The Existing Drug Nifuroxazide as an Antischistosomal Agent: In Vitro, In Vivo, and In Silico Studies of Macromolecular Targets

Abstract

Schistosomiasis is a parasitic disease that afflicts approximately 250 million people worldwide. There is an urgent demand for new antiparasitic agents because praziquantel, the only drug available for the treatment of schistosomiasis, is not universally effective and may derail current progress toward the WHO goal of eliminating this disease as a public health problem by 2030. Nifuroxazide (NFZ), an oral nitrofuran antibiotic, has recently been explored to be repurposed for parasitic diseases. Here, in vitro, in vivo, and in silico studies were conducted to evaluate the activity of NFZ on Schistosoma mansoni. The in vitro study showed significant antiparasitic activity, with 50% effective concentration (EC₅₀) and 90% effective concentration (EC₉₀) values of 8.2 to 10.8 and 13.7 to 19.3 μM, respectively. NFZ also affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg of body weight) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden (~40%). In patent infection, NFZ achieved a high reduction in the number of eggs (~80%), but the drug caused a low reduction in the egg burden of animals with prepatent infection. Finally, results from in silico target fishing methods predicted that serine/threonine kinases could be one of the potential targets for NFZ in S. mansoni. Overall, the present study revealed that NFZ possesses antischistosomal properties, mainly in terms of egg burden reduction in animals with patent S. mansoni infection. IMPORTANCE The increasing recognition of the burden imposed by helminthiasis, associated with the limited therapeutic arsenal, has led to initiatives and strategies to research and develop new drugs for the treatment of schistosomiasis. One of these strategies is drug repurposing, which considers low-risk compounds with potentially reduced costs and shorter time for development. In this study, nifuroxazide (NFZ) was evaluated for its anti-Schistosoma mansoni potential through in vitro, in vivo, and in silico studies. In vitro, NFZ affected worm pairing and egg production and induced severe damage to the tegument of schistosomes. In vivo, a single oral dose of NFZ (400 mg/kg) to mice harboring either prepatent or patent S. mansoni infection significantly reduced the total worm burden and egg production. In silico investigations have identified serine/threonine kinases as a molecular target for NFZ. Collectively, these results implied that NFZ might be a potential therapeutic candidate for the treatment of schistosomiasis.

Keywords: anthelmintic agent; antibiotic; drug discovery; drug repurposing; medicinal chemistry; parasitic diseases; schistosomiasis.

FIG 1

Viability and egg output of adult S. mansoni worms during exposure to nifuroxazide (NFZ) and praziquantel (PZQ). Adult worm pairs were obtained from animals by perfusion at 42 days after infection. Each concentration was tested at least in triplicate, with the highest concentration of DMSO serving as the control. Male (A) and female (B) schistosomes were monitored for up to 72 h, and results are expressed as the percent mortality recorded by Kaplan-Meier survival curves. Eggs released by paired adult worms exposed to NFZ (C). Data are presented as the mean ± SD from three independent experiments (n = 3). Control, drug-free medium; PZQ, praziquantel at 2 μM.

FIG 2

Scanning electron microscopy of adult S. mansoni following incubation with nifuroxazide (NFZ). Control male parasite showing intact tubercles (T) and spines on the surface (A), and control female worm (B) showing the sensory papillae (Sp). Schistosomes were exposed to NFZ at 12.5 μM (C and D), 25 μM (E and F), and 50 μM (G and H). Male (A, C, E, and G) and female (B, D, F, and H) schistosomes. The dorsal tegumental surface shows tubercle shortening (Ts), swelling (Sw), erosion (Er), sloughing (Sl), blisters (Bl), and tubercle disintegration (Td). Images were captured using a JEOL SM 6460LV scanning electron microscope after 72 h of incubation. Scale bars: 10 μm.

FIG 3

Effect of nifuroxazide (NFZ) and praziquantel (PZQ) on the parasite burden of mice with prepatent (A) and patent (B) Schistosoma mansoni infection. Vehicle (control), NFZ, and PZQ (400 mg/kg, single dose) were administered at 21 days (A) or 42 days (B) postinfection by oral gavage. On day 56 postinfection, all rodents were euthanized and parasite burdens were determined. Data are presented as the mean ± SD from five animals (n = 5 per group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values were compared with infected untreated control. WBR, worm burden reduction.

FIG 4

Effect of nifuroxazide (NFZ) and praziquantel (PZQ) on the egg burden in feces (A) and intestine (B) of mice with prepatent and patent Schistosoma mansoni infection. Vehicle (control), NFZ, and PZQ (400 mg/kg, single dose) were administered at 21 days (prepatent infection) or 42 days (patent) postinfection by oral gavage. On day 56 postinfection, all rodents were euthanized, and egg burden was determined by Kato Katz technique (egg in the feces) and oogram examination (immature eggs in the intestine). Data are presented as the mean ± SD from five animals (n = 5 per group). EBR, egg burden reduction.