-based phenotypic screening assay against Cryptosporidium

Alexander T. Chao1,2, Boon Heng Lee1, Kah Fei Wan1, Jeremy Selva1, Bin Zou1, Peter Gedeck1,4, David John Beer1, Thierry T. Diagana1,2, Ghislain MC Bonamy1,3* and Ujjini H. Manjunatha1,2*

Affiliations: 1

Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore, 138670

2

Novartis Institute for Tropical Diseases, 5300 Chiron Way, Emeryville, California 94608, United States

3

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669

*

to whom correspondence may be addressed:

[email protected] [email protected] Present address: 4Collaborative Drug Discovery, Inc. 1633 Bayshore Hwy, Suite 342 Burlingame, CA 94010

One Sentence Summary: Description of a simplified, high-throughput, high-quality in vitro screening assay to enable cryptosporidiosis drug discovery

1 ACS Paragon Plus Environment

ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Cryptosporidiosis is a diarrheal disease predominantly caused by Cryptosporidium parvum (Cp) and Cryptosporidium hominis (Ch), apicomplexan parasites which infect the intestinal epithelial cells of their human hosts. The only approved drug for cryptosporidiosis is nitazoxanide, which shows limited efficacy in immunocompromised children, the most vulnerable patient population. Thus, new therapeutics and in vitro infection models are urgently needed to address the current unmet medical need. Toward this aim, we have developed novel cytopathic effect (CPE)-based Cp and Ch assays in human colonic tumor (HCT-8) cells and compared them to traditional imaging formats. Further model validation was achieved through screening a collection of FDA-approved drugs, confirming many previously known anti-Cryptosporidium hits as well as identifying a few novel candidates. Collectively, our data reveals this model to be a simple, functional, homogenous, gain of signal format amenable to high throughput screening, opening new avenues for the discovery of novel anticryptosporidials. Keywords: Cryptosporidium, phenotypic screening, cytopathic effect, HCT-8, high-content imaging.

Globally, approximately two thousand children die every day from diarrheal-related diseases, estimated to be responsible for 11% of the overall mortality for children under the age of five. 1 Multiple pathogens are known to be the etiological agents, including viruses (e.g. rotavirus and norovirus), bacteria (e.g. enterotoxigenic E. coli, Campylobacter spp., Shigella spp. and Vibrio cholera) and protozoan parasites (e.g. Cryptosporidium spp. and Entamoeaba histolytica) 2. In the developing world, it is also a major cause of malnutrition, and malnourished children are more likely to succumb from diarrhea. Recently a few large-scale epidemiological analysis of the causes of life-threating cases in small children around the world has identified Cryptosporidium as one of the important diarrheal pathogens after rotavirus 3-5. In addition, clinical cases due to cryptosporidiosis are associated with a higher mortality compared to other studied pathogens and are estimated to contribute to more than 200,000 deaths in children less than two years of age every year 6. Cryptosporidium is an apicomplexan protozoan parasite responsible for acute enteritis with severe diarrhea as its primary clinical symptom. Cryptosporidiosis in humans is caused primarily by two species, Cryptosporidium parvum (Cp) and Cryptosporidium hominis (Ch)

7

, which typical result in a self-limiting infection in

immunocompetent adults but may lead to life-threatening clinical manifestations in immunocompromised patients and malnourished children 4. Cp has a broader range of hosts, and it can cause a zoonotic disease often acquired by humans through livestock contact, whereas Ch is strictly anthroponotic and potentially life-threating in children. Repeated infections in children may also be associated with long-term and debilitating growth-stunting 8-9. While an effective vaccine against rotavirus, the leading cause of diarrhea in children, is available and being widely implemented in many countries

10

, nitazoxanide (NTZ) is the only US Food and Drug Administration (FDA)-

approved drug for the treatment of cryptosporidiosis 11. While NTZ is efficacious in immunocompetent individuals, it has an unsatisfactory response in malnourished children and is completely ineffective for the treatment of cryptosporidiosis in immunocompromised patients

12-14

. Despite its significant burden and clinical impact,


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cryptosporidiosis is still an under-appreciated global health concern with limited drug discovery efforts, limited in part by the throughput of the cell-based Cryptosporidium-targeted in vitro assays currently available 15. Cryptosporidium can complete its entire life cycle in the lumen of the intestine, occupying a unique niche intracellularly but developing outside of the cytoplasm at the apical surface of intestinal epithelial cells. Various host cell lines including Caco-2, MDCK, HCT-8, T84 etc. have been used for in vitro Cp infection, of which in most studies the HCT-8 cell line has been shown to be superior for its ability to support growth 16-17. Previously, a highcontent imaging (HCI) assay with fluorescent Vicia villosa lectin (VVL) to score Cp growth was used to evaluate anti-Cryptosporidium compounds

