Table 1.
The Structural Genomics Consortium (SGC) epigenetic probes (EPs) and epigenetic inhibitors (EIs) used in this study.
Fig 1.
Epigenetic probes/inhibitors targeting histone modifying enzymes negatively modify schistosomula phenotype and motility metrics.
A collection of 37 EPs/EIs targeting histone modifying enzymes (readers, writers and erasers) as well as controls (PZQ and AUR) were screened against S. mansoni schistosomula at 10 μM for 72 h using the Roboworm platform as described in the Materials and Methods. (A) Parasite phenotype and (B) Parasite motility were both negatively affected by fourteen (38%) of these compounds upon repeat screening (each filled circle represents average phenotype or motility scores derived from ~ 80–120 schistosomula; n = 2–3; horizontal bars represent average scores derived from replicates). (C) Representative images of schistosomula phenotypes affected by the fourteen active EPs/EIs, compared to controls (Media only, DMSO, Auranofin and Praziquantel). Zˊ values obtained from these screens ranged from 0.27–0.48 (mean = 0.40) for phenotype and 0.41–0.56 (mean = 0.48) for motility. Table 1 (closest S. mansoni homolog) and S1 Table (structures, SMILES, MW, etc.) provide further information related to EPs/EIs screened and their putative S. mansoni targets.
Table 2.
Dose response titrations of the 14 active SGC compounds against schistosomula and adult schistosome pairs.
Fig 2.
The HMT inhibitors LLY-507 and BAY-598 significantly reduce adult worm H3K36me2 levels.
Homology modeling of Smp_000700, using H. sapiens SMYD3 (PDB 5EX3) as the template, was performed according to the Materials and Methods. (A) Domain architecture and homology model of Smp_000700 showing the tetratricopeptide repeat (TPR, gold, AA 76–195), the SET N-terminal domain (red, AA 279–312), the myeloid, nervy and DEAF-1 domain (MYND, blue, AA 313–370) and the SET C-terminal domain (green, AA 371–642). SAH (S-adenosyl homocysteine) and histone H3 are indicated. (B) Predicted binding of LLY-507 to Smp_000700 substrate binding pocket. (C) Predicted binding of BAY-598 to Smp_000700 substrate binding pocket. (D) Adult schistosome pairs (21 pairs/biological replicate; n = 3; 63 worm pairs in total) were co-cultured for 72 h in a sub-lethal concentration of LLY-507 (6.25 μM) or BAY-598 (25 μM) in 0.625% DMSO. After co-cultivation, schistosomes were separated by sex (males and females), histones extracted and total levels of H3K36me2 quantified by ELISA according to Methods.
Fig 3.
Schistosome motility, egg production and H3K36me2 are regulated by Smp_000700.
RNAi of adult schistosome pairs (21 worm pairs/biological replicate; n = 3 replicates) using siRNAs directed against smp_000700 and luc was performed as described in the Materials and Methods. (A) qRT-PCR analysis of smp_000700 RNA levels in siLuc vs siSmp_000700 treated worms at 48 h. (B) Quantification of adult schistosome worm motility at 168 h. (C) Enumeration of in vitro laid egg (IVLE) production at 168 h. (D) Detection of H3K36me2 in adult schistosome nuclear extracts at 168 h. Statistical significance is indicated (Student’s t test, two tailed, unequal variance). *** represents p < 0.001.
Fig 4.
The cell-permeable JMJD3 inhibitor GSK-J4, but not cell impermeable GSK-J1, significantly affects IVLE production and vitellocyte packaging.
Adult schistosome pairs (3 pairs/well; n = 3 or 6) were co-cultured for 72 h in GSK-J4 (50 μM– 0.05 μM), GSK-J1 (6.25 μM or 3.13 μM) or DMSO (0.625%) as described in the Materials and Methods. (A) Adult worm motility scores (red circles–males; blue circles–females). (B) Number of IVLEs produced. (C) Representative IVLE phenotypes (autofluorescence; Ex = 488 nm, Em = 519 nm and DAPI; Ex = 405 nm, Em = 458 nm) from schistosome pairs co-cultivated in GSK-J4 (0.2 μM), GSK-J1 (6.25 μM) or 0.625% DMSO for 72 h. (D) Quantification of egg volumes between treatment groups (n = 10 per group; GSK-J4–0.2 μM, GSK-J1–6.25 μM). (E) Number of vitellocytes per egg (n = 10 per group) between treatment groups (GSK-J4–0.2 μM, GSK-J1–6.25 μM). *, p < 0.05; ***, p < 0.001.