Screening the Pathogen Box for Identification of New Chemical Agents with Anti-Fasciola hepatica Activity

Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica. This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry.

ABSTRACT

Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica. This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry. For decades, triclabendazole has been the unique anti-Fasciola drug that can effectively treat this disease. However, triclabendazole resistance in fascioliasis has more recently been reported around the world, and thus, the discovery of novel drugs is an urgent need. The aim of this study was to investigate the fasciocidal properties of 400 compounds contained in the Pathogen Box. The first stage of the screening was carried out by measuring the fasciocidal activity on metacercariae at a concentration of 33 μM each compound (the standard dose). Subsequently, the activities of the most active compounds (n = 33) at their 50% inhibitory concentration (IC₅₀) values against metacercariae were assayed, and the results showed that 13 compounds had IC₅₀s of ≤10 μM. The second stage queried the activities of these compounds at 33 μM against adult flukes, with seven of the compounds producing high mortality rates of >50%. Four hit compounds were selected on the basis of their predicted nontoxic properties, and the IC₅₀ values obtained for adult worms were <10 μM; thus, these compounds represented the best fasciocidal compounds tested here. A cytotoxicity assay on four types of cell lines demonstrated that three compounds were nontoxic at their most active concentration. In conclusion, three hit compounds identified in this proof-of-concept study are potential candidates in the discovery of new fasciocidal drugs. Further studies are warranted.

INTRODUCTION

Fasciola hepatica is the etiological agent of fascioliasis, the most widespread trematodiasis that affects both humans and herbivorous mammals, such as sheep, cattle, goats, and other species (1, 2). In humans, fascioliasis can be acquired by the consumption of contaminated vegetables. Up to 17 million people in 51 countries are estimated to be infected with F. hepatica worldwide, and more than 91 million are at risk of infection by this parasite (3, 4). Among all the continents, the Andean region of South America is the most affected by Fasciola, where prevalence rates above 10% have been documented (5–8) and national treatment programs are being scaled up.

Triclabendazole (TCBZ) is the single most effective fasciocidal drug, with activity against both the infective larvae (metacercaria [MC]) and adult worms and efficacy that exceeds 90% in humans after a single oral dose (9, 10). Nonetheless, after decades of successful efficacy, TCBZ resistance has developed in both animals and humans (11). Cases of TCBZ-resistant Fasciola infection in both animals and humans have been reported in Australia, Europe, and Latin America (12–18). The development of TCBZ resistance represents an important public health concern throughout the world that mainly affects animal husbandry and leads to enormous economic losses (19). As a consequence, the discovery of novel drugs and vaccines effective against Fasciola is an urgent need for the global control of fascioliasis. Repurposing of praziquantel (PZQ) as an anti-Fasciola drug failed, whereas oxfebendazole was shown to be an effective drug in animals (20, 21). Currently, there is no other fasciocidal drug in clinical practice available for use in humans, and thus, TCBZ remains the unique treatment against this infectious disease.

Open-access drug discovery provides a substantial resource in research on those diseases that primarily affect people living in low-resource locations. The Medicines for Malaria Venture (MMV) foundation assembled a set of compounds, called Malaria Box, whose activities have been tested against various infectious agents, including Cryptosporidium parvum (22), Plasmodium falciparum (23, 24) Schistosoma mansoni (25, 26), Toxoplasma gondii (27), and mycobacteria (28, 29). Later, a new set of chemical entities was assembled and named the Pathogen Box collection. It contains 400 drug-like compounds that have shown inhibitory activity against various infectious diseases, such as hemonchosis, toxoplasmosis, tuberculosis, neosporosis, malaria, sleeping sickness, Chagas disease, leishmaniasis, and trypanosomiasis (30–36). The activities of the compounds in Pathogen Box against fungal diseases caused by Cryptococcus neoformans and Candida albicans have also been tested (37–39). The aim of this study was to identify the fasciocidal activity of 400 compounds contained in the Pathogen Box by in vitro testing.

RESULTS

In vitro activity of the Pathogen Box compounds against F. hepatica metacercariae.

In the first stage of the study, the 400 compounds contained in the Pathogen Box were screened in vitro for activity against F. hepatica metacercariae. A total of 33 compounds showed mean mortality rates above 25% at 33 μM, but all these compounds were less active than TCBZ (mortality rate, 90%), as shown in Table 1. The fasciocidal activity of these 33 compounds was then assessed by determining the 50% inhibitory concentration (IC₅₀) values (Table 1). As a result, 13 compounds showed potent inhibitory activities with IC₅₀ values of between 0.31 μM and 8.23 μM and were then assayed for their activities against adult worms, despite their low r values (Table 1).

TABLE 1.

Summary of the chemical compounds that showed the best biological activity against metacercariae