18

. More recently, a high-throughput phenotypic HCI Cp infection assay was

developed in HCT-8 cells and used to screen an FDA library and the MMV (Medicines for Malaria Venture) “malaria box” 19-20 and similar efforts led to the identification of clofazimine as a potential therapeutic intervention 21. Although the HCI-based screening assay has been widely-utilized, the assay setup is labor-intensive, requires multiple time-consuming post-endpoint washing steps and an expensive imaging platform as well as data processing expertise; making it less amenable to high throughput screening (HTS). Upon infection, Cryptosporidium spp. rapidly mature in vitro, leading to the death of intestinal host cells and subsequently resulting in the early termination of the Cryptosporidium life cycle 22-23. Based on the observation of infection-dependent host cell death, we report here a novel cytopathic effect (CPE)-based assay which derives parasite clearance by measuring viability of infected HCT-8 cells subsequent to compound treatment via a simple, functional, homogenous, gain of signal luminescence-based assay, amenable to ultra-high throughput. Additionally, we validated this model both by correlation analysis to the classic Cryptosporidium imaging assay formats and by screening a collection of 1,018 FDA-approved drugs, confirming several previously known anti-Cryptosporidium hits and identifying a few new candidates.

RESULTS AND DISCUSSION Disease relevant, cross-species imaging models for anticryptosporidials: To capture a disease-relevant condition where infection is pre-established, 384-well microtiter plate assays were developed for both Cp and Ch quantitating the percent infection of one-day-infected HCT-8 cells after a 48 hour drug exposure (72 hours total infection time) through standard immunolabeling methods. Briefly, Cp or Ch oocysts were excysted and inoculated onto confluent monolayers of HCT-8 cells. The multiplicity of infection (MOI), defined by the number of oocysts added per seeded HCT-8 cell for Cp and Ch was selected to reach an optimal 20-40% infection by three days, without a significant impact on host cells (0.5 and 2 respectively). The extent of Cp / Ch infection was monitored by staining with biotinylated VVL and streptavidin-conjugated Alexa Fluor 568 and the host cell nuclei counter-stained with DRAQ5. Images at 10× magnification were captured for each well and subsequently processed and quantified using Acapella® software. Under these conditions, percentage of HCT-8 cells infected were calculated to be 37 ± 8.5% and 18 ± 3.3% for Cp (Zꞌ= 0.27±0.10) and Ch (Zꞌ= 0.41±0.11) respectively. Images captured representing uninfected and infected monolayers treated with DMSO (Fig1A; i-iv) as well as images for 2.22 µM inhibitory concentrations of cladosporin and 2.22 µM sub-inhibitory concentrations of nitazoxanide for Cp and Ch are shown for reference



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purposes. (Fig 1A; v-viii) In ten-point dose response format, IC50 values for nitazoxanide were calculated to be 2.8 µM (95% CI) using Cp oocysts, correlating with previously published figures

19

, and 3.8 µM (95% CI) using Ch

oocysts. The Cp imaging IC50 for cladosporin was calculated to be 1.1 µM (95% CI) while the respective Ch IC50 was calculated to be 0.7 µM (95% CI). Concentration response curves for both species are shown for cladosporin and nitazoxanide (Fig 1B). Cryptosporidium infection-dependent cytopathic effect in HCT-8 cells: While optimizing the MOI of Cp infection in HCT-8 cells for imaging models, we observed a dose-dependent cytopathic effect (CPE) triggered by Cp infection, evident when visualizing HCT-8 cell nuclear staining between mock-treated wells with increasing initial oocyst inoculum (Fig 2A). Further quantitation of host cell nuclei per microscopic image revealed that with increasing excysted parasites from 10,000 to 80,000, there was a corresponding decrease in the number of host nuclei by up to 90% compared to uninfected controls within 3 days of infection (Fig 2B). At an MOI of 3, we observed a time-dependent decrease in host cell viability as measured by ATP levels using CellTiterGlo (Fig 2C). It was confirmed that the ATP contribution of replicating parasites was negligible to the overall luminescence signal measured in the growth medium. This observation is not surprising, considering Cryptosporidium is an obligate intracellular parasite that lacks the mitochondrial machinery responsible for ATP production 24. The time-dependent cytopathic effect (Fig 2C) correlated well with increased cellular detachment and gross morphological changes observable through differential interference contrast microscopy and a decrease in the quantity of DRAQ5-stained HCT-8 cell nuclei, unsurprising as previous groups have demonstrated that Cryptosporidium infection induces apoptosis in multiple cell lines22-23,

25-28

as well as inducing a decrease in

resistance across the epithelial monolayer and mucosa, suggesting damage to the tight junctions in intestinal cells or holes in the monolayer resulting from epithelial cell extrusion25, 27. Development and optimization of a CPE-based Cryptosporidium phenotypic screening assay: To develop a more efficient, robust and alternative host-centered anticryptosporidial assay we kept to a more disease-relevant in vitro 24 hour post-infection dosing point and established phenotypic human intestinal cell rescue assays based on the CPE we observed in infected HCT-8 cells in 384-well microtiter plate format for both Cryptosporidium spp.. To measure the efficacy of potential inhibitors, cladosporin, a commercially available isocoumarin shown to target the lysyl-tRNA synthetase in Plasmodium spp.

