Identifying and prioritizing the metabolic enzyme chokepoints in parasitic nematodes

The overall analysis process is outlined in Fig. 1 (for more details see Supplementary Fig. S1). A total of 669 unique ECs (Enzyme Commission IDs) were identified across 17 nematode species. Analysis of individually reconstructed metabolic networks led to identification of 389 ECs that were chokepoints in at least one species (Supplementary Table S1). Among these, 186 were taxonomically conserved20 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).

Figure 1 Flowchart outlining the overall analysis pipeline. ww = ”whipworm”. Full size image

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 ChEMBL19 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 pChEMBL19 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 PDB21 (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 sinensis22 and Taenia asiatica23. A wide variety of existing effective anthelmintics including Praziquantel, Levamisole, and Mebendazole have been found to result in LDH inhibition in trematode family Paramphistomidae24; (ii) Malate dehydrogenase (MDH; 1.1.1.37), inhibited by several major benzimidazole anthelmintics including Albendazole, although it is not their primary target25; (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 drugs26 and (iv) Dihydroorotate dehydrogenase (quinone) (DHODH; 1.3.5.2), studied as a target for protozoan (malaria and trypanosome) parasites27,28,29,30. 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-20th century as an anthelmintic31; (ii) Pantothenate kinase (PanK/CoA; 2.7.1.33), which is involved in CoA biosynthesis and possibly available at high levels in cells32 and (iii) 1-phosphatidylinositol 4-kinase (PI4K; 2.7.1.67), with several inhibitors under studies against malaria33 and Cryptosporidium34. 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 sites35; and Leucyl aminopeptidase (LAP-1/cathepsin III; 3.4.11.1), a potential drug target in Leishmania32, known to be an excretory/secretory (ES) product and a modulator of host immune system36, and previously studied as a potential vaccine candidate for the liver fluke, Fasciola hepatica37,38.

Table 1 Characteristics of the top 10 prioritized nematode chokepoint enzymes. Full size table

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) had deleterious 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).

Table 2 Compound efficacies in a phenotypic whole worm assay. * - Miconazole tested at 10 uM, not at 30 uM. ** - Worms looked very sick). Full size table

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.

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 ChemmineR105 package, and agglomerated using “complete linkage” method. Full size image

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.

Table 3 First and second generation active compounds and their targets in literature. Full size table

Screening of known azoles

Four of our active first-generation compounds were prioritized based on their known activities against MDH39,40 although they have numerous discrete canonical targets (Table 3). Gossypol is a polyphenol known to target lactate dehydrogenase41,42 and BCL2 family proteins involved in apoptosis and APE-1, which participates in DNA damage repair43,44,45. 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 membrane46,47. These azoles also display other activities, including inhibition of certain ion channels and receptors48,49. 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-demethylase48. 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 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, respectively50. 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 Omipalisib51 (105) and WYE-35452 (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-α53, 2. MLN1117 (Serabelisib; 125): selective p110α (a PI3K) inhibitor with an IC 50 of 15 nM54, and 3. XL388 (126): a highly potent ATP-competitive mTOR inhibitor with an IC 50 of 9.9 nM55 which simultaneously inhibits both mTORC1 and mTORC2. None of the three showed significant activity. Dual inhibitors such as PF-04979064 exist56 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 respectively57) in the worm assays and, subsequently, a more potent inhibitor Tadalafil58 (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 PDE1158. Subsequent screening of BC-11-38 (123), a selective PDE11 inhibitor60, 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.

Figure 3 Characterizing PDE as a potential target (a) Tadalafil docked to human (magenta) and A. ceylanicum (cyan) PDE5 homology model, showing the interacting residues. (b) Sequence alignment of PDE5 homologs from 6 worms (Ac = A. ceylanicum, Na = N. americanus, Ce = C. elegans, Bm = B. malayi, Ov = O. volvulus, Wb = W. bancrofti) and 3 paralogs each from the hosts human (Hs) and mouse (Mm). No homolog was identified in T. muris. The region shown here is selected to illustrate that most of the interacting residues from panel A are conserved among nematodes and divergent from host species (indicated with red asterisks). Residues at or near the active site that are different between filarial and non-filarial worms are highlighted with a black asterisk. The residue numbers are based on A. ceylanicum PDE5. The residues matching the aligned residue in A. ceylanicum are colored, with the color depicting the residue type. (c) A maximum-likelihood phylogenetic tree based on sequence similarity of PDEs present in A. ceylanicum, T. muris and C. elegans, and their expression levels in adult stages (FPKM – f ragments p er k ilobase per m illion mapped reads - plotted as bars in the outer track). PDE family clusters are indicated using shaded ellipses. The sequences were aligned using MAFFT106. The phylogenetic tree is estimated using PhyML 3.0107 and the node support values are calculated using “aLRT SH-like” option. All node support values are >=0.79. Full size image

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 parasites61, 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 purines62, complete or near-complete purine synthesis pathways were recently found in most members of clades I, IIIb (Ascaridomorpha) and V12. 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 sites35. 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 malaria63 and Staphylococcus aureus64. 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 activity65 and it was originally thought to be the possible target of benzimidazole anthelmintics, with Mebendazole shown to inhibit MDH activity in vitro66. Multiple enzymes from carbohydrate metbolism and glycolytic pathways, including MDH, have been studied for their potential as anthelmintic targets67,68,69. 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 drugs63,70,71,72,73,74,75,76,77,78,79,80.

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 anthelmintics81. 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).

Figure 4 Butaconazole, Tadalafil, Torin-1 reduced fecal egg count but not total worm load and rolipram did not reduce either fecal egg count or total worm load. (a) Treatment with each of the three drugs significantly reduced fecal egg count in Syrian hamsters infected with the hookworm A. ceylanicum compared with untreated control animals. (b) The fecal egg count reduction was not accompanied by a reduction of worm load, which was not statistically significant between control and treated animals. No reduction in fecal egg count (c) or worm burden (d) in Syrian hamsters infected with the hookworm A. ceylanicum when treated with rolipram compared to control animals. Full size image

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.