Table 1.
Targets selected for yeast-based screens.
Table 2.
B. malayi targets selected for our screening pipeline (column 1); yeast and human orthologs (columns 2 and 3, respectively); outcome of the cloning efforts of B. malayi (Bm) or human (Hs) genes (columns 4 and 5); outcome of the functional complementation in yeast (columns 6 and 7), where % complementation = 100 * growth score of strains with the heterologous target/ growth score of wild-type strains; source of coding region for each clone (column 8).
Fig 1.
CLUSTAL 2.1 multiple sequence alignment of diphospho mevalonate decarboxylases from Brugia malayi (BmMVD), Loa loa (LlMVD), Homo sapiens (HsMVD) and Saccharomyces cerevisiae (ScMVD), highlighting two regions in the published BmMVD sequences that diverge from the consensus between the orthologous protein sequences.
These could be due to the presence of different splice variants of the Brugia enzyme, natural insertions or errors in the published sequence.* conserved residues;: chemically conserved changes;. non-conserved changes.
Fig 2.
Partial CLUSTAL 2.1 multiple sequence alignment of publicly available Brugia malayi (Bm1_48165), Homo sapiens (HsSAH), Saccharomyces cerevisiae (ScSAH), and our cloned Brugia malayi (BmSAH) S-adenosyl homocysteinases, showing the “insert” missing from the publicly available nematode protein sequence.
The absence of the 27 amino acids in the publicly available Brugia sequence (Bm1_48165) could indicate a splice variant of the enzyme or simply a problem with the genome assembly.
Fig 3.
Functional complementation of yeast deletions by Brugia malayi (Bm) or Homo sapiens (Hs) orthologous genes.
Relative growth of yeast strains expressing the parasite or human target compared to the growth of yeast strains expressing the native gene. Blue bars indicate the full expression of the heterologous genes from the TetO2 promoter, and red and green bars indicate the growth of strains with a reduced expression of the essential enzyme.
Fig 4.
Scatter plot showing no correlation between the percentage identity between the human (Homo sapiens) or Brugia malayi proteins that do or do not complement the essential functions of the yeast (Saccharomyces cerevisiae) orthologues.
The average protein identity (in %) between heterologous proteins able to complement the yeast deletions was 52.5 ± 10.6, whereas the identity between heterologous proteins NOT able to complement the yeast deletions was 48.1 ± 10.0.
Table 3.
List of compounds that specifically inhibit the parasite targets at 10 μM: Bm = Brugia malayi, Sm = Schistosoma mansoni, NMT = N-myristoyl transferase, PGK = phosphoglycerate kinase, PIS = inositol 3-phosphatidyltransferase, SAH = S-adenosylhomocysteinase.
Smiles, or simplified molecular-input line-entry system, is a line notation for describing the structure of different chemical species. Ratio indicates the specificity of the compound for the parasite target = growth score for yeast strains expressing the parasite enzyme/growth score for the yeast strains expressing the human counterpart, Hence, a ratio of 1 indicates the absence of discrimination between parasite and human target, a ratio >1 indicates that the human enzyme is inhibited more than the parasite enzyme, and a ratio <1 indicates a specific inhibition of the parasite target. The last column indicates the concentration (in μM) in which the compounds specifically inhibited one or both of the parasite targets in dose-response experiments.
Fig 5.
Validation of the hit compounds using Brugia pahangi adult female worms.
Chemical structures were obtained from www.chemspider.com. The IC50 for each of the compounds in human fibroblasts (MRC5) is shown in the last column [26]. The IC50 of Geldanamycin against different human cell lines is a consensus of multiple data available in the literature (www.medchemexpress.com/Geldanamycin.html).