29

, was selected as a reference compound. At a 5 µM concentration,

intestinal cell rescue from Cryptosporidium-induced CPE was observed over multiple time points (Fig 2C) and efficacy was observed in all assay formats (Fig 1B, Fig 3B, Fig 4A). To simplify the infection step, HCT-8 cells were infected with Cryptosporidium spp. directly in tissue culture flasks for 3 hours (batch infection) followed by TrypLE dissociation and re-seeding into 384 well microtiter plates (25,000 cells per well), eliminating an additional handling and dispensing step. Additionally, this methodology improved the homogeneity of the infection in each well and across plates in a screening set, thus increasing assay robustness and simplifying logistics. For Cp at an MOI of 2, the host cell viability / luminescence measured with 5 µM cladosporin and DMSO control was 13,403 ± 1,304 and 3,699 ± 439 RLUs respectively (n =384) (Fig 3A). The Z-factor calculation, a standard measure of assay quality for this model was calculated to be 0.67 ± 0.04 (n=384). CPE doseresponse curves for cladosporin, floxuridine, and docetaxel with Cp and Ch are shown in Fig 3B & 4A respectively.


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The EC50’s measured for cladosporin against Cp (Fig 3B) and Ch (Fig 4A) were 0.68 ± 0.20 µM and 1.10 ± 0.29 µM respectively, correlating well with imaging IC50 measurements. Because floxuridine also possesses an inherent toxicity on mammalian cells, a bell-shaped dose response curve is observed with a peak at ~ 0.7 µM. At the higher drug doses, the cytotoxic activity of the compound potentially reduces the benefit induced through parasite clearance (Fig 3B). The HepG2 cell cytotoxicity of floxuridine was measured to be 0.73 µM (Table 1). The Cp CPE EC50’s for floxuridine and docetaxel were measured to be 0.04 µM and 1.41 µM (Fig 3B and Table 1) and in Ch were measured to be 0.08 µM and 2.79 µM (Fig 4A and table 1) respectively. Validation of the CPE model through screening a library of FDA-approved drugs: Examination of the in vitro efficacy of a collection of FDA approved drugs has been extensively explored in parasitic diseases 30. To search for candidates against cryptosporidiosis, Bessoff et al. have screened a library of 719 FDA- approved drugs against Cp using an imaging assay 19. This effort yielded 21 hits with an IC50 70% intestinal cell recovery, upon re-confirmation in full dose response, higher doses at 6.66 µM and 20.00 µM indicate significant HCT-8 monolayer cell death, producing a bell-shaped curve similar to that seen with floxuridine with cytotoxic points at the higher doses (Fig 3B, Fig 4A), indicative of efficacious but toxic compounds. In a Target Product Profile (TPP) for cryptosporidiosis where children under two years of age are the primary target population, safety is paramount. Elucidating the potential toxicity liabilities of chemotherapeutic scaffolds such as that of vorinostat towards the intestinal host cell early in the drug discovery flowchart quickly and efficiently provides relevant information which can be used to prioritize anticryptosporidial medicinal chemistry efforts. Efficacy correlation across different assay formats: To evaluate a dynamic relationship between the CPE model and the imaging assay, as well as efficacy correlations between Cp and Ch, we determined the in vitro potency of a small set of compounds in the different assay formats. One hundred compounds were selected leveraging hits obtained from the screening of the Selleckchem FDA library as well as additional scaffolds identified in the “Parasite box” screening efforts (to be described elsewhere). With compounds chosen to be structurally diverse and displaying a broad range of anticryptosporidial activity (EC50 measurements from 0.001 µM to 20 µM), we observed a positive correlation between Cp CPE and Cp HCI in vitro efficacy (r2 = 0.85), indicative that measuring intestinal host cell viability is a simple yet functional surrogate readout to measure Cryptosporidium infection. Likewise, we measured a strong correlation (r2=0.95) between Cp and Ch cellular activities in the CPE model (Fig 4B). Advantages and disadvantages of CPE-based assay: Any in vitro drug discovery assay used for screening should attempt to mimic the in vivo situation as close as technically possible. Most anti-Cryptosporidium drug screening assays described in literature to date administer compounds in a prophylactic fashion either prior to oocyst monolayer inoculation or three hours post-infection

18-19, 21

. Such methodology may be more likely to identify

chemical series and targets only critical during the early developmental stages (sporozoite or trophozoite) of the parasite lifecycle

23

. Transcriptome analysis has shown that 24 hours post-infection ~85% of the Cp genome is

expressed while only 65% of the Cp genome was expressed at 2 hours post-infection

34-35

. In addition, by 24 hours

post Cp infection the intestinal epithelial cells have been shown to have undergone a considerable degree of pathophysiological change, including secretion of pro-inflammatory cytokines; epithelial cell injury; etc.