Compound plate codea	MMV identifierb	Molecular formulac	Mol wtc (g/mol)	Mean % mortality for MCd	SD for % mortality for MCd	IC50 (μM)e	rf	Activity against other infectious microorganism(s)g	In silico toxicity featuresh
									Acute cytotoxicity (fathead minnow) (mg/liter)	Blood-brain barrier penetration (human)	Carcinogenicity (rodent)	Mutagenicity (S. enterica serovar Typhimurium)	Maximum recommended daily dose (human) (mg/kg bwi /day)
TCBZ	NA	C14H9Cl3N2OS	359.7	100j	0j	15k	NA	Schistosoma	4.57	Penetrating	Noncarcinogenic	Nonmutagenic	NA
PAA2	MMV010764	C14H16N4OS2	320.4	22	38.5	24.1	−0.3	Plasmodium	NA	NA	Noncarcinogenic	Nonmutagenic	NA
PAF4	MMV676388	C15H14N4O3S	330.4	29	24.7	16.9	0.8	Mycobacterium	254.0	Penetrating	Carcinogenic	Mutagenic	2.44
PAF5	MMV202553	C15H15N3O2	269.3	29	24.7	14.9	0.9	Kinetoplastids	7.58	Penetrating	Noncarcinogenic	Mutagenic	0.993
PAG6	MMV063404	C19H24N3OCl	345.9	54	7.2	5.3	1.0	Mycobacterium	NA	Penetrating	Carcinogenic	Mutagenic	NA
PAH6	MMV676539	C20H16N2O3	332.4	17	28.9	24.7	−1.0	Mycobacterium	25.9	Penetrating	Carcinogenic	Mutagenic	4.05
PBD3	MMV637953	C51H40N6O23S6	1,435.3	25	9.9	21.8	−0.6	Trypanosoma and Onchocerca	NA	Penetrating	Noncarcinogenic	Nonmutagenic	NA
PBD7	MMV019838	C18H10N4OF6	412.3	26	11.6	12.4	0.0	Plasmodium	NA	Penetrating	Noncarcinogenic	Mutagenic	NA
PBF4	MMV003270	C7H5N2OCl	168.6	26	25.1	8.2	−0.7	Ancylostoma	6.75	Penetrating	Noncarcinogenic	Nonmutagenic	15.5
PBF6	MMV688853	C19H23N5O2	389.9	25	22.5	31.9	−0.8	Cryptosporidium	NA	Nonpenetrating	Noncarcinogenic	Mutagenic	NA
PBF11	MMV085210	C22H24N3O3ClS	446.0	40	15.3	2.4	0.8	Plasmodium	NA	Penetrating	Noncarcinogenic	Nonmutagenic	1.64
PBH10	MMV676380	C18H15N4O3Cl	370.8	33	33.3	1.3	0.1	Plasmodium	132.0	Penetrating	Noncarcinogenic	Nonmutagenic	101.0
PCA2	MMV675997	C24H29N4O2F	424.5	22	38.4	18.1	−0.2	Kinetoplastids	NA	Penetrating	Noncarcinogenic	Mutagenic	1.51
PCA6	MMV688852	C16H17N5ClF	333.8	29	37.4	17.2	−0.7	Toxoplasma	NA	Penetrating	Noncarcinogenic	Mutagenic	NA
PCC2	MMV688508	C19H19N2O4F	358.4	26	3.7	16.9	−0.5	Mycobacterium	NA	Penetrating	Noncarcinogenic	Mutagenic	NA
PCC5	MMV687730	C22H32N4O2	384.5	28	13.4	0.4	−0.5	Mycobacterium	NA	Penetrating	Carcinogenic	Nonmutagenic	NA
PCC6	MMV687251	C8H9N3O4S2	275.3	30	12.0	0.3	−0.5	Mycobacterium	NA	Penetrating	Noncarcinogenic	Nonmutagenic	13.3
PCC9	MMV688361	C21H19N5O	357.4	32	11.5	17.2	−0.7	Kinetoplastids	NA	Penetrating	Carcinogenic	Mutagenic	NA
PCC10	MMV689029	C26H26N4O4S	490.6	33	19.1	10.5	0.8	Kinetoplastids	NA	Penetrating	Carcinogenic	Mutagenic	11.9
PCD11	MMV1030799	C20H18N4O	330.4	28	11.7	1.5	−0.3	Plasmodium	6.62	Nonpenetrating	Carcinogenic	Mutagenic	NA
PCE5	MMV687146	C19H26N2O	298.4	21	25.8	15.6	0.6	Mycobacterium	NA	Penetrating	Noncarcinogenic	Mutagenic	NA
PCE6	MMV687696	C29H28N4O2ClF3	557.0	26	20.6	18.2	−0.7	Mycobacterium	NA	Nonpenetrating	Carcinogenic	Mutagenic	NA
PCE7	MMV687170	C17H13N4O2Cl	340.8	34	25.3	13.1	0.0	Mycobacterium	NA	Penetrating	Carcinogenic	Mutagenic	NA
PCE8	MMV690102	C22H23N7O2	417.5	38	15.6	2.1	0.7	Kinetoplastids	NA	Penetrating	Noncarcinogenic	Mutagenic	3.27
PCE11	MMV1029203	C20H17N5OS	375.5	33	29.7	7.1	−0.4	Plasmodium	100.0	Penetrating	Carcinogenic	Mutagenic	NA
PCF2	MMV676053	C18H16N3O3Cl	357.8	38	12.5	1.9	0.6	Cryptosporidium	194.0	Penetrating	Noncarcinogenic	Mutagenic	0.991
PCF3	MMV688179	C18H16N6OCl2	476.2	35	32.0	3.1	−0.1	Kinetoplastids	4.62	Penetrating	Carcinogenic	Mutagenic	1.41
PCF4	MMV023969	C24H24N4OS	453.0	48	21.8	1.5	0.3	Mycobacterium	NA	NA	Carcinogenic	Mutagenic	NA
PCF5	MMV687138	C19H17NO3S	339.4	26	11.6	14.7	−0.2	Mycobacterium	524.0	Penetrating	Noncarcinogenic	Mutagenic	89.7
PCF11	MMV688921	C23H18N3O5Cl	451.9	31	43.0	2.4	−0.4	Aedes aegypti- chikungunya virus	NA	Penetrating	Carcinogenic	Mutagenic	NA
PCG9	MMV688891	C18H11NO4BrF3	442.2	25	10.9	25.7	−0.5	Mycobacterium	NA	Penetrating	Carcinogenic	Mutagenic	1.25
PDH11	MMV688980	C16H18N3O2FS	335.4	33	38.2	21.2	0.2	Plasmodium	NA	Penetrating	Carcinogenic	Mutagenic	NA
PEC8	MMV687765	C25H26N6O	463	28	25.5	20.6	−0.8	Mycobacterium	NA	Penetrating	Noncarcinogenic	Mutagenic	NA
PEG9	MMV084864	C17H12N6O	316.3	40	18.7	17.3	0.8	Plasmodium	14.2	Penetrating	Noncarcinogenic	Mutagenic	NA

^aCoordinates used to identify the compounds in each plate. TCBZ, triclabendazole.

