Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.

). Among these, 186 were taxonomically conserved 20 across at least Trichuris muris (Clade I), Ancylostoma ceylanicum (Clade V) (the two species used for screening -"target species") and at least one other Clade III or Clade IVa species (to ensure broad nematode conservation).
These 186 chokepoint ECs were ranked based on a weighted scoring method (Supplementary Fig. S1; Methods) which included phylogenetic conservation, number of metabolic pathways they are involved in, RNAi phenotype of the C. elegans ortholog, previous identification in literature, and RNAseq based expression levels in especially relevant developmental stage(s). Every nematode chokepoint from the two target species -intestinal nematodes representing the phylogenetic extremes of the phylum Nematoda (clade I, the whipworm T. muris and clade V, the hookworm A. ceylanicum) -was linked to 'drug-like' compounds in the ChEMBL 19 database based on similarity to ChEMBL targets, resulting in 448 and 606 unique ChEMBL targets corresponding to 664 A. ceylanicum proteins and 756 T. muris proteins, respectively. Using pChEMBL 19 values a total of 188,454 target:compound pairs were identified (pChEMBL score ≥5, corresponding to 10 μM IC 50 etc.). Of these, 83,134 pairs included both a gene with an EC assignment and a compound with a "Quantitative Estimate of Druglikeness" (weighted QED) score recorded in the database. Intersection of these with the 186 selected nematode chokepoint ECs resulted in 22,498 conserved chokepoint ChEMBL target:compound pairs (82 targets/64 ECs paired to 12,395 unique compounds). The final ChEMBL target:compound scores were calculated, prioritizing target:chokepoint pairs that have high drug likeness and high target affinity. Multiplying the final nematode chokepoint EC score and the final ChEMBL target:compound score ( Supplementary Fig. S1C) resulted in the final prioritization score. Thereafter, the 22,498 scored compound:target pairs (64 ECs) were reduced to 11,869 pairs (50 ECs) with a PDB 21 (Protein Data Bank) structural match to the target. In the final step, the top 3 target:compound pairs per EC were selected (142 total target:compound pairs, 128 compounds; Supplementary Fig. S1D, Supplementary  Tables S2, S3), and these were ranked according to the final prioritization score.
Out of the top 50 ECs, 10 ECs were prioritized based on associated compound availability and guided by literature review of the EC functions and potential for biological efficacy (Table 1). These ECs were categorized into five classes. The first class of target ECs was dehydrogenases, and included (i) L-lactate dehydrogenase (LDH; 1.1. 1.27), previously suggested to be a potential anthelmintic target in Clonorchis sinensis 22 and Taenia asiatica 23 . A wide variety of existing effective anthelmintics including Praziquantel, Levamisole, and Mebendazole have been found to result in LDH inhibition in trematode family Paramphistomidae 24 ; (ii) Malate dehydrogenase (MDH; 1.1.1.37), inhibited by several major benzimidazole anthelmintics including Albendazole, although it is not their primary target 25 ; (iii) Aldehyde dehydrogenase (NAD+) (ALDH; 1.2.1.3), previously explored as a potential target due to its impact on alcohol tolerance and cancer chemotherapy resistance. ALDH may be related to broad based drug resistance, and its inhibition might facilitate efficacy of other drugs 26 and (iv) Dihydroorotate dehydrogenase (quinone) (DHODH; 1.3.5.2), studied as a target for protozoan (malaria and trypanosome) parasites [27][28][29][30] . The second class of EC targets was kinases, and included (i) Hexokinase (HK; 2.7.1.1), a possible target of phenothiazine, which had been widely used in the mid-20 th century as an anthelmintic 31 ; (ii) Pantothenate kinase (PanK/CoA; 2.7.1.33), which is involved in CoA biosynthesis and possibly