36

. As a


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result, to more accurately mimic what may occur in a clinical setting, we developed our CPE-based model utilizing a 24 hour post-infection dosing point, documented to harbor both a significantly higher parasite load as well as a more heterogeneous population of parasitic stages. 23, 35 Although dosing point is important for closer clinical translation, the key advantage of the CPE-based assay format in general is its simplicity. The format is ideal for automated HTS as it involves a straightforward addition of CellTiter Glo reagent at the assay endpoint, followed by a luminescence measurement. In contrast, the imaging assay requires multiple post-endpoint processing steps including washing, fixing, blocking, staining, costly highcontent imaging platforms and intensive data processing (Supplementary Figure S1). Unlike imaging formats where every individual well is imaged multiple times at specified magnifications and the data is extracted from a cell population analysis, the CPE assay uses a luminescence-based measurement and provides a homogenous read-out for the entire well, essentially quantifying a gain in cellular viability when cells are cured of the parasite at specific compound concentrations; it is thus less prone to false positives. An additional advantage is that despite the simple assay setup, the CPE readout allows rapid filtering of potentially toxic scaffolds which may phenocopy active compounds in an imaging format. Recently by screening a library of compounds with anti-parasitic activity using CPE-based assay, we identified several scaffolds active against Cryptosporidium including KDU731, a novel CpPI(4)K inhibitors 37. One further logistical advantage we have noticed through analysis of both assay systems relates to the fragility of the infected HCT-8 cell monolayer. Through assay development with several oocyst lots, we have observed batch to batch variation in oocyst virulence, likely caused by the length of oocyst storage and variations in oocyst purification protocols, etc. More virulent batches of oocysts may cause increased fragility of infected HCT-8 cell monolayers leading to loss of these cells during multiple washing steps, potentially causing inconsistencies using imaging platforms. To evaluate whether the variability observed in the HCI assay would translate in the CPE model, we analyzed the respective Z’ measurements, parameters commonly used to assess the robustness of assay models where a score between 0.5 - 1 is considered to be robust 38. The Zꞌ of the HCI assay was measured to be 0.27 ± 0.10, similar to the values reported in literature 19. By comparison, the Zꞌ measured for the CPE assay was 0.67 ± 0.04, indicating that this screening modality is significantly more robust. Some challenges inherent in the CPE model are the higher requirement for oocysts per HCT-8 cell, 4-fold higher than for the HCI assay, which translates into increased cost when running large screens. Also, the model will not distinguish between direct-acting antiparasital drugs and compounds that may potentially inhibit cell death pathways, therefore further imaging assay analysis enumerating parasites should be performed to confirm scaffold targets and in vitro efficacy. In conclusion, widespread use of effective vaccines for rotavirus may soon bring cryptosporidiosis to the forefront as a leading threat globally to children and the immunocompromised suffering from severe diarrheal disease, making it imperative that new tools are developed and new therapies are quickly brought to clinic. Here, we present and validate novel cryptosporidiosis in vitro efficacy models which we believe provide several key technical advantages such as higher throughput, simpler setup, amenability to ultra-high throughput screening, and improved reproducibility. Taken together, our work identifies the Cp and Ch CPE efficacy assays as new and important tools



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for drug discovery that we hope will aid in quickly delivering safe and novel therapies to patients suffering from the potentially dire consequences of cryptosporidiosis.

METHODS Cells and parasites: Human ileocecal colorectal adenocarcinoma cells (HCT-8 [HRT-18] ATCC, CCL-34) were maintained in T-175 flasks (Corning, 431080) in complete growth medium (RPMI-1640 medium (Thermo Fisher Scientific, 11875) supplemented with 10% heat-inactivated horse serum (Thermo Fisher Scientific, 26050), 1× MEM non-essential amino acids (Thermo Fisher Scientific, 11140), 10 mM HEPES (Thermo Fisher Scientific, 15630), 100 units/mL penicillin, and 100 units/mL streptomycin) at 37°C and 5% CO2. Cultures were passaged twice weekly using 10 mL of 1× Phosphate-Buffered Saline (PBS) without Ca2+ and Mg2+ (Thermo Fisher Scientific, 20012) for washing and 3-5 mL per T-175 flask of TrypLE Express Enzyme (Thermo Fisher Scientific, 12604) for adherent cell dissociation. Cp oocysts, purchased from the Sterling Laboratory, University of Arizona (Iowa isolate, originally obtained from Dr. Harley Moon) were purified from infected calf feces using discontinuous sucrose and cesium chloride centrifugation gradients and stored in an antibiotic solution containing 0.01% Tween 20, 100 units/mL penicillin and 100 units/mL gentamicin. The purified Ch (TU502) oocysts, purchased from the Tufts University Cummings School of Veterinary Medicine, were propagated in the gnotobiotic piglet model 39 and stored in an antibiotic solution containing 0.01% Tween 20, 100 units/mL penicillin and 100 units/mL gentamicin. Both Cp and Ch oocysts less than three months old from date of shedding were used in subsequent infection experiments. Excystation and infection protocols were developed following established methods with some modifications

40-42

.