^bIdentification codes assigned by the Medicines for Malaria Venture (MMV). NA, not applicable.

^cMolecular formulas and molecular weights were obtained from www.mmv.org. For TCBZ, these values were obtained from ChEMBL (https://www.ebi.ac.uk/chembl/).

^dMeasured at 72 h after drug exposure for the metacercaria (MC) stage. Results are the mean and standard deviation for triplicate experiments at a concentration of 33 μM.

^eCompounds were serially diluted and tested in culture. Results are the means from triplicate experiments. Fasciolicidal activity values were determined with CompuSyn software.

^fr, correlation coefficient.

^gActivity shown against other agents causing infectious diseases, obtained from www.mmv.org.

^hPredictions were obtained using the lazar program (https://lazar.in-silico.de/predict). NA, not available.

ⁱbw, body weight.

^jData represent the mean and standard deviation for 10 individual experiments performed in 5 plates.

^kData were obtained from https://drugs.ncats.io.

In vitro activity of selected compounds against F. hepatica adult worms and in silico toxicology prediction.

The activities of the 13 selected compounds listed in Table 2 at 33 μM against adult worms were assayed. Seven compounds produced moderate or high mean mortality rates (>50%) (Table 2). These were MMV003270, MMV676380, MMV690102, MMV1029203, MMV063404, MMV1030799, and MMV688921. Six compounds showed low mortality rates (<50%), and for that reason, these were not considered in the next assays. Before we proceeded with the IC₅₀ assay, the in silico safety profiles of the seven selected compounds were predicted by use of the lazar (lazy structure-activity relationship) program (Table 1). Whereas MMV003270 and MMV676380 were predicted noncarcinogenic and nontumorigenic compounds, MMV690102 was deemed noncarcinogenic and tumorigenic (Table 1). MMV1029203, MMV063404, MMV1030799, and MMV688921 were predicted to be carcinogenic and tumorigenic substances. Thus, the three compounds deemed noncarcinogenic as well as MMV1029203, a predicted carcinogenic substance that produced the highest mean mortality rate (78%), were tested in adult worms. These four compounds constituted our hit compounds.

TABLE 2.

Biological activity of the compounds screened on adult worms^a

Compound plate codeb	MMV identifierc	Mean % mortality for adultsd	SD for % mortality for adultsd
TCBZ	NA	100e	0
PAG6	MMV063404	67	33.3
PBF4	MMV003270	67	0
PBF11	MMV085210	0	0
PBH10	MMV676380	78	19.2
PCC5	MMV687730	11	19.2
PCC6	MMV687251	33	33.3
PCD11	MMV1030799	67	33.3
PCE8	MMV690102	56	19.2
PCE11	MMV1029203	78	19.2
PCF2	MMV676053	0	0
PCF3	MMV688179	22	19.2
PCF4	MMV023969	33	33.3
PCF11	MMV688921	67	33.3

^aCompounds in italics were selected for evaluation of the IC₅₀ for adult worms and for use in the cytotoxicity assay with cell lines. NA, not applicable.

^bCoordinates used to identify the compounds in each plate. TCBZ, triclabendazole.

^cIdentification codes assigned by the Medicines for Malaria Venture (MMV).

^dMeasured at 48 h after drug exposure for adult worms. Results are means and standard deviations from triplicate experiments at a concentration of 33 μM.

^eMean and standard deviation from 6 individual experiments performed in 3 plates.

To determine which of the four hit compounds were the most potent at inhibiting the growth of F. hepatica adult worms, the IC₅₀ values were determined. The hit compounds had IC₅₀ values of <10 μM in adult worms (Table 3; see also Fig. S1 and Table S1 in the supplemental material). These four hit compounds were tested in the cytotoxicity study on cell cultures.

TABLE 3.

Hit compounds selected as new effective drugs for their fasciocidal activity against F. hepatica

^aIdentification codes were assigned by MMV.

^bA measurement of the octanol/water partition coefficient. Data were obtained from www.mmv.org.

^cCompounds were serially diluted and tested in culture. The results are means from triplicate assays.

^dCytotoxicity assays were performed on 3T3 cells and cells of the cancer cell lines H460, DU145, and HT-29. Results are the means from duplicate tests.

^eObtained from www.mmv.org. HepG2, hepatocellular carcinoma; HL60, human promyelocytic leukemia cells; MRC5, fibroblasts derived from lung. CC₂₀ and CC₅₀, 20% and 50% cytotoxic concentrations, respectively; NA, not available.

^fData were obtained from https://drugs.ncats.io.

In vitro cytotoxicity for cell lines.

The cytotoxicity of the four hit compounds for cell lines was evaluated in culture (Table 3). The 50% growth inhibitory concentration (GI₅₀) values ranged from 0.95 and >23.73 μM across the four types of cell lines assayed (Table 3). MMV003270, MMV676380, MMV1029203, and TCBZ presented GI₅₀ values above their IC₅₀ values, meaning that these compounds are not toxic at their active concentrations. In one of the four cell lines, MMV690102 had a GI₅₀ value below its IC₅₀ value, thus suggesting that it may cause a level of toxicity in certain cell types when it is used at its active concentration (Table 3).

