Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) induces global transcriptional deregulation and ultrastructural alterations that impair viability in Schistosoma mansoni

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ) and, due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935, which was used to treat schistosomula and adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-Seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors

Evaluation of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole as a Novel Anti-Inflammatory Drug Candidate

BACKGROUND: 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole (DIC) is a five-membered heterocyclic compound containing a N-O bond. The anti-inflammatory effects of this compound were studied both in vitro and in vivo. PRINCIPAL FINDINGS: DIC effectively decreased TNF-α and IL-6 release from LPS-stimulated macrophages in a dose dependent manner. DIC diminished the levels of COX-2 with subsequent inhibition of PGE(2) production. DIC also compromised HMGB1 translocation from the nucleus to the cytoplasm. Moreover, DIC prevented the nuclear translocation of NF-κB and inhibited the MAPK pathway. In vivo, DIC inhibited migration of neutrophils to the peritoneal cavity of mice. CONCLUSIONS: This study presents the potential utilization of a synthetic compound, as a lead for the development of novel anti-inflammatory drugs

CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

parasite resides in mesenteric veins where fecundated female worms lay hundred of eggs daily. Some of the egg antigens are trapped in the liver and induce a vigorous granulomatous response. High Mobility Group Box 1 (HMGB1), a nuclear factor, can also be secreted and act as a cytokine. Schistosome HMGB1 (SmHMGB1) is secreted by the eggs and stimulate the production of key cytokines involved in the pathology of schistosomiasis. Thus, understanding the mechanism of SmHMGB1 release becomes mandatory. Here, we addressed the question of how the nuclear SmHMGB1 can reach the extracellular space. eggs of infected animals and that SmHMGB1 that were localized in the periovular schistosomotic granuloma were phosphorylated.We showed that secretion of SmHMGB1 is regulated by phosphorylation. Moreover, our results suggest that egg-secreted SmHMGB1 may represent a new egg antigen. Therefore, the identification of drugs that specifically target phosphorylation of SmHMGB1 might block its secretion and interfere with the pathogenesis of schistosomiasis

Schistosoma mansoni histone acetyltransferase GCN5: linking histone acetylation to gene activation

Univ Fed Rio de Janeiro, CCS, ICB, Dept Bioquim Med, BR-21941590 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Effect of DIC on LPS-induced PGE₂ production.

<p>(<b>A</b>) RAW 264.7 macrophages were pretreated with DIC 200 µM for 2 h prior to addition of LPS (1 µg/mL) for 24 h and then PGE₂ levels were determined by EIA. The values shown are means ± SD of three independent experiments. NS, non-significant <i>vs</i> CTRL+; * p<0.05 <i>vs</i> vehicle; ** non-significant <i>vs</i> vehicle. Significances between treated groups were determined using unpaired t-test. (<b>B</b>) Protein levels of COX-2 were determined by western blot analysis of cellular protein extract (upper panel). A representative immunoblot out of three independent experiments were shown.</p

Effect of DIC on macrophage viability.

<p>RAW 264.7 macrophages were treated with DIC (from 10 µM to 500 µM) for 24 h. Cell viabilities were determined by LDH release (<b>A</b>) and MTT assay (<b>B</b>). Values represent means ± SD of three independent experiments. * Significant differences (p>0.05) between treated and untreated cells (250–500 µM), using unpaired t-test.</p

Effect of DIC on nuclear translocation of HMGB1.

<p>RAW 264.7 macrophages were pretreated with DIC 200 µM for 2 h prior to addition of LPS (1 µg/mL) for 24 h. Intracellular HMGB1 was visualized with green immunofluorescent FITC-staining. Untreated cells (UT); LPS-stimulated cells (LPS); DIC-treated cells stimulated with LPS (LPS + DIC).</p

Effect of DIC on the MAPK pathway.

<p>RAW 264.7 macrophages were pretreated with 200 µM of DIC for 2 h prior to addition of LPS (1 µg/mL) for 15 min, and then the whole cell lysate was analyzed by western blot using antibodies against the phosphorylated (activated) and unphosphorylated MAPK. The data shown are representative of three independent experiments.</p

Effect of DIC on LPS-induced TNF-α and IL-6 production.

<p><b>A</b> and <b>B</b>, following pretreatment with Polymyxin B (Pol B, 15 µg/mL), vehicle (DMSO 0.25%) or DIC (10−200 µM) for 2 h, the cells were treated with LPS (100 ng/mL) for 4 h (A) or 24 h (B). Negative control (CTRL −): cell medium only; Positive control (CTRL +): cells stimulated with LPS, only. TNF-α and IL-6 levels were assayed by ELISA. Values represent means ± SD of three independent experiments. NS, non-significant <i>vs</i> CTRL +; * p<0.05 <i>vs</i> vehicle; ** non-significant <i>vs</i> vehicle. Significances between treated groups were determined using unpaired t-test.</p

Effect of DIC on cell migration in thioglycollate-induced peritonitis in mice.

<p>(<b>A</b>) DIC (5 mg/kg) or vehicle (DMSO 2.4%) was administered intraperitoneally 30 min before the thioglycollate administration. Mice were sacrificed after 4 h of thioglycollate-induced peritonitis. Total cell migration was counted using a Neubauer chamber. (<b>B</b>) Differential cell count was evaluated by Cytospin. Data represent mean ± S.D. from at least 8 animals per group. * P<0.05 (significances between treated groups were determined using unparied t-test).</p