available at high levels in cells 32 and (iii) 1-phosphatidylinositol 4-kinase (PI4K; 2.7.1.67), with several inhibitors under studies against malaria 33 and Cryptosporidium 34 . The other classes were represented by one EC each, and included Fructose-bisphosphatase (FBPase; 3.1.3.11), a key enzyme in gluconeogenesis that has not been identified as a target in helminths; 3′,5′-cyclic-nucleotide phosphodiesterase (PDE; 3.1.4.17), inhibitors for which have been suggested to be good potential drugs for targeting parasitic nematodes due to their ability to disrupt the C. elegans life cycle and nematode-specific active binding sites 35 ; and Leucyl aminopeptidase (LAP-1/cathepsin III; 3.4.11.1), a potential drug target in Leishmania 32 , known to be an excretory/secretory (ES) product and a modulator of host immune system 36 , and previously studied as a potential vaccine candidate for the liver fluke, Fasciola hepatica 37,38 . First generation compound screening with parasitic nematodes in a phenotypic motility assay. To test the knowledge-based compound predictions for the 10 prioritized ECs (15 ChEMBL targets; 2,167 compound:target pairs; 1,581 drugs), 94 compounds were purchased and assayed in adult hookworm. Eleven (Table 2)  www.nature.com/scientificreports www.nature.com/scientificreports/ effects in hookworm (Motility Index, MI ≤0.75 for severe phenotype, 0.76 to 1.8 for moderate phenotype or had noticeable phenotype; 1.81 to 3, no phenotype) and were putatively targeting six of the 10 enzymes (success rate of 60% based on the target numbers), representing 3 different enzyme classes (2 dehydrogenases, 3 kinases, and a phosphodiesterase; Table 2). The 11 compounds causing severe or moderate phenotype in hookworm were also assayed in whipworm and seven were hits (i.e. had moderate to severe motility inhibition), hence classified as potential pan-intestinal compounds. Four of the seven compounds were potential pan-intestinal hits resulting in severe phenotypes (MI < 1.3) in both species, and two (33 and 69) had severe phenotype only in whipworm. One compound (9) showed moderate/ noticeable phenotype in both species. Based on our analysis ( Supplementary Fig. S1) the active compounds were linked to drug-like compounds shown to target MDH, PDE and PI4K/PI3K/mTOR in other species. The eight compounds (out of the 11 hits) associated with these enzymes ( Table 2) were screened in non-intestinal nematode, adult stage of the filarial nematode Brugia pahangi, to examine their pan-phylum potential. Four (15,47,58,59) were effective at day 2, and 3 additional (20, 30, 33) at day 6 in B. pahangi, hence showing pan-Nematoda potential (motility inhibition of at least 80% at day 2 or day 5 was used as a cut-off for activity).
Identification of second generation compounds with pan-phylum potential and screening with parasitic nematodes in a phenotypic motility assay. Based on the results from the 94 first generation compounds we identified three effective target classes -MDH, PDE and PI4K/PI3K/mTOR -and expanded the compound list by structural similarity and pharmacophore searches, resulting in identification and screening of 26 second generation compounds (Supplementary Table S4). This compound expansion and screening in the intestinal and filarial species led to the identification of four (102, 105, 108, 113) further active potential pan-intestinal molecules, of which two (102 and 105) have pan-Phylum molecules (Fig. 2, Supplementary Table S4) in this series. The putative target-associated compounds that were not active in this assay, were not necessarily structurally related to the first-generation inhibitors.
Overall the screening of the first and second generation compounds resulted in identification of twelve active compounds that putatively target three homologous therapeutic targets -MDH, PI3K/mTOR and PDEs (Table 3) in at least one parasitic species.