Computational recognition of targets.

As a result of a search of the ChEMBL database, a total of 27 targets were recognized for TCBZ, whereas MMV003270 was found to have 19 known targets, most of which were in humans (Table 4). MMV003270 and TCBZ have common human targets that comprise nuclear factor erythroid 2-related factor 2, microtubule-associated protein tau, and TAR DNA-binding protein 43. According to the data deposited in the ChEMBL database, MMV003270 targets a number of cytochrome P450 members of families 1, 2, and 3. MMV676380 and MMV023969 have identical cell targets that include the human glucose transporter and the hexose transporter of Plasmodium falciparum and Leishmania mexicana (Table 4). MMV1029203 and MMV676053 were also shown to have known targets, including human ferrochelatase and the IMP dehydrogenase of Cryptosporidium parvum, respectively. The remaining eight compounds had no known targets, according to the ChEMBL database (Table 4).

TABLE 4.

Potential targets of the 13 hits and TCBZ tested in adult worm assays

Compound plate codea	MMV codeb	Target detailsc
		No. of targets predicted	CHEMBL identifier	Preferred name	Organism	Protein target classification
PBH10	MMV676380	3	CHEMBL2535	Glucose transporter	Homo sapiens	Transporter > electrochemical transporter > slc superfamily of solute carriers > slc02 family of hexose and sugar alcohol transporters
			CHEMBL4697	Hexose transporter 1	Plasmodium falciparum
			CHEMBL3431938	Glucose transporter	Leishmania mexicana	Transporter
PCE11	MMV1029203	1	CHEMBL3879831	Ferrochelatase	Homo sapiens	Unclassified protein
PCF2	MMV676053	1	CHEMBL6145	IMP dehydrogenase, probable	Cryptosporidium parvum	Enzyme
PCF4	MMV023969	3	CHEMBL2535	Glucose transporter	Homo sapiens	Transporter > electrochemical transporter > slc superfamily of solute carriers > slc02 family of hexose and sugar alcohol transporters
			CHEMBL4697	Hexose transporter 1	Plasmodium falciparum
			CHEMBL3431938	Glucose transporter	Leishmania mexicana	Unclassified protein
PBF4	MMV003270	19	CHEMBL340	Cytochrome P450 3A4	Homo sapiens	Enzyme > cytochrome P450 > cytochrome P450 family 3 > cytochrome P450 family 3A > cytochrome P450 3A4
			CHEMBL289	Cytochrome P450 2D6	Homo sapiens	Enzyme > cytochrome P450 > cytochrome P450 family 2 > cytochrome P450 family 2D > cytochrome P450 2D6
			CHEMBL3397	Cytochrome P450 2C9	Homo sapiens	Enzyme > cytochrome P450 > cytochrome P450 family 2 > cytochrome P450 family 2C > cytochrome P450 2C9
			CHEMBL3622	Cytochrome P450 2C19	Homo sapiens	Enzyme > cytochrome P450 > cytochrome P450 family 2 > cytochrome P450 family 2C > cytochrome P450 2C19
			CHEMBL3356	Cytochrome P450 1A2	Homo sapiens	Enzyme > cytochrome P450 > cytochrome P450 family 1 > cytochrome P450 family 1A > cytochrome P450 1A2
			CHEMBL4040	MAP kinase ERK2	Homo sapiens	Enzyme > kinase > protein kinase > CMGC protein kinase group > CMGC protein kinase MAPK family > CMGC protein kinase ERK subfamily
			CHEMBL2903	Arachidonate 15-lipoxygenase	Homo sapiens	Enzyme
			CHEMBL2756	Monoamine oxidase B	Bos taurus	Enzyme
			CHEMBL3254	Monoamine oxidase A	Bos taurus	Enzyme
			CHEMBL1075094	Nuclear factor erythroid 2-related factor 2	Homo sapiens	Unclassified protein
			CHEMBL1293224	Microtubule-associated protein tau	Homo sapiens	Unclassified protein
			CHEMBL2362981	TAR DNA-binding protein 43	Homo sapiens	Unclassified protein
			CHEMBL1293235	Prelamin-A/C	Homo sapiens	Unclassified protein
			CHEMBL1781865	78-kDa glucose-regulated protein	Homo sapiens	Unclassified protein
			CHEMBL1977	Vitamin D receptor	Homo sapiens	Transcription factor > nuclear receptor > nuclear hormone receptor subfamily 1 > nuclear hormone receptor subfamily 1 group I > nuclear hormone receptor subfamily 1 group I member 1
			CHEMBL1947	Thyroid hormone receptor beta-1	Homo sapiens	Transcription factor > nuclear receptor > nuclear hormone receptor subfamily 1 > nuclear hormone receptor subfamily 1 group A > nuclear hormone receptor subfamily 1 group A member 2
			CHEMBL1697668	Solute carrier organic anion transporter family member 1B1	Homo sapiens	Transporter > electrochemical transporter > slc superfamily of solute carriers > slc21/slco family of organic anion transporting polypeptides
			CHEMBL1743121	Solute carrier organic anion transporter family member 1B3	Homo sapiens
			CHEMBL1741193	Chromobox protein homolog 1	Homo sapiens	Epigenetic regulator > reader > methyl-lysine/arginine binding protein > chromodomain
TCBZ	NA	27	CHEMBL1293278	Geminin	Homo sapiens	Unclassified protein
			CHEMBL1075094	Nuclear factor erythroid 2-related factor 2	Homo sapiens	Unclassified protein
			CHEMBL1293224	Microtubule-associated protein tau	Homo sapiens	Unclassified protein
			CHEMBL1293258	Mothers against decapentaplegic homolog 3	Homo sapiens	Unclassified protein
			CHEMBL2362981	TAR DNA-binding protein 43	Homo sapiens	Unclassified protein
			CHEMBL2146310	Aberrant vpr protein	Human immunodeficiency virus type 1	Unclassified protein
			CHEMBL2029198	Rap guanine nucleotide exchange factor 4	Homo sapiens	Unclassified protein
			CHEMBL6152	Alpha-synuclein	Homo sapiens	Unclassified protein
			CHEMBL1293191	Transcriptional regulator ERG	Homo sapiens	Unclassified protein
			CHEMBL2007624	Peripheral myelin protein 22	Rattus norvegicus	Unclassified protein
			CHEMBL1795086	HSP90	Plasmodium falciparum 3D7	Unclassified protein
			CHEMBL5567	Luciferin 4-monooxygenase	Photinus pyralis	Enzyme
			CHEMBL2007625	Isocitrate dehydrogenase (NADP), cytoplasmic	Homo sapiens	Enzyme
			CHEMBL3563	Cruzipain	Trypanosoma cruzi	Enzyme > protease > cysteine protease > cysteine protease CA clan > cysteine protease C1A family
			CHEMBL1293248	4′-Phosphopantetheinyl transferase FFP	Bacillus subtilis	Enzyme
			CHEMBL1795087	Ubiquitin carboxyl-terminal hydrolase 1	Homo sapiens	Enzyme
			CHEMBL1293234	Putative fructose-1,6-bisphosphate aldolase	Giardia intestinalis	Enzyme
			CHEMBL1293228	Streptokinase A	Streptococcus pyogenes serotype M1	Enzyme > kinase
			CHEMBL2524	Alpha-galactosidase A	Homo sapiens	Enzyme
			CHEMBL1784	Glucagon-like peptide 1 receptor	Homo sapiens	Membrane receptor > family B G protein-coupled receptor > peptide receptor (family B GPCR) > glucagon-like receptor > glucagon-like peptide receptor
			CHEMBL1793	Parathyroid hormone receptor	Homo sapiens	Membrane receptor > family B G protein-coupled receptor > peptide receptor (family B GPCR) > parathyroid hormone receptor > parathyroid hormone receptor
			CHEMBL5162	Neuropeptide S receptor	Homo sapiens	Membrane receptor > family A G protein-coupled receptor > peptide receptor (family A GPCR) > short peptide receptor (family A GPCR) > neuropeptide receptor
			CHEMBL1293231	Nuclear receptor ROR-gamma	Mus musculus	Transcription factor > nuclear receptor > nuclear hormone receptor subfamily 1 > nuclear hormone receptor subfamily 1 group F > nuclear hormone receptor subfamily 1 group F member 3
			CHEMBL1871	Androgen receptor	Homo sapiens	Transcription factor > nuclear receptor > nuclear hormone receptor subfamily 3 > nuclear hormone receptor subfamily 3 group C > nuclear hormone receptor subfamily 3 group C member 4
			CHEMBL3880	Heat shock protein HSP90-alpha	Homo sapiens	Other cytosolic protein
			CHEMBL6032	Histone-lysine N-methyltransferase, H3 lysine-9 specific 3	Homo sapiens	Epigenetic regulator > writer > protein methyltransferase
			CHEMBL4377	Guanine nucleotide-binding protein G(s), subunit alpha	Homo sapiens	Other membrane protein