Cryptosporidium spp. oocysts were treated with 1 mL of 10 mM hydrochloric acid in 1× Hank’s Balanced Salt Solution (HBSS) (Thermo Fisher Scientific, 14025) for 10 minutes with agitation at 1,000 rpm, 37°C on an Eppendorf thermomixer, then washed twice with 1 mL of room temperature non-acidic 1× HBSS by centrifugation at 13,000 rpm for 3 minutes at 25°C. Oocysts were excysted at a concentration of 1 × 106 primed oocysts/µL in parasite infection medium consisting of a pre-warmed (37°C) and pre-gassed (5% CO2) 1:1 formulation of Leibovitz’s L-15 medium (Thermo Fisher Scientific, 11415) and UltraCULTURE medium (Lonza, 12-725F) supplemented with 2 mM sodium taurocholate (Sigma, 86339-1), 10% heat-inactivated horse serum, and 200 µM Lascorbic acid (Sigma, 95210) at 25°C for 10 minutes before further in situ excystation and infection onto HCT-8 monolayers. All dilutions for subsequent assays were performed in parasite infection medium without sodium taurocholate. Pre-excysted oocysts were enumerated microscopically using a C-Chip disposable hemocytometer (NanoEnTek, DHC-N01).Compound preparation: The Selleckchem FDA library (Selleckchem, L1300) was received as 10 mM compounds in dimethyl sulfoxide (DMSO). The compounds were further diluted by a factor of 1:3 in 100% DMSO, arrayed into the wells of V-bottom polypropylene 384-well source plates (Whatman), and stored at 4°C until use for single concentration screening. Compound powders were dissolved in 100% DMSO (Fisher, D4121) to 10 mM and stored at 4°C prior to dilution into source plates. Three-fold, 10- or 8-point serial dilutions laid out horizontally (HDR) or vertically (VDR) respectively were carried out using a Microlab STAR liquid handler (Hamilton) to obtain compound source plates with duplicates of each concentration. Source plates


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were stored at 4°C prior to spotting into assay plates. Before administration, all compound source plates were equilibrated to room temperature.

Assay controls: Each HDR compound source plate contained thirty-two active control compound wells (wells I1-P1, A12-H12, I13-P13 and A24-H24) and thirty-two DMSO-treated negative control wells (wells A1-H1, I12-P12, A13-H13 and I24-P24). Each VDR assay source plate contained eight active control compound wells (wells I24-P24) and eight DMSO-treated negative control wells (wells A24-H24). Active controls in full HDR 10point dose response in duplicate or VDR 8-point in duplicate were included in all compound source plates. All control wells in the downstream assays were treated with either the active control compound (Cladosporin, 5.0 µM final) or DMSO at the same volume as assay wells and used to calculate a Z’-value for each plate. All data was normalized on a per-plate basis. Cytopathic effect (CPE) assay: Cytopathic effect assays were optimized with reference to established methods in infectious disease with several modifications.

43-44

HCT-8 cells were grown to confluence in T-175 flasks, at which

spent growth medium was removed from flasks allowing for a 10 mL remaining volume. A non-infected control flask was used to enumerate quantity of host cells per T-175 flask using a NucleoCounter. (Chemometec, NC-100) Primed Cp oocysts at a ratio of two oocysts per one HCT-8 cell or primed Ch oocysts at a ratio of four oocysts to one HCT-8 cell in 1 mL of parasite growth medium were allowed to further excyst in situ on HCT-8 monolayers for 3 hours at 37°C and 5% CO2 before subsequent cell dissociation. Infected monolayers were washed once with 10 mL of 1 × PBS before dissociation with 3-5 mL per T-175 flask of TrypLE. Infected cell pellets were resuspended in 90% complete growth medium and 10% parasite infection medium without sodium taurocholate. 2.5×104 batchinfected HCT-8 cells were seeded in each well of a 384-well plate (Greiner, 789091) in a total well volume of 30 µL using a MultiDrop liquid handler (Thermo Fisher Scientific, 5840300) and standard tube dispensing cassette (Thermo Fisher Scientific, 24072670) at the high speed setting and incubated at room temperature for one hour to allow for negation of plate edge effects. All plates were subsequently incubated for 24 hours at 37°C and 5% CO2 prior to compound administration. 60 nL of compounds were transferred from the compound plate into the assay plate using an Echo Acoustic liquid handler (LABCYTE, 550). Compound treatment was allowed to proceed for 48 hours at 37°C and 5% CO2. Following compound treatment, assay plates were allowed to equilibrate to room temperature for 20 minutes in a biosafety cabinet to minimize temperature gradient effects. Cells were lysed and host cell viability measured by addition of 20 µL per well of Cell-Titer Glo 2.0 (Promega, G9243) using the Multidrop at the high-speed setting, followed by luminescence measurement at 0.1 seconds per well on a Clarity Luminometer (BioTek). Raw data files were exported and results were expressed as percent stimulation where 100% stimulation was equal to the median of the active control wells and 0% stimulation was equal to the median of the DMSO-treated negative control wells. Cell viability curves were analyzed using Novartis software and Z’-factors were calculated using the following equation: Z’ = 1 - 3 × (standard deviation of active control compound treated wells + standard deviation of DMSO treated wells) / |(median of active control compound treated wells - mean of DMSO treated wells).