Screening of known azoles. Four of our active first-generation compounds were prioritized based on their known activities against MDH 39,40 although they have numerous discrete canonical targets (Table 3). Gossypol is a polyphenol known to target lactate dehydrogenase 41,42 and BCL2 family proteins involved in apoptosis and APE-1, which participates in DNA damage repair [43][44][45] . The other three were azoles (Miconazole, Econazole and Sulconazole), which are imidazole derivatives known to be antifungal agents. They inhibit P-gp and multiple CYP proteins. One of the CYP proteins (14 alpha-demethylase) is thought to be the main target that leads to antifungal activity by inhibiting synthesis of ergosterol, an important steroid necessary for permeability of the fungal cellular membrane 46,47 . These azoles also display other activities, including inhibition of certain ion channels and receptors 48,49 . We included Butaconazole, also an azole, among our second-generation compounds based on the successful motility inhibition shown by these three azoles. Like the other three azoles, Butaconazole is also predicted to inhibit 14 alpha-demethylase 48 . In our in vitro assays, Sulconazole had IC 50 s of 11.8 µM against the whipworm T. muris and 4.6 µM against the filarial worm B. pahangi.

Screening of known PI3K, PI4K lipid kinases and mTOR inhibitors.
Our discovery that the mixed mTOR/PI3K/PI4K inhibitor Torin1 (15) showed efficacy in inhibiting worm motility in both hookworms and whipworms prompted us to test other known and more selective inhibitors of each of these kinases. The high  www.nature.com/scientificreports www.nature.com/scientificreports/ prioritization score of 15 was based on its high potency as an inhibitor of mTOR with an IC 50 of 2 nM and 10 nM in the protein complexes mTORC1 and mTORC2, respectively 50 . In the same study, it exhibited 1000-fold selectivity of mTOR over PI3K in mammalian cells. In our assays, the IC 50 for 15 was 13.3 µM in the whipworm T. muris and 3.9 µM in the filarial worm B. pahangi. Intriguingly, the structurally related inhibitor Torin-2 (23) did not exhibit similar activity, nor did the other PI4K kinase inhibitors (95, 106, 109, 116, and 120) ( Supplementary  Table S4). Therefore, we identified and evaluated additional mTOR inhibitors and found that Omipalisib 51 (105) and WYE-354 52 (113) showed equivalent or better activity against both these nematodes (Fig. 2). Interestingly, 105 showed better activity than 113, a more selective inhibitor of mTOR. Because the structurally related inhibitor Torin-2 did not exhibit activity and Omipalisib did, we screened more selective PI3K inhibitors and additional mTOR inhibitors to help better indicate the mechanism of action ( Supplementary Fig. S3). The three additional drugs were: 1. CC-223 (124): a potent, selective, and orally bioavailable mTOR inhibitor with IC 50 of 16 nM, >200-fold selectivity over the related PI3K-α 53 , 2. MLN1117 (Serabelisib; 125): selective p110α (a PI3K) inhibitor with an IC 50 of 15 nM 54 , and 3. XL388 (126): a highly potent ATP-competitive mTOR inhibitor with an IC 50 Figure 2. Structural similarity of the 17 active compounds, identified in the primary, secondary and tertiary screen of compounds for the identified targets. The clustering was based on (1 − Tanimoto similarity measure) as distance metric, calculated using ChemmineR 105 package, and agglomerated using "complete linkage" method.  www.nature.com/scientificreports www.nature.com/scientificreports/ of 9.9 nM 55 which simultaneously inhibits both mTORC1 and mTORC2. None of the three showed significant activity. Dual inhibitors such as PF-04979064 exist 56 and testing them can provide further clarification.

Screening of known PDE inhibitors.