^aCoordinates used to identify the compounds in each plate. For the following hits, no target was identified: MMV063404, MMV687730, MMV687251, MMV1030799, MMV690102, MMV085210, MMV688179, and MMV688921.

^bIdentification codes assigned by MMV except for TCBZ.

^cObtained by consulting ChEMBL. Abbreviations: MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; ERK2, extracellular signal-regulated kinase 2; ERK extracellular signal-regulated kinase; slc, solute carrier; CMGC, cyclin-dependent kinase; MAPK, mitogen-activated protein kinase; GSK, glycogen synthase kinase; CLK, and CDC-like kinase; GPCR, G protein-coupled receptors; NA, not available.

DISCUSSION

In the present study, the Pathogen Box was queried to identify compounds with in vitro activity against both metacercariae and adult worms of Fasciola (Fig. 1). We found 13 compounds with potent inhibitory activity against metacercariae (IC₅₀ < 10 μM), meaning that 3% of the substances within the Pathogen Box are effective against the infective form of F. hepatica. Two out of the 13 compounds (MMV687730 and MMV687251) had the most potent activity against metacercariae, with IC₅₀ values being below 1 μM, but showed mild effects on adult worms (Tables 1 and 2). Since we were interested in identifying hit compounds that were active against the larval and adult stages, these two compounds were not further studied (Table 2). When assayed on adult worms, seven promising compounds showed mortality rates above 50% (Table 2). As a criterion for hit prioritization during the screening on adult worms, we prepared a list of hit compounds that mostly excluded the predicted carcinogenic/tumorigenic compounds (Table 3). Thus, three (MMV676380, MMV003270, and MMV690102) of the seven most promising candidates were included in the list of hit compounds since they were predicted noncarcinogenic agents (Table 1). One additional compound (MMV1029203) that was predicted to be a carcinogenic compound was also included due to its very strong effect on adult worms. According to our results, the four hit compounds were potent molecules that inhibited both the MC and adult stages (Table 3). The cytotoxicity assay revealed that three hit compounds (MMV676380, MMV003270, and MMV1029203) were nontoxic agents when assayed at their most active concentrations on cell lines (Table 3). In contrast, MMV690102 may cause cell cytotoxicity at its most active concentration, meaning that it is not a primary candidate for drug development (Table 3). Our results are consistent with those of previous cytotoxicity assays on HepG2, HL60, and MRC5 cells, as shown in Table 3 (data provided by the MMV as part of the supporting information for the open-access Malaria Box).