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High Content Imaging (HCI), immunofluorescence assay: Imaging assays were developed following established Cryptosporidium spp. labeling and in vitro infection models with some modifications 19, 41. 2.0 × 104 HCT-8 cells per well were seeded into 384-well, flat black clear-bottom OPERA plates (Greiner, 789071-G) in a similar fashion as the dispensing of infected cells for the CPE assay. The cells were subsequently inoculated using the Multidrop with 10 µL per well of 1.0 × 104 primed Cp oocysts or 10 µL per well of 4.0 × 104 primed Ch oocysts in parasite infection medium at 37°C and 5% CO2 for 24 hours prior to compound administration. Compounds were spotted as described in the CPE assay and treatment proceeded for 48 hours at 37°C and 5% CO2. All following aspiration steps allow for a 15 µL residual volume. Following compound treatment, monolayers were washed twice with 65 µL of PBS; fixed with 40 µL of 4% paraformaldehyde (Electron Microscopy Sciences, 15710) in PBS for 20 minutes at 25°C; washed once with 65 µL of PBS; washed once with 65 µL of PBS-FT (0.05% Tween-20 and 1% fetal bovine serum in PBS); then permeabilized and blocked with 65 µL of PBS-FT for 30 minutes at 25°C. Streptavidin-conjugated Alexa Fluor® 568 (Life Technologies, S11226) was diluted in 5 mM sodium azide in PBS and incubated with biotinylated Vicia villosa lectin (Vector Laboratories, B-1235) in PBS-FT at 25°C for 1 hour (4.0 µg/mL Alexa Fluor® 568, 2.0 µg/mL VVL). Subsequently, the bound label was filtered through a pre-equilibrated (10 mL of PBS-FT) syringe filter (Sartorius Stedim, 16534-K). Intracellular parasitic life stages were labeled with 20 µL of the pre-bound, prefiltered Alexa568-VVL and allowed to incubate for 1 hour at 25°C. Following an additional 65 µL wash with PBSFT and one 65 µL wash with PBS, HCT-8 host cell nuclei were counterstained with 5 µM DRAQ5™ cell permeable DNA dye (Abcam, ab108410) diluted in PBS. Detection: Once labeled, the plates were imaged using an Opera QEHS. (PerkinElmer™) Imaging was performed at 10× using a Nikon UPlan Apo lens. Nine images were collected in each well which covered more than 80% of the well surface. The samples were exposed to 561 nm and 635 nm laser lines to excite respectively the Alexa Fluor® 568 conjugated lectins and DRAQ5™. The laser power was set to the maximum power for both lasers, the exposure time was set to 800 milliseconds and focal height set to 5 µm. The fluorescence signal was then collected on cooled CCD cameras after passing the emitted light through a quad-band primary dichroic (405/488/561/635) and a detection dichroic (510) followed by emission filters 600/40 and a 690/50 to collect the light emitted respectively by the labeled parasite and nuclei. Analysis: Images were analyzed using a custom analysis script written in Acapella® (PerkinElmer™). Quantitative data measured for each biological object was created and the feature files compressed via gzip. In brief, nuclei were detected and the mask obtained was then dilated to encompass the cell cytoplasm. These objects were thereafter referred to as cell bodies. The average signal from the images collected for the parasite channel was measured for each cell body. Cells were then classified as infected or non-infected by applying a calculated intensity cut-off. The intensity cut-off was set as the intensity threshold which maximized the Z’ factor

38

of the positive and negative

controls for a given plate. This optimization step was done using the statistical tool, R version 3.1.0. Z’ factors were calculated using the following equation: Z’ = 1 - 3 × (standard deviation of active control compound treated wells + standard deviation of DMSO-treated wells) / |(median of active control compound treated wells - mean of DMSOtreated wells)|. For each well, the number of cells and the percentage of infected cells were then calculated. The


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percentage of infected cells were expressed as percent inhibition where 100% inhibition was equal to the median of the active control wells and 0% inhibition was equal to the median of the DMSO-treated negative control wells. After manual curation to address any potential screening patterns or artifacts, data from each well was normalized by Novartis in-house software using the control wells such that no inhibition was set to 0% and full inhibition was set to -100%. The data was then curve-fitted as parasite inhibition curves to calculate the active concentration which resulted in having 50% of the cells infected. Methods used by Novartis in-house software have been previously published 45-49. Cell Cytotoxicity Assay: Cytotoxicity against HepG2, (ATCC# HB-8065) a human liver carcinoma cell line was determined as previously described with some modifications 50. Briefly, cells were seeded at a density of 400 cells per well of a 384 well plate, incubated at 37°C for 24 hours and exposed to three-fold serially diluted compounds for 96 hours. Cell viability was monitored using the Cell Counting Kit-8 (Dojindo, CK04-20).