We identified a weak PDE1/PDE5 inhibitor (69) (IC 50 1.9 µM/0.7 µM respectively 57 ) in the worm assays and, subsequently, a more potent inhibitor Tadalafil 58 (108) among the second-generation compounds. The additional seven PDE inhibitors tested (Supplementary Table S4) are from several chemical series, which encompass varied PDE family selectivity profiles. Intriguingly, Tadalafil was the most active PDE inhibitor, while other classes including Sildenafil (110) and Vardenafil (89) did not show activity against the representative hookworm or whipworm (Supplementary Table S4). The compounds we tested so far are largely selective for PDE5, PDE1 and PDE6 although our lead Tadalafil also shows significant activity for PDE11 (IC 50 15 nM) 59 , for which a direct ortholog is not found in nematodes even though PDE5 is the most closely related nematode enzyme to mammalian PDE11s (Supplementary Fig. S4). Tadalafil is a PDE5 inhibitor with IC 50 of 9.4 nM and was at least 1000 times more selective for PDE5 than most of the other families of PDEs, with the exception of PDE11 58 . Subsequent screening of BC-11-38 (123), a selective PDE11 inhibitor 60 , showed no activity against whipworm (Supplementary Table S4). To further explore the Tadalafil scaffold, and to test the hypothesis that we are hitting PDE (and that worm PDE is similar to human), Tadalafil and N-cyclopropyl Tadalafil were docked into A. ceylanicum PDE5 homology model (Tadalafil docking shown in Fig. 3a). Acknowledging that uptake may be differentiated, we chose two compounds N-Desmethyl Tadalafil (121) and N-Desmethyl N-cyclopentyl Tadalafil (122) with varying ClogP (1.864 and 3.7944, respectively) for screening in whipworm. Both compounds showed activity (Supplementary Table S4). We note that most of the residues important for interactions with Tadalafil are conserved among Nematoda, but are divergent from the hosts (Fig. 3b). Additional studies are needed to determine the target, in light of the difference in PDE geneset among species (e.g. lack of PDE5 and two additional PDE2 in whipworm; Supplementary Fig. S4) and the PDE expression profiles in adult worms ( Fig. 3c; Supplementary Table S5). Both 69 and 108 were not active against the filarial nematode B. pahangi. Taxonomically restricted residues in PDE5 exist among intestinal and filarial nematodes (Fig. 3c) and further focused studies are needed to determine their role in the observed differential activity.
Putative nematode targets are involved in diverse metabolic pathways. The compounds that caused the most severe phenotypes putatively target three nematode enzymes that are involved in six metabolic pathways. PIK (EC2.7.1.67) and PDE (EC3. 1.4.17) are involved in inositol phosphate metabolism and purine metabolism, respectively and MDH (EC1.1.1.37) is involved in four other pathways (described below).
PIK is a transferase that transfers phosphorus-containing groups (phosphotransferases with an alcohol group as acceptor) which is involved in carbohydrate metabolism (inositol phosphate metabolism). A previous study has identified another enzyme in the inositol phosphate metabolism pathway, inositol polyphosphate multikinase (IPMK, EC2.7.1.151), as a druggable and lethal target in kinetoplastid parasites 61 , showing vulnerability of this pathway in other parasites.
PDEs are hydrolases that cleave ester bonds (phosphoric-diester hydrolases) and are involved in purine nucleotide metabolism. Despite the widespread notion that nematodes cannot synthesize purines 62 , complete or near-complete purine synthesis pathways were recently found in most members of clades I, IIIb (Ascaridomorpha) and V 12 . This critical function highlights purine metabolism as a promising drug target, as evidenced here by the efficacy of targeting PDE; notably, inhibitors of PDE have been suggested as promising potential drugs against parasitic nematodes given their ability to disrupt the C. elegans life cycle and existence of nematode-specific active binding sites 35 . Ten other high priority chokepoints are part of the purine metabolism pathway (Supplementary Table S6). For example, previously we have prioritized pyruvate kinase (EC 2.7.1.40), an enzyme upstream of PDE, as a top promising conserved chokepoint drug target, 1 and due to its critical function and diversification in the active site compared to the human host it has also been identified as a promising drug target for malaria 63 and Staphylococcus aureus 64 . Pyruvate kinase is also a chokepoint in pyruvate metabolism pathway.
