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

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.

(B) Structure and alignment of MEK1 and MEK-2. Structure of PDB: 3PP1 (Human MEK1) in green with allosteric inhibitor TAK-733 bound (in orange) aligned to C. elegans MEK-2 homology model in blue above. Structure of PBD:5EYM (Human MEK1) with ATP-competitive inhibitor BI-847325 bound (in yellow) aligned to C. elegans MEK-2 homology model below. The purple arrow in the BI-847325-bound structure alignment highlights the C. elegans leucine residue that may sterically hinder BI-847325 from binding C. elegans MEK-2. This steric hindrance is further visualized in the surface representations of the Human MEK1 crystal structure and C. elegans MEK-2 homology model. The human and C. elegans kinase domains share 60% identity. All residues proximal to the ATP-competitive inhibitor binding site (identified in PDB structures 5EYM, 5HZE) and allosteric site (identified from PDB structures 3EQH, 3PP1) with side-chains facing inwards are highlighted on the alignment. The orthologous MEK-2 sequence from the parasitic nematode B. malayi was modeled to the human MEK-1 structures (PDB: 3PP1 above, PDB: 5EYM below) to confirm the favourable position of the identified divergent residues within the inhibitor binding site. Residues of interest are indicated on the structures in dark pink (nematode residue) and light pink (human residue). (C) Structure and alignment of PLK1 and PLK-1. Structure of PDB: 2RKU (Human PLK1) in green with BI2536 bound (in yellow), aligned to C. elegans PLK-1 homology model in blue. The human and C. elegans kinase domains share 64% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 2RKU, 3FC2, 2YAC, 4J52 and associated C. elegans homology models). The orthologous PLK-1 sequence from the parasitic nematode B. malayi was modeled to the human PLK1 structure (PDB:2RKU) to confirm the favourable position of the identified divergent residues within the inhibitor binding site. Residues of interest are indicated on the structures in dark pink (nematode residue) and light pink (human residue).  (D) Structure and alignment of BRAF and LIN-45. Structure of PDB: 5CSW (Human BRAF) in green with dabrafenib bound (in yellow), aligned to C. elegans LIN-45 homology model in blue. The human and C. elegans kinase domains share 62% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 5CSW, 5CT7 and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).

I463V
(E) Structure and alignment of AURKB and AIR-2. Structure of PDB: 5EYK (Xenopus laevis AURKB) in green with BI-847325 bound (in yellow), aligned to C. elegans AIR-2 homology model in blue. The human and C. elegans kinase domains share 64% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from human structure PDB: 4AF3 and X. laevis structure PDB: 5EYK and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (X. laevis residue). (F) Structure and alignment of MTOR and LET-363. Structure of PDB: 4JSX (Human MTOR) in green with Torin 2 bound (in yellow), aligned to C. elegans LET-363 homology model in blue. The human and C. elegans kinase domains share 62% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 4JSX, 4JSV and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).
(G) Structure and alignment of CDK1 and CDK-1 Structure of PDB: 4Y72 (Human CDK1) in green with inhibitor bound (in yellow), aligned to C. elegans CDK-1 homology model in blue. The human and C. elegans kinase domains share 67% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 4Y72, 6GU4 and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue). , aligned to C. elegans PMK-1 homology model in blue. The human and C. elegans kinase domains share 71% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 6SFO, 3ZS5, 1DI9, 1KV2 and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).

I146
(I) Structure and alignment of SRC and SRC-1. Structure of PDB: 6ATE (Human SRC) in green with inhibitor bound (in yellow), aligned to C. elegans SRC-1 homology model in blue. The human and C. elegans kinase domains share 63% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 6ATE, 4MXO and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).
Human SRC crystal structure Human residue C. elegans SRC-1 homology model C. elegans residue  All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 5FXS, 4D2R and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue). The human and C. elegans kinase domains share 56% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 6EIM, 4EQU and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue). The human and C. elegans kinase domains share 80% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 5MQV, 5OKT and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).