SUPPORTING INFORMATION PARAGRAPH Supplementary Figure S1: Comparison of the steps involved in the Cryptosporidium CPE assay and the Cryptosporidium HCI assay. Steps noted below CPE assay (2) and Imaging assay (24) bars constitute technical steps affecting individual assay wells. CTG: cell-titer glo, PBS: phosphate buffered saline, PFA: paraformaldehyde; FT: fetal bovine serum, Tween-20. Supplementary Table S1: CpCPE 3-point primary screening data with FDA library at 0.33, 1 and 3 µM.

ABBREVIATIONS Cp, Cryptosporidium parvum; Ch, C. hominis; HCI, High-Content Imaging; CPE, Cytopathic effect; HCT-8, Human Colonic Tumor cell line; NTZ, nitazoxanide;VVL, Vicia villosa lectin; HTS, high throughput screening; .

AUTHOR INFORMATION Corresponding Authors: Ujjini H. Manjunatha, ORCID : 0000-0002-7461-9303, Email: [email protected] Ghislain M.C. Bonamy, ORCID : 0000-0002-2826-5225, Email: [email protected]

AUTHOR CONTRIBUTIONS:



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A.C., T.D., G.B. and U.M. conceived and designed the study; A.C., B.L., K.W., J.S., G.B. and U.M. designed and performed experiments; A.C., B.Z., P.G., D.B., G.B. and U.M. analyzed and interpreted assay development and screening results; A.C., G.B. and U.M. wrote the manuscript; all authors reviewed the manuscript.

CONFLICT OF INTEREST The authors declare the following competing financial interest(s): The authors are all current or former employees of Novartis and some own shares in Novartis.

ACKNOWLEDGEMENTS: We would like to thank Saul Tzipori and Donald Girouard (Cummings School of Veterinary Medicine, Tufts University) for Ch oocysts. This work was supported by the Novartis Institutes for Biomedical Research (NIBR). We would also like to thank Yi Hua Chan, Chang Bok Lee and other colleagues from Novartis Institute for Tropical Diseases

(NITD)

for

their

support.

All

authors

on

this

paper

are

employees

of

Novartis.


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FIGURE LEGENDS Fig 1. C. parvum and C. hominis high-content imaging (HCI) 384-well microtiter plate screening assay: Confluent HCT-8 cells in a 384-well microtiter plate were infected with excysted C. parvum (MOI: 0.5) or C. hominis (MOI: 2) oocysts and infection was allowed to proceed for 24 hours before the addition of compounds in 10-point dose response. After two days of compound exposure the extent of Cryptosporidium infection was monitored by staining with biotinylated Vicia villosa lectin and streptavidin-conjugated Alexa Fluor 568 (green) and the host nuclei counter-stained with DRAQ5 (red). A, representative 10× magnification images captured for each well; images were subsequently processed and quantified using Acapella® software. B, dose response curves representing in vitro efficacy of cladosporin and nitazoxanide against C. parvum (blue) and C. hominis (red). The respective EC50 values are given in the parenthesis.Fig 2. Cytopathic effect of C. parvum infection on HCT-8 monolayers. A & B, HCT-8 cells (20,000 per well in a 384 microtiter plate) infected with C. parvum at an MOI (0, 0.5, 1, 2, 4). One day post-infection 0.1 µM NCE-1 or DMSO was added and incubated for 2 days. Cells were subsequently fixed and labeled with DRAQ5, and captured and quantified using an Opera QEHS and Acapella software. (A) Representative 10× magnification images captured for each well at different MOI with DMSO control. The number of oocysts added and MOI are indicated below. (B) Quantitation of a number of host-nuclei per microscopic image at 10× magnification at different MOI. (C) Time-dependent C. parvum-induced cytopathic effect without treatment (blue line) or with 5 µM cladosporin (red line), as measured by using CellTiter Glo over 5 days. Host cell viability is plotted as a % function of viability in uninfected cells (black line). Fig 3.

Validation of C. parvum CPE assay: (A) C. parvum CPE single-point screening assay. HCT-8 cells

were infected with excysted C. parvum oocysts (MOI: 2) for 24 hours, then treated with 5 µM cladosporin or DMSO (n=384) for 48 hours distributed over three 384 well plates. After 3 days of incubation cell viability was measured by adding CellTiter-Glo reagent and plotted as relative luminescence units (RLUs). The upper dotted line at 9,490 RLUs) represents the mean -3× SD of the cladosporin treated wells and the lower dotted line at 5,017 RLUs represents the mean +3× SD of the DMSO-treated wells. (B) C. parvum CPE dose response curves. Dose response curves for cladosporin, docetaxel, floxuridine and vorinostat. The data points that show a dose-response gain of signal in viability are used for EC50 analysis; other data points which represent a decrease in host cell viability potentially suggest cytotoxic effects on HCT-8 cells. (C) Distribution of FDA library single-point screening hits. 1,018 FDA-approved drugs were screened at 3 µM and the distribution of hits is displayed against % host-cell recovery in a histogram analysis. The vertical dotted lines represent the mean ± 3× SD. (D) Scatter plot of FDA library screen. 1,018 FDA approved drugs screened at 3 µM are displayed in a scatter plot with selected compounds highlighted. The dotted line represents the mean + 3× SD for all compounds tested. (E) Correlation between C. parvum anti-cryptosporidial activity as measured by CPE and HCI based assays. A selected set of compounds with diverse chemical structures spanning 4-log orders of anti-cryptosporidial activity are plotted.