MDH is an oxidoreductase that acts on the CH-OH group of donors with NAD+ as acceptor. It is involved in three carbohydrate metabolism pathways (pyruvate, glyoxylate/dicarboxylate, and TCA cycle) and also in amino acid metabolism (cysteine and methionine). It is essential to energy production through both aerobic and anerobic pathways, making it essential for parasite's survival even in very diverse niches. Some anthelmintics appear to target MDH activity 65 and it was originally thought to be the possible target of benzimidazole anthelmintics, with Mebendazole shown to inhibit MDH activity in vitro 66 . Multiple enzymes from carbohydrate metbolism and glycolytic pathways, including MDH, have been studied for their potential as anthelmintic targets [67][68][69] . There are 7 other enzymes among our top 50 chokepoints list (Supplementary Table S2) that are also part of pathways that involve MDH (4 in pyruvate metabolism and 4 in cysteine/methionine metabolism; one enzyme -EC 1.1.1.27 -is involved in both these pathways). Many of these have been suggested and explored as potential anti-parasitic, antifungal or anthelmintic drugs 63,70-80 . In vivo efficacy of lead compounds. To ascertain whether the newly discovered classes of pan-nematode chokepoint inhibitors possessed therapeutic utility in parasitic nematodes, Syrian hamsters (Mesocricetus auratus) infected with A. ceylanicum were treated with representative drugs from each of the three target classes at 100 mg/kg per os, based on previously reported effective dosage of currently used major anthelmintics 81 . Three (Butaconazole, Tadalafil and Torin-1) of the four tested compounds that were active in vitro had significant and noteworthy impact on eggs per gram of feces indicating treatment affected parasite fecundity, although the treatment had no marked effect on worm burden (Fig. 4a,b). Rolipram, a drug that was not active in vitro, did not have an impact on eggs per gram or on worm burden (Fig. 4c,d). www.nature.com/scientificreports www.nature.com/scientificreports/ In conclusion, omics-driven knowledge-based identification of drug targets is a powerful approach to identify targets essential for organisms survival. Undertaking this approach we were able to prioritize 10 of the 186 identified conserved chokepoint enzymes and undertake a target class repurposing approach to test and identify drugs with broad spectrum anthelmintic activity. First, we tested 94 compounds using an in vitro phenotypic assay, and identified 11 actives. Based on the findings and SAR, we tested 32 additional compounds, identifying 6 more actives. Among the 17 actives 5 are potential pan-intestinal and 6 are potential pan-Nematoda drugs. The active compounds target three different target classes. Using representative drugs from each target class, we demonstrated efficacy in hamsters infected with hookworm, as characterized by negative effects on parasite fecundity. While the leading scaffolds require optimization, the study identified target classes and essential pathways for parasitic nematode survival.

Materials and Methods
Enzyme annotation, nematode chokepoints identification and taxonomic classification. High confidence annotation of nematode metabolic enzymes was performed as described 12 . In short, the multi-step process assigns Enzyme Commission (EC) numbers to sequences in proteomes of interest. Multiple EC-annotation approaches that were employed included KAAS 82 12 . Second, chokepoint ECs were assigned a score with a maximum value of 6, based on five different criteria: (i) ECs with higher levels of pan-Nematoda conservation received a higher score, calculated using (2× [Number of species/17]), with a maximum value of 2; (ii) Chokepoints annotated to multiple KEGG 95 pathways received a higher score, calculated using (number of pathways/4), to a maximum value of 1; (iii) If the C. elegans ortholog has a "lethal" or "sterile" phenotype annotated by WormBase 96 , it received 1 point; (iv) The chokepoints among the top 25% targets scored in the "Comparative genomics of the major parasitic worms" study (International Helminth Genomes Consortium 12 ) received a score calculated by (score/137), maximum value 1. This score was based on multiple factors, including lack of human homolog, presence of a C. elegans or Drosophila melanogaster homolog with a deleterious phenotype, expression in key life cycle stages, widespread conservation among helminths, lack of paralogs, presence of a related structure in PDB, etc. The top quartile among all the scored proteins were given a positive score by us, as they represented potentially good anthelmintic targets; and (v) RNA-Seq-based gene expression levels were used to prioritize ECs based on data from A. ceylanicum (0.5 x the percentile of the EC's adult-stage expression compared to all other genes) and T. muris (0.5 x the percentile of the EC's [adult stage/free-living larval stages] fold change value compared to all other genes), with a maximum value of 1. The distribution of all of the contributing scores and the final scores are shown in Supplementary Fig. S2.
