Discovery and Characterization of Novel Anti-schistosomal Properties of the Anti-anginal Drug, Perhexiline and Its Impact on <i>Schistosoma mansoni</i> Male and Female Reproductive Systems

<div><p>Background</p><p>Schistosomiasis, one of the world’s greatest human neglected tropical diseases, is caused by parasitic trematodes of the genus <i>Schistosoma</i>. A unique feature of schistosome biology is that the induction of sexual maturation as well as the maintenance of the differentiation status of female reproductive organs and egg production, necessary for both disease transmission and pathogenesis, are strictly dependent on the male. The treatment and most control initiatives of schistosomiasis rely today on the long-term application of a single drug, praziquantel (PZQ), mostly by campaigns of mass drug administration. PZQ, while very active on adult parasites, has much lower activity against juvenile worms. Monotherapy also favors the selection of drug resistance and, therefore, new drugs are urgently needed.</p><p>Methods and Findings</p><p>Following the screening of a small compound library with an ATP-based luminescent assay on <i>Schistosoma mansoni</i> schistosomula, we here report the identification and characterization of novel antischistosomal properties of the anti-anginal drug perhexiline maleate (PHX). By phenotypic worm survival assays and confocal microscopy studies we show that PHX, <i>in vitro</i>, has a marked lethal effect on all <i>S</i>. <i>mansoni</i> parasite life stages (newly transformed schistosomula, juvenile and adult worms) of the definitive host. We further demonstrate that sub-lethal doses of PHX significantly impair egg production and lipid depletion within the vitellarium of adult female worms. Moreover, we highlighted tegumental damage in adult male worms and remarkable reproductive system alterations in both female and male adult parasites. The <i>in vivo</i> study in <i>S</i>. <i>mansoni</i>-patent mice showed a notable variability of worm burdens in the individual experiments, with an overall minimal schistosomicidal effect upon PHX treatment. The short PHX half-life in mice, together with its very high rodent plasma proteins binding could be the cause of the modest efficacy of PHX in the schistosomiasis murine model.</p><p>Conclusions/Significance</p><p>Overall, our data indicate that PHX could represent a promising starting point for novel schistosomicidal drug discovery programmes.</p></div

Release of oocytes, spermatozoa and vitelline cells by PHX treated worm couples in culture.

<p>Representative images of oocytes (oc), spermatozoa (sp), mature spermatozoa (ms) and vitelline cells (vc) in tissue culture medium of worm couples treated with DMSO (a), PHX at 2.5 μM (b), and 5 μM (c, d) are shown. Scale bars = 100 μm (a-c) and 25 μm (d) are shown.</p

Survival of <i>S</i>. <i>mansoni</i> schistosomula after <i>in vitro</i> PHX treatment.

<p>Schistosomula (100/well) were incubated with serial dilutions of PHX, 5 wells at each concentration. Data, evaluated 24 hours post treatment, were normalized based on 50 μM GA-treated control (0% survival) and DMSO treated schistosomula (100% survival) values at each time point as described in methods. The error bars represent the SEM. The LD₅₀ was 4.25.</p

Vitellarium alterations in adult female worms treated with PHX.

<p>Representative confocal laser scanning microscope images of the vitellarium of adult female worms. <i>S</i>. <i>mansoni</i> worm couples were treated for 3 and 6 days with DMSO (A, E) or different doses of PHX (B-D, F-H) for 3 and 6 days as indicated and stained with carmine red. Scale bars = 25 μm in the low magnification panels and 2.5 μm in the high magnification insets indicated with yellow boxes.</p

BSA and human AGP proteins supplementation impair PHX antischistosomal activity <i>in vitro</i>.

<p>Adult male worms (7–8 weeks old) were incubated with the indicated compounds in complete culture medium alone (A) or supplemented with SA (B) or AGP (C). Viability of parasites was scored as described under methods. The mean data ± SEM of three independent experiments are shown.</p

Effect of PHX on adult <i>S</i>. <i>mansoni</i> worm couples and on egg production <i>in vitro</i>.

<p>Adult couples were incubated with the indicated doses of PHX and viability and total egg counts assessed as described in the methods section. A) viability curves of adult worm couples; B) total egg counts normalized to worm couples. The mean values ± SEM are shown, the data are representative of three independent experiments. * indicates p values < 0.05, ** indicates p values < 0.01 and *** indicates p values < 0.001.</p

PHX activity on worm burden in mice infected with <i>S</i>. <i>mansoni</i>.

<p>The histograms show the % of worm burdens in mice treated with DMSO, PZQ and PHX. In white is represented the female and in black the male worm burden contribution. Worm count data are represented as a percentage of the mean worm count ± SD of experimental control (DMSO) group. In Figure A and B the average of worm burden percentages of mice treated with the same compound and at the same dose regimen are representative respectively of four and two independent experiments. * indicates p values < 0.05 and **** indicates p values < 0.0001.</p

Percentage of unbound fraction of PHX across tested species.

<p>Percentage of unbound fraction of PHX across tested species.</p

Ovary alterations in adult female worms treated with PHX.

<p>Representative confocal laser scanning microscope images of adult female ovaries. <i>S</i>. <i>mansoni</i> worm couples were treated for 3 and 6 days with DMSO (a, b) or different doses of PHX (c-h) for 3 and 6 days as indicated and stained with carmine red. Immature oocytes (io), mature oocytes (mo), receptaculum seminis (rs) are indicated. Scale bars = 50 μm (a-c), and 25 μm in all other PHX treated samples (d-h) are shown.</p

Tegumental damage in adult male worms treated with PHX.

<p>Representative confocal laser scanning microscope images of adult <i>S</i>. <i>mansoni</i> male worms treated for 7 days with DMSO (a) or 10 μM PHX (b). The worms were fixed and incubated with tetramethylrhodamine B-isothiocyanate (TRITC)-labeled phalloidin (546). Scale bar = 25μm.</p