FIG 1.

Flow chart of the study.

Repurposing of hits with activity against F. hepatica obtained using analysis of the compounds in the Pathogen Box is highly relevant since TCBZ is the only existing drug effective against Fasciola, but resistance to this agent is known (40–42). Previous works tried to repurpose albendazole, nitroxinil, and closantel as candidate fasciocidal drugs, but treatment failed (43, 44). In the present study, 4 out of 400 compounds contained in the Pathogen Box showed potent inhibitory activity against the infective form of F. hepatica as well as against its adult form (Table 3). Such a finding represents a relevant contribution to the identification of dual drug candidates that are able to act against the initial stages of the infective larvae (metacercaria) and adult forms of liver flukes, similar to TCBZ. Additionally, 13 other compounds showed biological activity at <20 μM against metacercaria (Table 1). Since MC represents the initial infective form of the parasites, it should be primarily controlled through potent compounds such as those identified here (Table 1). Future exploration of the activities of the compounds in the Pathogen Box against newly juvenile metacercaria is desirable, given that some compounds may not have penetrated the cyst wall of the larvae. By testing the activities of the compounds on juvenile worms, some additional molecules that are active against adult worms might be recognized.

The four hit compounds identified in this study have previously been characterized to have activity against Plasmodium falciparum, Ancylostoma ceylanicum, Trypanosoma cruzi, and Leishmania donovani (data provided by the MMV as part of the supporting information for the open-access Malaria Box). Therefore, a common mechanism of action or target among the hit compounds across such pathogens is plausible. For instance, MMV676380 has previously been shown to have a lethal effect on P. falciparum and here was found to be an inhibitory compound with potent activity against F. hepatica (36, 45). Known targets of MMV676380 are the glucose and hexose transporters, suggesting that such a mechanism may be affected in both parasites in the presence of such a compound (Table 4). On the other hand, MMV003270 (zoxazolamine), which is also active against A. ceylanicum, was found to have 19 targets, including 3 human proteins that are also targeted by TCBZ (Table 4). Two of these proteins are transcription regulators (nuclear factor erythroid 2-related factor 2 and TAR DNA-binding protein 43) whose disruption may affect gene expression. Such a finding is in accordance with a hypothetical mechanism of action of TCBZ that involves a direct effect of the drug on protein synthesis (11, 46). Similarly, the microtubule-associated protein tau is a known target both of TCBZ and of MMV003270. TCBZ is a benzimidazole derivative that disrupts the assembly of microtubules in helminths by binding to tubulin molecules (47). Our results suggest that MMV003270 also affects the microtubule formation mechanism. Common targets of TCBZ and MMV003270 may be partially explained by the similar scaffold structures. MMV1029203, one of the four hit compounds, targets a human ferrochelatase that is a mitochondrial factor involved in protoheme biosynthesis. The latter is a vital process that also exists in F. hepatica and whose disruption may be lethal. Some known targets of the hit compounds identified here correspond to human proteins, which suggests that a level of toxicity may exist in humans. However, according to our results with cell lines, the compound concentrations needed to kill F. hepatica (IC₅₀) were considerably less than those needed to cause cell death (GI₅₀), which means that all these compounds except MMV690102 are nontoxic (Table 3). Although no F. hepatica target was recognized for our hit compounds, the demonstration of the inhibitory activity of such chemical agents against both the metacercaria and adult forms suggests that common targets may exist in both liver fluke stages. The identification of drug targets becomes an important step that drives the discovery of novel antiparasitic agents administered in various ways (34). For that reason, further studies to identify the potential F. hepatica targets of hit compounds are desirable. Such studies should consider the recognition of human homologs in F. hepatica, according to our results (Table 4).

Our study has some limitations. First, TCBZ metabolites (TCBZ sulfoxide and TCBZ sulfone) that are quickly released in vivo were not included in this pilot study. However, given that TCBZ has moderate in vivo and in vitro fasciocidal effects, it is suitable for use as a positive control in bioassays (48, 49). A second limitation is that live F. hepatica worms were collected from a local abattoir, where some animals may have been infected by various other pathogens or may have been treated with TCBZ. To guarantee the best quality of adult worms for bioassays, we performed a quality control on adult fasciolas before using these in the experiments. Thus, only worms that presented an intense brown or red color and that had active motility were selected. All the remaining worms were discarded. A third limitation is the low number of parasites used for the assays, which did not allow formal statistical comparisons of the activities between TCBZ and the test drugs to be performed. Obtaining MC and adults was a challenging task since both MC and adult worms were collected from natural reservoirs. Therefore, we had limited access to parasites for bioassays. However, our exploratory study aimed to identify fasciocidal compounds, and we found that the use of negative controls was enough for such purposes.

In conclusion, we identified three promising noncytotoxic drug-like compounds, MMV003270, MMV676380, and MMV1029203, that showed potent biological activity against F. hepatica metacercariae and adult worms. Such compounds represent new lead candidates to potentially become future anti-F. hepatica drugs. By acting both on the infective form and on adult worms, such agents may provide an appropriate treatment against fascioliasis.