For the ChEMBL target:drug pair prioritization, ChEMBL 19 targets were annotated among the two target species (minimum 40% identity across 75% of the protein's length, P ≤ 10 −10 ) and subsequently ChEMBL was used to match drugs, identifying target:drug pairs with a pChEMBL score ≥5. The drugs that had a "Quantitative Estimate of Druglikeness" (weighted QED) score available and were associated with one of the prioritized 186 nematode metabolic chokepoints were identified. The final ChEMBL target:drug scores were calculated by multiplying (i) the pChEMBL drug:target affinity scores, scaled between 0.5 and 1, and (ii) the weighted QED scores, scaled between 0 and 1. Final prioritization score was calculated by multiplying the final nematode chokepoint EC score and the final ChEMBL target:drug score ( Supplementary Fig. S1). Last, significant sequence similarity to either a human or a mouse protein with a structure in PDB (sequence match with an E value < 10 −10 ) was required for a drug:target pair to be included for further consideration.
For each of the 50 target ECs with available PDB structure, the nematode sequence was aligned with that of the PDB structure using MOE (Chemical Computing Group, Inc.). A two-step visual inspection of the ligand binding site ranked targets based on (i) identification of a 'druggable' binding site 97 , with compound bound where possible, and (ii) protein sequence differences between the nematode and host sequences in the region of the binding site (this step was preceeded by manual improvement of the nematode gene models guided by transcriptional evidence). This process resulted in 10 priority targets.

Identification of small molecule inhibitors.
First generation compounds: For the 10 prioritized EC targets, we identified all reported active compounds in ChEMBL. These were assessed for drug-likeness; PAINS 98 were eliminated. Of these, 94 compounds were available for purchase. Both hookworm and whipworm assays were carried out for an initial batch of 30 compounds. Based on the results of these, it was decided to employ a stepwise approach for the remaining first generation compounds i.e. hookworm, then whipworm, then Brugia, contingent on activity in the previous step(s). Second generation compounds: Based on the results from 94 first bars in the outer track). PDE family clusters are indicated using shaded ellipses. The sequences were aligned using MAFFT 106 . The phylogenetic tree is estimated using PhyML 3.0 107 and the node support values are calculated using "aLRT SH-like" option. All node support values are >=0. 79. www.nature.com/scientificreports www.nature.com/scientificreports/ generation compounds, five proteins were reasoned as the potential biologically relevant targets for our active hits and thus more readily commercially available compounds known to target each of these were included (n = 26). These were five PI4K/PI3K inhibitors, nine mTOR inhibitors, five PDE inhibitors, two potential malate dehydrogenase inhibitors, and five Econazole analogs. The mTOR inhibitors were included based on CHEMBL1256459 (Torin1) -a potent inhibitor of mTORC1/2. Third generation compounds: Based on the results from the 26 second generation compound, additional compounds were identified and screened (n = 6). These included three PDE inhibitors, two mTOR and one PI3K (p110α) inhibitor.

Compound screening with adult stage of intestinal nematode Ancylostoma ceylanicum. In
vitro assays were carried out as described 81 . Briefly, young adult A. ceylanicum were harvested from the small intestine of infected hamsters on day 11 post-inoculation (p.i.) and washed three times using prewarmed medium (RPMI1640, 100 U penicillin, 100 μg/ml streptomycin, 10 μg/ml amphotericin). Three worms were manually picked into each well of the 96-well, 4 wells/test drug, containing 99 μl of medium. 1 μl of 3 mM (100% DMSO) drugs were transferred into the corresponding well giving a final concentration of 30 µM (1% DMSO) and 100 µl final volume. Assay plates were incubated for 48 hours at 37 °C and 5% CO 2 . Drug activity was determined every 24 hours using the standard motility index. Motility index of 3 were given to vigorous worms, 2 for motile worms, 1 for motile after stimulation by touching and 0 for dead worms. Pyrantel pamoate 30 µM was used as positive control prepared exactly as the test drugs, while 1% DMSO was used as negative control.
