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. 2021 Apr 28;11(1):9161.
doi: 10.1038/s41598-021-88150-6.

A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics

Jessica Knox  1   2 , Nicolas Joly  3 , Edmond M Linossi  4 , José A Carmona-Negrón  4 , Natalia Jura  4   5 , Lionel Pintard  3 , William Zuercher  6 , Peter J Roy  7   8   9
Affiliations

A survey of the kinome pharmacopeia reveals multiple scaffolds and targets for the development of novel anthelmintics

Jessica Knox et al. Sci Rep. .

Abstract

Over one billion people are currently infected with a parasitic nematode. Symptoms can include anemia, malnutrition, developmental delay, and in severe cases, death. Resistance is emerging to the anthelmintics currently used to treat nematode infection, prompting the need to develop new anthelmintics. Towards this end, we identified a set of kinases that may be targeted in a nematode-selective manner. We first screened 2040 inhibitors of vertebrate kinases for those that impair the model nematode Caenorhabditis elegans. By determining whether the terminal phenotype induced by each kinase inhibitor matched that of the predicted target mutant in C. elegans, we identified 17 druggable nematode kinase targets. Of these, we found that nematode EGFR, MEK1, and PLK1 kinases have diverged from vertebrates within their drug-binding pocket. For each of these targets, we identified small molecule scaffolds that may be further modified to develop nematode-selective inhibitors. Nematode EGFR, MEK1, and PLK1 therefore represent key targets for the development of new anthelmintic medicines.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
A screen of vertebrate kinase inhibitors reveals 17 druggable nematode kinases. (A) Schematic of small molecule screening methodology. (B) The pipeline used to identify candidate nematode kinase targets for structure analysis.
Figure 2
Figure 2
Structure analysis identifies LET-23, MEK-2 and PLK-1 as candidate targets for anthelmintic development. C. elegans homology models (blue) for LET-23, MEK-2 and PLK-1 aligned with human crystal structure (green) for EGFR (PDB: 1XKK), MEK1 (PDB: 5EYM) and PLK1 (PDB: 2RKU) are shown in (A–C) respectively. Key divergent residues proximal to the inhibitor binding sites are highlighted in dark pink (C. elegans residue) and light pink (human residue) within the structure diagrams (A–C). Residues are labeled according to their position in the human kinase, with the first letter indicating the identity of the vertebrate residue and the latter indicating the identity of the nematode residue(s). The conservation of these residues among free-living nematodes, parasitic nematodes and vertebrates is displayed in the corresponding sequence alignments (A′–C′). The vertical line indicates a discontinuous break in the sequence. The horizontal line indicates the separation between the nematode and vertebrate sequences. Yellow residues in the sequence alignments highlight those nematode residues that differ in identity from both vertebrate and C. elegans sequence at the location of these divergent residues of interest. Residues that have distinct physicochemical properties between vertebrates and nematodes are indicated with an asterisk below the alignment.
Figure 3
Figure 3
The conserved C. elegans EGFR/Ras/MAPK pathway controls vulval induction signaling. (A) A schematic of the conserved EGFR signaling pathway in nematodes. The vertebrate orthologs for each pathway component are shown in brackets. Changes in signaling levels through this pathway result in the Vulvaless (Vul) and Multivulva (Muv) vulva induction phenotypes shown in (B). Filled arrows indicate primary functional vulva, clear arrows indicate ectopic vulval protrusions. Scale bar 0.1 mm.
Figure 4
Figure 4
Structural divergence in EGFR/LET-23 impacts response to EGFR inhibitor gefitinib. The Vulvaless phenotype observed in the let-23(sy1) mutant is rescued by the expression of a chimeric protein whereby the kinase domain of LET-23 is replaced with human EGFR (LET-23::hEGFR). Gefitinib induces a vulvaless phenotype in the humanized strain, but not in wild type animals. DMSO is used as the solvent control. Error bars indicate SEM.
Figure 5
Figure 5