MATERIALS AND METHODS

Study design. The study was conducted in three stages: (i) bioassays on metacercariae, (ii) bioassays on adult worms, and (iii) assays on cytotoxicity for cells (Fig. 1). The best fasciocidal compounds, based on in vitro biological activity, were selected at each stage to be tested in the next phase. To complement our knowledge on the active compounds obtained by the experimental assays, computational resources were consulted to describe the chemical properties as well as the in silico toxicology features and biological targets of these active compounds.

Drugs and media.

The Pathogen Box compounds were provided by MMV (Geneva, Switzerland) and manufactured by Evotec (USA). The 400 drug-like molecules were supplied in 96-well plates as stock solutions of 10 mM dissolved in dimethyl sulfoxide (DMSO). Full data on the Pathogen Box compounds is available at https://www.pathogenbox.org (50). TCBZ was purchased from Sigma-Aldrich (Buchs, Switzerland). All of the compounds in the Pathogen Box were dissolved in DMSO (Sigma-Aldrich, Irvine, UK) to make drug stock solutions of 200 μM. Additional vials of MMV063404, MMV003270, MMV085210, MMV676380, MMV687730, MMV687251, MMV1030799, MMV690102, MMV1029203, MMV676053, MMV688179, MMV023969, and MMV688921 were manufactured by Evotec (France). RPMI 1640 culture medium (Sigma-Aldrich, St. Louis, MO, USA) was used for both stages, metacercariae and adult worms, and was supplemented with penicillin (100 U/ml) and streptomycin (100 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA).

Parasites.

Metacercariae of F. hepatica were obtained, following the protocol described by Ortiz et al. (16), at the Immunology and Research Laboratory of the Faculty of Veterinary Sciences of the Universidad Nacional de Cajamarca in Peru. Eggs of F. hepatica were collected directly from the gallbladder of sheep slaughtered in a popular abattoir in the city of Cajamarca, Peru (an area of endemicity for fascioliasis where TCBZ resistance has been seen). Miracidia were collected from Fasciola eggs that had been incubated for 15 days at 25°C. Afterwards, they were used to infect Lymnaea sp. snails (5 to 6 mm) in a proportion of two miracidia per snail. The infected snails were kept in plastic containers for 45 to 60 days at room temperature. After this time, the snails were stimulated by direct solar exposure and with water at 4 to 8°C to produce metacercaria. Approximately 20,000 metacercariae were obtained for this study and stored in distilled water in cryovials at 4 to 8°C. Adult worms were collected from the bile ducts of infected cattle from a slaughterhouse in Lima, Peru, and maintained at 37°C until usage (within 2h). Before incubation, three washes with phosphate-buffered saline (PBS; HiMedia, India) and one additional wash with supplemented RPMI 1640 medium were performed to remove host debris. All the incubations for both metacercariae and adults were carried out at 37°C with 5% CO₂.

In vitro screening of activity against metacercariae.

The activities of the 400 compounds against F. hepatica metacercariae were initially tested at 33 μM. Drug stock solutions were diluted in 96-well plates (BD Falcon, USA) with RPMI 1640 medium supplemented with antibiotic up to a final volume of 180 μl. In all in vitro assays, positive and negative controls were run in parallel for each assay batch. A range of 7 to 10 metacercariae that had previously been analyzed microscopically to confirm their viability (microscopic features intact) were added to each well. Some physical properties of the parasite, determined by microscopy, as described previously (51, 52), were considered to determine the viability of the metacercariae. MC viability was surveyed as a function of both damage to the membrane and fluke color (translucence). Therefore, low viability corresponded to heavy damage and high translucence. The viability scale was scored as follows: +++, total damage (dead parasite, shattered membrane, and mostly translucent); ++, partial damage (partial membrane damage and highly translucent); +, mild damage (partial membrane damage, poorly translucent); and no damage (intact membrane, dark metacercariae, a lack of translucence).

Positive-control wells contained TCBZ at 10 μM, whereas F. hepatica metacercariae incubated in the presence of the highest concentration of DMSO tested served as negative controls. Each test was performed in triplicate. Culture plates were incubated at 37°C in a humidified 5% CO₂ atmosphere for 72 h. First, metacercariae were evaluated by inverted microscopy (PhotoZoom microscope; Cambridge Instruments) at magnifications of ×10 and ×20 at 24, 48, and 72 h after drug exposure to determine their viability. Only the compounds that caused, on average, at least 25% metacercaria mortality at 72 h were considered for 50% inhibitory concentration (IC₅₀) determination. Experiments were run in sets of triplicates. The mean percent mortality caused by the study compounds was compared to that caused by DMSO. A standard deviation (SD) was also estimated.

In the second part, we determined the IC₅₀s of the selected compounds chosen in the previous bioassay. Drugs were tested at concentrations of 2.1, 4.2, 8.4, and 33 μM using supplemented culture medium. The incubation was done under the conditions described above, in triplicate and by considering TCBZ and DMSO as controls. Antiparasite activity was evaluated at 24, 48, and 72 h postexposure, using the above-mentioned metacercaria viability scale. Viability (the mean percentage of viable parasites) at 72 h was considered for the estimation of the IC₅₀. The IC₅₀ values of the test compounds were determined by linear regression analysis using CompuSyn software (version 3.0.1, 2007; ComboSyn Inc., USA). The linear correlation coefficient (r) was obtained.

Assessment of in vitro activity against adult Fasciola worms.

