Fig 1.
(A) Compounds identified, synthesized and tested for inhibition of parasite motility. (B) Flow diagram illustrating screening, synthesis and evaluation of compounds.
Fig 2.
Effects of GSK-J4 and demethylase analogs on adult B. malayi viability.
Female adult worms were cultured with 0.1% DMSO or 1 μM of compounds 6–38, 6–44, 6–104, 7–006, or GSK-J4 for 48 hours before evaluation by MTT assay. At least seven worms were used in each treatment group. Bars represent mean absorbance and error bars show SEM. Statistical significance was determined by ANOVA and post-hoc pairwise t-tests and are indicated by asterisks if p < 0.0083 (Bonferroni correction).
Table 1.
Percent parasite motility by various synthetic inhibitors and GSK-J4.
Parasites were treated with indicated compounds at 1 μM for 48 hours. All experiments were repeated at least three times n = 3. Except for MSU-TSR-6-104 which is a structurally inactive analog and MSU-TSR-6-38, all compounds are selective towards adult parasitic stages. The compounds are also active against the closely related B. pahangi and the rodent nematode L. sigmodontis. All reported percent inhibition are within a ≤ 10% standard error. All compounds are inactive against C. elegans at 100 μM.
Fig 3.
Residues in red represent contact sites for GSKJ1.
Triangles represent contact sites for N-Oxalylglycine (NOG).
Fig 4.
Left: B. malayi female western blot, treated with indicated compounds and vehicle control for 18 hours. Histones were extracted and purified and run on a 12% Bis-Tris gel. H2B ubiquitination on Lys120 was used as a loading control as it has been found to be constant regardless of experimental treatment in previous experiments. 6–104, an inactive structural analog was used as a negative control. 6–104, GSK-J4 and Emodepside do not appear to inhibit H3K27 demethylation. Right: Graphical representation of the western blot by normalization of H3K27me3 (top right) and H3K4me3 (bottom right) to loading control. Image: Odyssey CxL Image Studio.
Fig 5.
Recombinant B. malayi UTX-1 (E) serial diluted incubated with total bovine histone (H) constant.
Recombinant B. malayi UTX-1 activity assay. Purified histones (600 ng) were used for each reaction with varying amounts of enzyme (420–7 ng). An anti-H3K27me3 was used to detect the methylation status after an overnight incubation. BmUTX-1 demethylates H3K27me3.
Fig 6.
Biological functions affected by treatment of B. malayi males with 100 nM of MSU-TSR-6-44 (A and B) or MSU-TSR-6-38 (C and D). Gene classification was conducted with the Panther Classification System Gene Analysis tool, using the B. malayi Genome as a reference. (Mi H et. al. 2018; Mi H et. al. 2019).