Three scaffolds induce LET-23 loss-of-function phenotypes in C. elegans. (A) The structurally related anilino thienopyrimidine (ATOP), 4-anilino quinazoline (4AQ), and quinazoline benzimidazole (QBI) scaffolds induce LET-23 loss-of-function phenotypes in our C. elegans chemical screens. (B) Exemplar worm-active structures from each scaffold are shown along with the associated dose–response in wild type C. elegans. These dose–response analyses reveal phenotypes relevant to EGFR/MAPK pathway inhibition including sterility (Ste) and the bag-of-worms phenotype (Bag). Additional phenotypes including lethality (Let) and larval arrest (Lva) are shown and the resulting population growth defects are indicated by the colour coded scale (nb, no bacteria remaining in the well). Dose–response analyses for GSK306886A and GW583373A were performed in neutral media, GW576484X and GW272142A in acidic media. (C) Four worm-active inhibitors were tested for their ability to inhibit C. elegans LET-23 kinase activity in vitro. LET-23 inhibition is expressed as a the percentage of total kinase activity observed in no drug controls averaged across 3–5 replicates for each inhibitor condition. The ATOP and 4AQ scaffold compounds tested were able to inhibit LET-23 kinase activity (Student’s T-test; *p < 0.01). Error bars indicate SEM.
Figure 6
Figure 6
Allosteric MEK inhibitors induce sterility, embryonic lethality and vulva development phenotypes in C. elegans. 18 unique MEK inhibitors from the commercial libraries screened were included in our screen. These inhibitors were enriched for hits that induce the expected MEK-2 loss-of-function phenotypes including sterility (Ste) and the bag-of-worms phenotype (Bag), resulting from vulval induction defects preventing egg-laying (A-A′′, scale bar 0.1 mm). The resulting population growth defects are indicated by the colour coded scale (nb, no bacteria remaining in the well). (B) The allosteric inhibitor binding site of C. elegans MEK-2 (in blue) is well conserved with that of vertebrate MEK1 (in green, seen co-crystalized with allosteric inhibitor TAK-733 (PBD: 3PPI). The allosteric site contains only one amino acid difference, highlighted in pink (I99V).
Figure 7
Figure 7
Vertebrate allosteric MEK inhibitors target worm MEK-2, inducing loss-of-function phenotypes across nematode species. (A–C) Three structurally distinct allosteric MEK inhibitors suppress the Multivulva phenotype of upstream let-60(n1046) gain-of-function mutants, but have no effect on the Multivulva phenotype in downstream lin-1(e1275) loss-of-function mutants. The average number of vulva protrusions observed per worm in each condition quantified over 3 biological replicates is shown. Error bars indicate SEM. (A′–C′) Phenotypes induced by allosteric MEK inhibitors in free-living nematode species C. elegans, C. briggsae and P. pacificus. Sterility (Ste), embryonic lethality (Emb) and bag-of-worms (Bag) phenotypes are reported along with the resulting population growth defects indicated by the colour coded scale (nb, no bacteria remaining in the well).
Figure 8
Figure 8
ATP-competitive MEK inhibitor BI-847325 does not engage nematode MEK-2. (A) The ATP-competitive MEK inhibitor BI-847325 significantly suppresses vulva induction in Wild Type and both mutants at the highest concentration tested (120 μM) relative to solvent controls (Student’s T-test: p < 0.001). The average number of vulva protrusions observed per worm in each condition quantified over 3 biological replicates is shown. Error bars indicate SEM. (A′) BI-847325 induces sterility (Ste) and embryonic lethality (Emb) phenotypes in C. elegans and C. briggsae resulting in the population growth defects indicated by the colour coded scale (nb, no bacteria remaining in the well).
Figure 9
Figure 9
PLK1 inhibitors induce embryonic lethality and sterility phenotypes in nematodes. (A) PLK1 inhibitors from three core scaffolds including the 2,4-Dianilinopyrimidines (GSK1520489A), the 2,4-Dianilino pyrrolopyrimidines (GSK2220400A) and Benzimidazole N-thiophenes (GSK580432A, GSK479719A, GSK483724A, and GSK448459A) induce phenotypes consistent with loss of C. elegans PLK-1 including sterility (Ste) and embryonic lethality (Emb) resulting in the population growth defects indicated by the colour coded scale (nb, no bacteria remaining in the well). (B) The three Benzimidazole N-thiophene PLK1 inhibitors tested (GSK483724A, GSK479719A and GSK580432A) inhibit C. elegans PLK-1 kinase activity in vitro with IC50 values of 91 nM, 39 nM and 24 nM respectively.
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