Fig 1.
7-keto-sempervirol is a diterpenoid phenol derived from Lycium chinense.
This compound possesses a diterpenoid (C20) (4 isoprene units) scaffold consisting of a single phenyl group, one hydroxyl group (red) and one carbonyl group (red). In light of Lipinski’s rule of 5 (RO5) [57], this compound only possesses one violation to the rule (log p > 5), however, three out of the four core rules are satisfied.
Fig 2.
The diterpenoid 7-keto-sempervirol displays lethal activity against Schistosoma mansoni schistosomula.
Schistosomula (96 well plate format; 1000 parasites/well; triplicate wells for each concentration assessed) were co-cultivated with a 50% dilution series of 7-keto-sempervirol (100, 50, 25, 12.5, 6.25, 3.125, 1.563 μM) at 37°C and 5% CO2 for 24 hr and subjected to the helminth fluorescent bioassay (HFB). A) Fluorescent data derived from wells were converted into corresponding Log10 values and the mean percentage viability was transformed into probit values to create a dose-response curve as previously described [25]. An LD50 of 19.1 μM was calculated from this dose-response curve. Error bars represent the standard deviation of the mean (SD). B) High content imaging of fluorescently labelled parasites (fluorescein diacetate, green and propidium iodide, red) co-cultured with titrated 7-keto-sempervirol supports the HFB measurements. The concentration of fluorophores employed for high content imaging were the same as previously described for the HFB [25]. Dead control = parasites co-cultured with 70 μM auranofin. DMSO control = parasites co-cultured with 1% (v/v) DMSO. Parasites were imaged at 10 x magnification on a high content imaging system processed by the MetaXpress version 5.10.46 software utilising both FITC and TRITC filters.
Fig 3.
Adult Schistosoma mansoni motility is reduced in the presence of 7-keto-sempervirol.
World Health Organisation motility metrics were used for scoring adult S. mansoni male and female worm phenotypes (5 worm pairs/ml) co-cultured (37°C and 5% CO2) with 7-keto-sempervirol (100 μM final concentration) for 24 hr, 48 hr and 72 hr. Phenotypes derived from a control group (media containing 1% v/v DMSO) of male and female worms cultivated for 24 hr (worm pairs cultured for 48 hr or 72 hr were identical to the 24 hr worms, S2 Fig.) is also illustrated. Mean motility values are indicated as histograms (n = 5 replicates/time-point) and the error bars represent the standard deviation of the mean (SD). Different letters indicate a significant difference between the mean phenotypic scores calculated by ANOVA and Tukey’s post-hoc tests.
Fig 4.
The diterpenoid 7-keto-sempervirol induces surface tegumental damage in adult Schistosoma mansoni males.
Scanning Electron Microscopic (SEM) images of adult S. mansoni male worms cultured for 72 hr at 37°C and 5% CO2 in the presence (100 μM) or absence (1% v/v DMSO) of 7-keto-sempervirol. A) (left) 1.00K x magnification of a control (1% v/v DMSO) adult male surface (anterior end) and corresponding (right) 6.00K x magnification of normal tubercles. B) (left) 1.00K x magnification of a 7-keto-sempervirol treated (100 μM), adult male surface (anterior end) and enlarged (right) 9.00K x magnification of two indicated (black boxes) areas of tegumental damage. Holes in the surface tegument/tubercles are indicated by white arrows. These images are representative of 5 adult worms/condition.
Fig 5.
Schistosoma mansoni egg-shell formation is inhibited by 7-keto-sempervirol.
Adult S. mansoni female worms were cultured (+/- 100 μM 7-keto-sempervirol) for 24 hr at 37°C and 5% CO2. A) 40x laser scanning confocal microscopic image of an egg derived from a control female (1% v/v DMSO) demonstrating regular auto-fluorescence and a well-formed lateral spine (normal architecture of a S. mansoni egg). B) 40x laser scanning confocal microscopic image of an egg derived from one adult female worm treated with 100 μM 7-keto-sempervirol. Images are representative of typical in utero egg phenotypes obtained from 5 worms/condition.
Fig 6.
Schistosoma mansoni oviposition is inhibited by 7-keto-sempervirol.
Adult worm pairs (5 pairs/ml) were co-cultured (37 oC and 5% CO2) with 1% v/v DMSO (DMSO control), 10 μM 7-keto-sempervirol or 100 μM 7-keto-sempervirol for 72 hr. A) Bar charts represent the mean percentage of abnormal eggs (lacking a fully formed lateral spine) produced after 72 hr (n = 5 replicates/treatment). Error bars represent the standard deviation of the mean (SD). After 72 hr, a total of 68.4 +/- 74.4 eggs (normal and abnormal) were enumerated in the worm cultures containing 1% DMSO, 73.2 +/- 46.4 eggs (normal and abnormal) were observed in the cultures containing 10 μM 7-keto-sempervirol and zero eggs were counted in the wells containing 100 μM 7-keto-sempervirol. Student’s t-test indicates that a significant difference exists between the percentage of abnormal eggs laid between the 1% v/v DMSO and 10 μM 7-keto-sempervirol treatments (p = 0.01). B) Fluorescent images of representative eggs deposited in vitro demonstrate normal egg architecture in the DMSO control wells (DMSO control) and abnormal egg architecture in the 10 μM 7-keto-sempervirol wells (7-keto-sempervirol).
Fig 7.
The diterpenoid 7-keto-sempervirol displays lethal activity against Fasciola hepatica newly excysted juveniles (NEJs).
Complementary phenotypic-based [31,32] and fluorescent-mediated tests [25] were used to assess NEJ viability. A) The phenotypic assay involved co-culturing 20 NEJs (96-well plate format) with a 50% dilution series of 7-keto-sempervirol (100, 50, 25, 12.5, 6.25, 3.125, 1.563 μM) at 37°C and 5% CO2 for 24 hr. Mean phenotypic score values (11 = severely affected, 0 = not affected) are indicated by bar charts (n = 20 individuals scored/experimental treatment) with error bars representing the standard deviation of the mean (SD). A collection of fluorescent high content images (10 x) was obtained similar to those collected for schistosomula (Fig. 2). B) The fluorescent assay utilised the HFB [25] and co-cultured 100 NEJs/experimental treatment (96-well plate format) with the same dilution series of 7-keto-sempervirol used in the phenotypic-based test. Each concentration was converted into corresponding Log10 values and the percentage viability was transformed into probit values to create a dose-response curve. An LD50 of 17.7 μM was calculated from this dose-response curve. One representative HFB is illustrated here. Dead control = NEJs co-cultured with 70 μM auranofin. DMSO control = NEJs co-cultured with 1% v/v DMSO.
Fig 8.
The diterpenoid 7-keto-sempervirol induces damage, shortening and loss of adult Fasciola hepatica tegumental spines.
SEM images of adult F. hepatica 48 hr post-treatment with culture media containing DMSO (1% v/v) or 100 μM 7-keto-sempervirol. A) Normal fluke ventral surface architecture including oral sucker (OS) and ventral sucker (VS) is unaffected after cultivation with culture media containing 1% (v/v) DMSO. B) Fluke dorsal surface architecture is also unaffected after cultivation with culture media containing 1% (v/v) DMSO. C) Fluke ventral spine phenotype after cultivation with culture media containing 1% (v/v) DMSO. D) Dorsal spine sockets (ss) and folding (fo) after cultivation with 100 μM 7-keto-sempervirol. E) Ventral spines after cultivation with 100 μM 7-keto-sempervirol. F) Higher magnification of (ss) region outlined by black box in (D).