Effects of structurally distinct human HDAC6 and HDAC6/HDAC8 inhibitors against S. mansoni larval and adult worm stages

Abstract

Schistosomiasis is a major neglected parasitic disease that affects more than 240 million people worldwide caused by Platyhelminthes of the genus Schistosoma. The treatment of schistosomiasis relies on the long-term application of a single safe drug, praziquantel (PZQ). Unfortunately, PZQ is very effective on adult parasites and poorly on larval stage and immature juvenile worms; this can partially explain the re-infection in endemic areas where patients are likely to host parasites at different developmental stages concurrently. Moreover, the risk of development of drug resistance because of the widespread use of a single drug in a large population is nowadays a serious threat. Hence, research aimed at identifying novel drugs to be used alone or in combination with PZQ is needed. Schistosomes display morphologically distinct stages during their life cycle and epigenetic mechanisms are known to play important roles in parasite growth, survival, and development. Histone deacetylase (HDAC) enzymes, particularly HDAC8, are considered valuable for therapeutic intervention for the treatment of schistosomiasis. Herein, we report the phenotypic screening on both larvae and adult Schistosoma mansoni stages of structurally different HDAC inhibitors selected from the in-house Siena library. All molecules have previously shown inhibition profiles on human HDAC6 and/or HDAC8 enzymes. Among them we identified a quinolone-based HDAC inhibitor, NF2839, that impacts larval and adult parasites as well as egg viability and maturation in vitro. Importantly, this quinolone-based compound also increases histone and tubulin acetylation in S. mansoni parasites, thus representing a leading candidate for the development of new generation anti-Schistosoma chemotherapeutics.

Fig 1. Examples of hydroxamic acid based SmHDAC8 inhibitors reported in literature.

J1038, TH65 [16,17]; Compound 9 [22]; NF2624, NF2886 [18,21] are shown.

Fig 2. Structure, type of cap, and original reference of HDAC inhibitors tested against S. mansoni.

Fig 3. Schistosome viability assays.

Panel A: Representative dose-response curves of the inhibitors on NTS. The y-axis indicates the percentage of viability normalized against DMSO (100%) and gambogic acid 10 μM (0%). The calculated LD₅₀ (μM) average +/- SD are shown. Panel B: dose-response curve of the inhibitors on adult schistosome worm pairs. DMSO (vehicle) was used as negative control (100% viability), the indicated compounds were assayed at 5 (full triangle) 10 (inverted full triangle), 20 (full square) and 50 μM (full circle). Data are expressed as a % severity score (viability) relative to DMSO. Each point represents the average ± standard deviation of three independent experiments.

Fig 4. Quinolone-based HDAC inhibitors impair egg viability and maturation.

Panel A. Representative microscopy images of S. mansoni eggs laid by worm pairs exposed to DMSO, NF2836 (20 or 50 μM) or NF2839 (5, 10 μM). Panel B. Histograms represent the percentages of eggs at different maturation stages +/- SEM counted at 72 h after treatment; 5–6 microscopy images of at least 3 independent experiments were analyzed. Filled red triangles indicate viable eggs at stages I–II (immature); empty red triangles indicate damaged/morphological abnormal eggs at stages I–II (immature); filled red arrows indicate viable eggs at stages III–V (intermediate/developed); empty red arrows indicate damaged/morphological abnormal eggs at stages III-V; ef, egg fragments; sp, sperm cells; oc, oocyte. Scale Bars = 200 μm.

Fig 5. Confocal microscopy of carmine-red stained adult worm pairs treated with quinolone-based HDAC inhibitors.

The images are representative of 3–4 worm pairs treated with DMSO, NF2836 (20 and 50 μM) or NF2839 (5 and 10 μM) at 72 h in two independent experiments. The ovary, ootype and gut are shown. Immature oocytes (io), mature oocytes (mo), ootype (ot) and gastrodermis (ga) are labeled. In the Figure, red filled triangles indicate degenerated mo; empty red triangles indicate egg fragments in the ootypes; asterisks indicate deformed egg in the ootype and carmine red aggregates in the gut lumen, filled red arrows indicate the ga in the gut. Scale bars: 50 μm.

Fig 6. Effects of selected compounds on histones and tubulin acetylation.

Representative immunoblots of the histone-enriched and cytosolic protein fractions extracted from S. mansoni adult worm pairs incubated with anti-acetylated lysine antibody (acetylated-K) or anti-acetylated tubulin. Worm pairs were treated for 48 or 72 h at the concentration indicated for each compound. DMSO (vehicle) and of the HDAC pan- inhibitor, TSA (1 μM), were used respectively as negative and positive controls.