Figure 1.
Workflow for identification, characterization, and prioritization of chokepoint drug targets and drug-like compounds.
A. & B. The chokepoint compounds are shown in yellow. A “chokepoint reaction” either consumes a unique substrate or produces a unique product. In A., the chokepoint reaction (red) consumes a unique substrate (yellow). Five compounds are involved in reactions (blue) that produce the substrate for the chokepoint reaction. In B., the chokepoint reaction (red) produces a unique substrate, which is subsequently used in other reactions to create five new compounds (grey). C. Workflow diagram outlining the major steps in this study.
Figure 2.
Proteins with Enzyme Commission (EC) classification and chokepoint enzyme mapping.
Intersection of A. Proteins with EC classification and B. Chokepoint enzymes from CommNem, D. melanogaster, and H. sapiens. The 487 proteins that are referred to in the text from CommNem are derived from Figure 2A (7+465+15), and the 169 proteins from CommNem that are conserved chokepoints are derived from Figure 2B (5+163+1).
Figure 3.
Chemical structures of drug-like compounds and results from screening in C. elegans and H. contortus.
A. The drug-like compounds tested in the C. elegans, H. contortus, and O. lienalis screens. 1 (perhexiline; DB01074), 2 (DB00190), 3 (LT00255846 - DB00993 is a similar compound), 4 (DB00988), 5 (DB01032), 6 (DB01033), 7 (DB00548). B. Comparison of 8 (DB00190) from this study and 9 (Methyldopa), which was shown to inhibit L-DOPA decarboylase in S. mansoni. Dose-response curve for perhexiline (1) in C. C. elegans and D. H. contortus.
Table 1.
Prioritized list of drug-like compounds from DrugBank that can potentially be repositioned or further optimized for nematodes.
Figure 4.
Heatmap indicating enriched and depleted KEGG metabolic pathways.
The extreme blue color indicates that the enzyme category was significantly depleted and the extreme red color indicates the enzyme category was significantly enriched relative to either all chokepoint enzymes or all the EC values from KEGG using Fisher's Exact Test. The intermediate color shades indicate enrichment or depletion, but are not statistically significant. Enrichment or depletion of metabolic pathways in UniNem, CommNem, DrugBank, and KEGG Drug compared to A. AllKEGG and B. KEGGChoke. CommNem, intersection of nematode ECs; UniNem, set of all nematode ECs; KEGGChoke, chokepoint enzymes within KEGG; AllKEGG, all enzymes within KEGG; DrugBank, ECs from DrugBank; KEGG Drug, ECs from KEGG Drug.
Table 2.
Top prioritized chokepoint enzymes.
Figure 5.
Oxygen consumption rates in C. elegans after exposure to varying concentrations of Perhexiline (PER), Etomoxir (ETO) and Ivermectin (IVM).
A. Average basal oxygen consumption rates (OCR) of adult C. elegans incubated with vehicle (1% DMSO, 0 µM) or different concentrations of PER (CPT-2 inhibitor) (25, 50 and 100 µM) over 90 minutes. B. Average OCR of adult C. elegans incubated with vehicle (1% DMSO, Ctrl), ETO (CPT-1 inhibitor) (ETO, 50 and 100 µM), PER (50 and 100 µM), or 100 µM ETO plus 100 µM PER (ETO+PER) over 90 minutes. C. Average based OCR of adult C. elegans incubated with vehicle (1% DMSO, 0 µM) or different concentrations of IVM (binds to glutamate-gated chloride channels) over 40 minutes. D. Average OCR of adult C. elegans incubated with vehicle (1% DMSO, Ctrl), IVM (10 µM), PER (100 µM), ETO (100 µM), or 100 µM PER+100 µM ETO, over 40 minutes. Data are representative of at least 2 individual experiments. Bars represent the ±SEM of 15 OCR readings from 4 independent replicates per experiment. The experiment was repeated twice.
Figure 6.
Transcriptional response of C. elegans in the presence of Perhexiline (PER), Etomoxir (ETO) and Ivermectin (IVM).
A. Hierarchical clustering of samples based on gene expression patterns across all genes, and a heatmap based on differential expression profiles of 1,908 genes which were upregulated in at least one of the four samples relative to the control. B. Distribution of Gene Ontology enriched categories among the upregulated genes in each of the four samples. The list of GO categories is provided as Table S10.
Figure 7.
Docking of Perhexiline to rat carnitine palmitoyltransferase 2 and sequence alignment of carnitine palmitoyltransferase 2 from mammals and nematodes.
A. Part of the fatty acid metabolic pathway in KEGG that includes the chokepoint reaction (chokepoint enzyme 2.3.1.21, with its substrate (L-Palmitoylcarnitine) and product (Palmitoyl-CoA)) shown in red. Perhexiline is believed to bind to 2.3.1.21. B. Docked structure of perhexiline (green) to CPT-2 (PDB ID: 2H4T), superimposed onto a CPT-2 structure with a bound drug, ST1326 (yellow) (PDB ID: 2FW3). Residues that differ between mammals and nematodes are shown in gray (L335, S445, Q447, V597, S598, L599, A615, W620, C623, N624), and the catalytic H372 is shown in orange. C. Interactions perhexiline make with CPT-2 (PDB ID: 2H4T) (P133, F134, M135, H372, D376, G377, V378, L381, S590, L592, G601). D. The C. elegans protein was used to find similar mammalian sequences with BLASTP and the non-redundant (NR) database. Residues shown in gray in B are labeled with red asterisks below the sequence. The alignment (using MUSCLE) of the following proteins is shown: gi|294805368|gb|ADF42518.1 (S. scrofa), gi|296489058|gb|DAA31171.1 (B. Taurus), gi|4503023|ref|NP_000089.1 (H. sapiens), gi|162138915|ref|NP_034079.2 (M. musculus), gi|1850592|gb|AAB48047.1 (R. norvegicus), 2FW3 chain A, Tsp_06820 (T. spiralis), Mh10g200708_Contig108_46414_50093 (M. hapla), prot_Minc00582 (M. incognita), R07H5.2a (C. elegans), gi|308491342|ref|XP_003107862.1 (C. remanei), gi|341894296|gb|EGT50231.1 (C. brenneri), gi|324506871|gb|ADY42921.1 (A. suum), 14424.m00388 (B. malayi).
Figure 8.
KEGG metabolic pathways containing chokepoint reactions for which a drug-like compound showed activity.
Chokepoint reactions (chokepoint enzymes, with its substrate and product) are highlighted in red. A. Pathway Maps for Steroid Hormone Biosynthesis (ko00140). Chokepoint enzyme 1.3.99.5 is involved in a chokepoint reaction in this pathway. 5α-Androstane-3,17-dione (C00674) and 5α-Dihydro-testosterone (C03917) are chokepoint compounds. Azelic acid (drug-like compound 7, DB00548) targets 3-oxo-5-alpha-steroid 4-dehydrogenase (EC 1.3.99.5) as well as other targets. Exposing C. elegans to azelic acid led to a movement-impaired phenotype. B. Pathway Maps for Tryptophan Metabolism (ko00380). Enzyme 4.1.1.28 is involved in a chokepoint reaction in this pathway. 5-Hydroxykynurenamine (C05638), Serotonin (C00780), and Trypamine (C00398) are chokepoint compounds. Carbidopa (drug-like compound 2, DB00190) targets DOPA decarboxylase (EC 4.1.1.28). Exposing C. elegans to carbidopa produced a movement-impaired phenotype.