Nematodes
Vertebrates Nematodes Vertebrates 6 8 * I68 (M) Structure and alignment of GSK3B and GSK-3. Structure of PDB: 6HK3 (Human GSK3B) in green with inhibitor bound (in yellow), aligned to C. elegans GSK-3 homology model in blue. The human and C. elegans kinase domains share 82% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 6HK3, 5HLN and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue). (N) Structure and alignment of BUB1 and BUB-1. Structure of PDB: 6F7B (Human BUB1) in green with BAY-1816032 bound (in yellow), aligned to C. elegans BUB-1 homology model in blue. The human and C. elegans kinase domains share 30% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 6F7B, 4QPM and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).  (O) Structure and alignment of CSNK2A1 and KIN-3. Structure of PDB: 3R0T (Human CSNK2A1) in green with inhibitor bound (in yellow), aligned to C. elegans KIN-3 homology model in blue. The human and C. elegans kinase domains share 86% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structures 3R0T, 3NSZ and associated C. elegans homology models). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).   homology model in blue. The human and C. elegans kinase domains share 90% identity. All residues that are proximal to the inhibitor binding site with side-chains facing inwards are highlighted on the alignment (identified from PDB structure 6GZD and associated C. elegans homology model). Residues of interest are indicated on the structure above in dark pink (C. elegans residue) and light pink (human residue).  Compound ID R 1 R 2 R 3 R 4 R 5 X 1 X 2 X 3 X 4 pH=7 pH=4.5   Figure 4. Conservation of essential nematode-specific kinases across species. Matrices show the percent sequence identity between the kinase domain of the C. elegans essential nematode-specific kinase and that of the most similar kinase found across nematode species along with the kinase domain sequence of the best human kinase match and its ortholog across vertebrate species. C. elegans kinases from nematode-specific families (A-P) and nematode-expanded families (Q-Z) are shown. Sequence identity matrices for the kinase domain of the well-conserved kinases MEK1/MEK-2, PLK1/PLK-1 and EGFR/LET-23 are included for comparison (AA-AC). Kinase sequences were identified using NCBI BLASTP. Percent identity matrices were generated using Clustal Omega.  Figure 4. Conservation of essential nematode-specific kinases across species. Matrices show the percent sequence identity between the kinase domain of the C. elegans essential nematode-specific kinase and that of the most similar kinase found across nematode species along with the kinase domain sequence of the best human kinase match and its ortholog across vertebrate species. C. elegans kinases from nematode-specific families (A-P) and nematode-expanded families (Q-Z) are shown. Sequence identity matrices for the kinase domain of the well-conserved kinases MEK1/MEK-2, PLK1/PLK-1 and EGFR/LET-23 are included for comparison (AA-AC). Kinase sequences were identified using NCBI BLASTP. Percent identity matrices were generated using Clustal Omega. Sequence % Identity 0 100 Supplementary Figure 4. Conservation of essential nematode-specific kinases across species. Matrices show the percent sequence identity between the kinase domain of the C. elegans essential nematode-specific kinase and that of the most similar kinase found across nematode species along with the kinase domain sequence of the best human kinase match and its ortholog across vertebrate species. C. elegans kinases from nematode-specific families (A-P) and nematode-expanded families (Q-Z) are shown. Sequence identity matrices for the kinase domain of the well-conserved kinases MEK1/MEK-2, PLK1/PLK-1 and EGFR/LET-23 are included for comparison (AA-AC). Kinase sequences were identified using NCBI BLASTP. Percent identity matrices were generated using Clustal Omega.  Figure 4. Conservation of essential nematode-specific kinases across species. Matrices show the percent sequence identity between the kinase domain of the C. elegans essential nematode-specific kinase and that of the most similar kinase found across nematode species along with the kinase domain sequence of the best human kinase match and its ortholog across vertebrate species. C. elegans kinases from nematode-specific families (A-P) and nematode-expanded families (Q-Z) are shown. Sequence identity matrices for the kinase domain of the well-conserved kinases MEK1/MEK-2, PLK1/PLK-1 and EGFR/LET-23 are included for comparison (AA-AC). Kinase sequences were identified using NCBI BLASTP. Percent identity matrices were generated using Clustal Omega.  Figure 4. Conservation of essential nematode-specific kinases across species. Matrices show the percent sequence identity between the kinase domain of the C. elegans essential nematode-specific kinase and that of the most similar kinase found across nematode species along with the kinase domain sequence of the best human kinase match and its ortholog across vertebrate species. C. elegans kinases from nematode-specific families (A-P) and nematode-expanded families (Q-Z) are shown. Sequence identity matrices for the kinase domain of the well-conserved kinases MEK1/MEK-2, PLK1/PLK-1 and EGFR/LET-23 are included for comparison (AA-AC). Kinase sequences were identified using NCBI BLASTP. Percent identity matrices were generated using Clustal Omega.