Table 1.
Preparatory summary of compounds 1–9.
Fig 1.
The screening pipeline utilised in this anti-infective study.
A total of 14 compounds were initially screened against S. mansoni schistosomula at a final concentration of 10 μM. Hits were subsequently subjected to dose response titrations with the most effective anti-schistosomula compound (Compound 1) subsequently being used as a template for the preparation of further derivatives (compounds 2–9). Compounds 2–9 were subsequently subjected to dose response titrations against schistosomula and adult worms. The most effective compounds (original compound 1 and analogue 3) were next titrated against juvenile worms. Finally, compounds 1, 3, 5 and 6 were additionally titrated against both Gram+ (S. aureus) and Gram- (E. coli) bacterial exemplars.
Fig 2.
Anti-schistosomula activity of the eight analogues compared to parent compound 1.
A total of 120 mechanically transformed schistosomula were co-cultured with each compound, titrated at doses between 10 and 0.625 μM. Test plates were incubated at 37°C for 72 hrs in an atmosphere containing 5% CO2. At 72hrs, schistosomula were scored using the Roboworm platform for both motility (A) and phenotype (B). Any compound that induced a score of below -0.35 for motility (A) and -0.15 for phenotype (B) were considered a hit. Black squares indicate the most positive effect on motility or phenotype; grey scale from dark grey to lighter shades of grey indicates a progressive reduced compound efficacy; white squares indicate no effect on either phenotype or motility. Z´ values for this screen was 0.41741 for motility and 0.57275 for phenotype. (C) Phenotypes of schistosomula when treated with 10μM of each of the test compounds and controls.
Fig 3.
Adult schistosome motility and egg production are differentially affected by the nine N-acyl homoserine lactones.
A) Adult S. mansoni worm pairs were cultured in decreasing compound concentrations of between 20 μM and 1.25 μM (not all concentrations were used for all compounds) for 72 hrs at 37°C, 5% CO2. Parasite motility was evaluated for each sex and scored using the WHO-TDR scoring system (0 = Dead parasite, 4 = Normal/Healthy movement). B) Culture media were collected from some adult co-cultures at 72 hrs and eggs present in the media were counted. p values are indicated as follows: * <0.05, ** <0.01, *** <0.001.
Table 2.
Calculated EC50, CC50 and subsequent selectivity indices of N-acyl homoserines.
Fig 4.
Three week juvenile worms are immobilised by N-Acyl homoserine lactones.
Three-week old juvenile S. mansoni worms (n = 13–33 per well) were co-cultured with compounds (1) and (3) at concentrations spanning 15 μM—0.23 μM for 72 hrs at 37°C in a humidified atmosphere containing 5% CO2. Parasite motility was scored between 0–4 (0 = no movement/dead, 4 = full movement/healthy). DMSO negative controls were also included (1.25% final concentration) as well as two positive controls (15 μM PZQ and 15 μM auranofin; both in 1.25% DMSO). A) Effect of compound (1) on juvenile worm motility. (B) Effect of compound (3) on juvenile worm motility. p values are indicated as follows: * <0.05, ** <0.01, *** <0.001.
Fig 5.
N-Acyl homoserine lactones kill juvenile stage schistosomes.
Three week juvenile schistosomes, incubated with compounds (1), (3) or controls for 72 hrs, were subsequently cultured with PI at a final concentration of 2 μg/mL for 15 minutes at 37 oC in an environment containing 5% CO2. PI positive parasites (dead) were counted and the percent live vs dead in each well is indicated. A) Quantification of compound (1) mediated juvenile death. B) Quantification of compound (3) mediated juvenile death. C) Quantification of juvenile deaths in co-cultures containing 15 μM PZQ (in 1.25% DMSO), 15 μM auranofin (in 1.25% DMSO) and 1.25% DMSO.
Fig 6.
In silico approach to identify potential targets of compound 1 within S. mansoni.
A) FragFP output in DataWarrior demonstrates that Compound 1 is structurally related to C4-HSL (formerly called PAI-2 [34]); C4-HSL is an N-acyl homoserines involved in quorum sensing within P. aeruginosa. Substitution of the methyl and bromine groups (found in compound 1) is observed with propyl and stereochemistry modifications. B) Pathways in P. aeruginosa that utilise both C4- and C12- HSLs (LasI = acyl-homoserine-lactone synthase; LasR = transcriptional activator; RhlR = regulatory protein RhlI = acyl-homoserine-lactone synthase). Highlighted (red rectangles) are those P. aeruginosa proteins used in BLASTp analysis of the S. mansoni genome (v 7.0).
Table 3.
Antibacterial activity (MIC) of the tested compounds and antibacterial selectivity of compound 3 (IC50).