Compound screening with adult stage of intestinal nematode Trichuris muris. In vitro assays were carried out as described 81 . Briefly, T. muris adult whipworms were harvested from the cecum and large intestines of infected STAT6 −/− mice. Three worms were manually picked into each well of a 24-well plate, 2 wells/ test drug, containing 990 µl. 10 µl of 3 mM (100% DMSO) drugs were transferred into the corresponding well giving a final concentration of 30 µM (1% DMSO) and a final volume of 1 ml of medium. Test plates were incubated for 48 hours at 37 °C and 5% CO 2 . Drug activity was assayed using the standard motility index as per hookworm adults. Levamisole 30 µM was used as positive control prepared exactly as the test drugs, while 1% DMSO was used as negative control. IC 50 values at 48 hours were established using a non-linear regression with Prism 7 (Graphpad, La Jolla, CA). Only IC 50 s displaying R 2 values ≥ 0.7 are reported.
Compound screening with adult stage of the filarial nematode Brugia pahangi. Adult female B. pahangi worms were obtained from Dr. Brenda Beerntsen, University of Missouri, Columbia, MO. Individual females were placed in each well of a 24-well plate in culture medium (RPMI-1640 with 25 mM HEPES, 2.0 g/L NaHCO3, 5% heat inactivated FBS, and 1X Antibiotic/Antimycotic solution.
Initial screening of the compounds was performed at 10 μM for Miconazole, Econazole, and Sulconazole. Gossypol, Torin2, PP121, and Torin1 were initially screened at 30 μM and 10 μM. Four worms were used as replicates at each concentration in 500 uL of media. Worms treated only with 1% DMSO served as the negative control. The cultures were maintained at 37 °C, 5% CO 2 incubator for seven days -the duration of the assay.
The Worminator, a visual imaging software, was used to determine the effect of each treatment on worm motility 99 . Movements of individual worms were calculated by determining the number of pixels displaced per second per well. Worm movements were averaged over the time of recording (60 seconds) and mean movement units (MMUs) were determined for individual worms. Percent inhibition of motility was calculated by dividing the MMUs of the treated worms by the average MMUs of the 1% DMSO treated control worms, subtracting the value from 1.0, flooring the values to zero and multiplying by 100%. Videos of the worms during the assay were www.nature.com/scientificreports www.nature.com/scientificreports/ recorded on days 0, 1, 2, 3, and 6 of the incubation. Compounds showing ≥ 75% inhibition of motility at either concentration on day 3 of the assay were investigated further to determine IC 50 s. Worms were treated in the same fashion as above and motility was measured each day for three days using the Worminator. IC 50 values on day 3 (Torin 1 and Sulconazole) were established using a non-linear regression curve fit with Prism 7 (Graphpad, La Jolla, CA). All IC 50 s with R 2 values ≥ 0.7 are reported.
In vivo A. ceylanicum assay. Assays were conducted essentially as previously 81,100 . Briefly, 5 weeks old golden Syrian hamsters were infected with 120 third-stage larvae of A. ceylanicum. Infected rodents were grouped based on the fecal egg count on day 18 p.i. Each group had five hamsters. Drugs were delivered in water suspension after completely dissolved in 100% DMSO giving a final dose of 100 mg/kg and 0.1% DMSO volume to body weight. Control group was dosed only with water and 0.1% DMSO. Fecal samples were collected 21 days p.i. for fecal egg counts. On day 22 p.i., the hamsters were euthanized, and the small intestine was opened longitudinally. Adult parasites within the intestine were counted under a dissecting microscope.
A 100 mg/kg dosing for in vivo experiments was chosen based on similar studies from other groups as maximum single dosing for anthelmintic compounds 81,[101][102][103] . For comparison, with current clinically approved anthelmintics high efficacy is seen with doses at 5-10 mg/kg 103

Data Availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.