The calculated

2

correlation r value is 0.85. Different shapes and colors correspond to different chemical classes. The dotted line represents a diagonal 1:1 relationship.



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Fig 4. Validation of C. hominis CPE assay: (A) C. hominis CPE dose response curves. Dose response curves for cladosporin, docetaxel, floxuridine and vorinostat. The data points that show a dose-response gain of signal are used for EC50 analysis; other data points which represent a decrease in host cell viability suggest cytotoxic effects on HCT-8 cells. (B) Correlation between growth inhibition (EC50) of C. parvum and C. hominis measured by CPE-based assays. A selected set of compounds with diverse chemical structures spanning 4-log orders of antiCryptosporidium activity are plotted. The calculated correlation r2 value is 0.95. Different shapes and colors correspond to different chemical classes. The dotted line represents a diagonal 1:1 relationship.


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"For Table of Contents Use Only" Table of Contents Graphic:


ACS Infectious Diseases 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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TABLE 1: FDA library confirmed screening hits with anti-cryptosporidium in vitro efficacy (µM with 95% CI) Compound name Floxuridine Vorinostat Cabazitaxel Clofazimine Daunorubicin Emetine Doxorubicin Docetaxel Posaconazole Dasatinib Fluvastatin Sodium Atorvastatin Calcium Paclitaxel Chlorquinaldol Cyclosporine Crystal violet Ibrutinib Ciclopirox ethanolamine Imatinib Mesylate Bufexamac Clofarabine Dabrafenib Cephalomannine Crizotinib Idarubicin HCl Pitavastatin Calcium Lovastatin Cladosporinb

Indication / Target Cancer / DNA/RNA Synthesis Cancer / HDAC Neurological Disease / Microtubule Infection / ND Cancer / Telomerase Infection / ND Cancer / Topoisomerase Cancer / Microtubule Infection / sterol-14α-demethylase Cancer / Src, Bcr-Abl, c-Kit Cardiovascular Dis., / HMG-CoA Cardiovascular Dis., / HMG-CoA Cancer / Microtubule Infection / ND Immunology / Isomerase Infection / Mitochondrial Respiration Neurological Disease / Src Infection / ATPase Cancer / PDGF-R, c-Kit, Bcr-Abl Metabolic Disease / COX Cancer / DNA/RNA Synthesis Infection / Raf Cancer / ND Cancer / c-Met, ALK Cancer / Topoisomerase Cardiovascular Disease / HMG-CoA Respiratory Disease / HMG-CoA Pf Lysyl-tRNA synthetase

Cp CPE EC50 0.037 0.134 0.167 0.364 0.711 0.725 1.325 1.412 1.791 2.493 2.821 3.805 4.410 5.729 5.839 5.982 6.240 6.354 7.365 7.385 7.411 8.900 8.735 10.064 11.101 12.963 16.100 0.65

Ch CPE EC50 0.084 0.283 ND 1.620 ND 1.074 3.497 2.789 3.184 > 20.000 6.452 8.633 ND 15.405 3.707 2.977 >20.000 6.667 10.603 2.717 12.441 10.674 ND 11.026 ND ND > 20.000 0.827

HepG2 CC50 0.731 6.630 < 0.003 2.030 0.042 0.026 0.030 < 0.003 3.000 12.200 25.800 34.300 0.005 2.170 4.380 0.168 9.910 0.836 1.830 > 50.000 12.400 13.200 0.009 1.900 0.004 17.500 32.000 > 50.000

SIa 19.757 49.478 0.018 2.570 0.059 0.036 0.023 0.018 1.675 4.894 9.146 9.014 0.001 0.379 0.750 0.028 1.588 0.132 0.248 6.770 1.673 1.480 0.001 0.189 < 0.001 1.350 1.989 >60

Cp HCI IC50 0.023 0.083 ND 0.252 ND 0.231 ND 0.143 0.444 ND 2.844 1.512 ND 2.448 ND 1.448 5.120 2.787 2.155 ND 5.674 ND ND 2.366 ND ND > 20.000 1.310

a

SI, selectivity index, SI calculated as CC50 in HepG2 cells divided by Cp CPE EC50; bidentified by NITD “parasite box screening”; ND, not determined


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Figure 1 110x146mm (600 x 600 DPI)

ACS Paragon Plus Environment


Figure 2 105x63mm (300 x 300 DPI)


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Figure 3 106x133mm (600 x 600 DPI)



Figure 4 133x242mm (600 x 600 DPI)


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Development of a Cytopathic Effect-Based Phenotypic Screening

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