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

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 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 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 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

Chemical structure of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole, DIC.

<p>Chemical structure of 3-(3-chloro-phenyl)-5-(4-pyridyl)-4,5-dihydroisoxazole, DIC.</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

Zika virus infection drives epigenetic modulation of immunity by the histone acetyltransferase CBP of Aedes aegypti.

Epigenetic mechanisms are responsible for a wide range of biological phenomena in insects, controlling embryonic development, growth, aging and nutrition. Despite this, the role of epigenetics in shaping insect-pathogen interactions has received little attention. Gene expression in eukaryotes is regulated by histone acetylation/deacetylation, an epigenetic process mediated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). In this study, we explored the role of the Aedes aegypti histone acetyltransferase CBP (AaCBP) after infection with Zika virus (ZIKV), focusing on the two main immune tissues, the midgut and fat body. We showed that the expression and activity of AaCBP could be positively modulated by blood meal and ZIKV infection. Nevertheless, Zika-infected mosquitoes that were silenced for AaCBP revealed a significant reduction in the acetylation of H3K27 (CBP target marker), followed by downmodulation of the expression of immune genes, higher titers of ZIKV and lower survival rates. Importantly, in Zika-infected mosquitoes that were treated with sodium butyrate, a histone deacetylase inhibitor, their capacity to fight virus infection was rescued. Our data point to a direct correlation among histone hyperacetylation by AaCBP, upregulation of antimicrobial peptide genes and increased survival of Zika-infected-A. aegypti