Those compounds that showed activity with an IC₅₀ of ≤10 μM for metacercariae were subsequently tested for their activity against the adult stage of F. hepatica. In all in vitro assays, positive and negative controls were run in parallel for each assay batch. First, the selected compounds were tested at 33 μM in triplicate, using drug stock solutions diluted in supplemented RPMI 1640 medium on a 6-well plate to a final volume of 4 ml. Adult worms were thoroughly washed with PBS to remove host debris, and then three worms were placed in each well. The incubation was done under the same conditions applied in bioassays with metacercariae. The positive control consisted of 50 μM TCBZ, and the negative control was DMSO at the highest concentration tested. The viability of the adult flukes was scored after 24 and 48 h using a motility criterion described previously (48) and also the color and rigidity criteria previously applied by our team (data not published). Motility was assessed only in adults and not in MC because the latter has no movements. Rigidity was a parameter used to confirm the damage caused by the drug once the incubation time finished. In general, a low motility level corresponded to transparent and rigid worms. Those changes were attributed to the damage caused by a drug. The viability scale was determined as follows: (i) for worm motility, a score of 3 was assigned for normal movements, a score of 2 was assigned for reduced movements, a score of 1 was assigned for very weak movements, and a score of 0 was assigned for the absence of movements (i.e., death of worm); (ii) for worm color, +++ was assigned for dark red, ++ was assigned for pink, + was assigned for slightly transparent, and − was assigned for totally transparent; and (iii) for worm rigidity, − was assigned for no rigidity, + was assigned for rigidity, and ++ was assigned for cell breakage when the cell was touched. Assessments were not done at 72 h after drug exposure because the death of the worms always occurred at ≤48 h. Experiments were run in triplicate. The mean percent mortality and SD of the study compounds were estimated. The selected compounds were those that caused an average mortality of >50% in adult parasites. Then, IC₅₀ assays were conducted by testing the selected compounds at five different concentrations of 0.31, 0.93, 2.78, 8.33, and 25.0 μg/ml. DMSO and TCBZ were used as negative and positive controls, respectively. Parasite viability at 24 h was estimated on the basis of survival in DMSO. The IC₅₀s and 95% confidence intervals (CI) were estimated, using GraphPad Prism (version 7.0) software, using the variable slope of the sigmoidal curve from the normalized percent activity values and log₁₀-transformed concentrations. The top and bottom values were constrained to 100 and 0, respectively. The fasciocidal activity was determined by considering the adult viability scale described above.

Computational analysis.

(i) Evaluation of biological targets of small compounds. To learn about biological targets, those compounds that showed promising anti-Fasciola activity in the adult stage as well as TCBZ were entered into the ChEMBL database (https://www.ebi.ac.uk/chembl/) (53). First, the SMILES (simplified molecular-input line-entry system) notation of each of the selected compounds was obtained from the supplemental material provided by MMV (also available at www.mmv.org). Then, the SMILES notations were entered into ChEMBL, and known targets of each compound were retrieved. ChEMBL compares the query compound to a large database of compounds and their targets available from multiple sources, including the projects funded by MMV (54). The target name, organism, and protein target classification were collected.

(ii) In silico cytotoxicity prediction.

lazar (lazy structure-activity relationships), a modular framework for predictive toxicology, was consulted to predict the toxic effects of the selected compounds that showed activity on the metacercariae (55–57). lazar was accessed through https://lazar.in-silico.de/predict, and the SMILES notation of each compound was entered. Relevant data, including carcinogenicity in rodents, mutagenicity in Salmonella enterica serovar Typhi, acute toxicity for the fathead minnow, blood-brain barrier penetration, and the maximum recommended daily dose in humans, were predicted.

Cell growth inhibition bioassay.

The cytotoxicity of the compounds was evaluated in tumor and nontumor cell lines using the sulforhodamine B (SRB) assay method (58, 59). The cell lines tested included BALB/3T3 (nontumorigenic, BALB/c mouse embryo cells), H460 (human lung large cell carcinoma), DU145 (human prostate carcinoma), and HT-29 (human colon adenocarcinoma).

To determine the cytotoxicity of the compounds, cells were plated into 96-well tissue culture plates and in their corresponding growth medium, Dulbecco’s modified Eagle medium (DMEM), at approximately 10% confluence (BALB/3T3 cells at 3,500 cells/well, H460 cells at 1,500 cells/well, DU145 cells at 3,500 cells/well, and HT-29 cells at 3,000 cells/well) and incubated at 37°C in a 5% CO₂ and 95% air humidified atmosphere for 24 h to allow the cells to attach. A plate containing each of these cells was fixed in situ with trichloroacetic acid (TCA) in order to obtain the cell values at zero time before adding the compounds. The rest of the plates containing the different cell lines received serial dilutions of the compound to be tested at the following final concentrations: 4, 1, 0.25, and 0.0625 μg/ml. The plates were then incubated at 37°C in a 5% CO₂ and 95% air humidified atmosphere for 48 h. The assay was terminated by the addition of cold TCA. TCA-treated plates were incubated at 4°C for 1 h and then washed five times with tap water to remove TCA and air dried. Background optical densities were measured in wells incubated with growth medium without cells. TCA-fixed cells were stained for 20 min with 0.4% (wt/vol) SRB dissolved in 1% acetic acid. At the end of the staining period, unbound dye was removed by washing four times with 1% acetic acid. After air drying the plates, bound dye was solubilized with 10 mM Tris base (pH 10.5) and the absorbance was read on an automated plate reader at a wavelength of 550 nm. The GI₅₀ value was defined as the concentration of test sample resulting in a 50% reduction of the absorbance compared with that for the untreated controls that received a serial dilution of the solvent in which the test samples were dissolved and was determined by linear regression analysis. The optical density values obtained were used to determine the cell growth and cytotoxicity from each compound.

Ethics.

This study was approved by the Animal Ethics Committee of the Universidad Peruana Cayetano Heredia (approval identification code 41-07-16).