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The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

Edwards J, Brown M, Peak E, Bartholomew B, Nash RJ, Hoffmann KF - PLoS Negl Trop Dis (2015)

Bottom Line: This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology.As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM).Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss. 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, United Kingdom.

ABSTRACT

Background: Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control.

Methodology/ principle findings: Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss.

Conclusions/ significance: 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.

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Related in: MedlinePlus

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.
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pntd.0003604.g007: 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.

Mentions: To assess 7-keto-sempervirol’s activity on F. hepatica NEJs, two complementary methodologies were employed (Fig. 7). The first methodology, using well-established motility and phenotypic metrics [31,32], indicated that 7-keto-sempervirol induced a negative concentration-dependent effect on NEJ movement and viability (Fig. 7A). This finding was supported by fluorescent microscopy of NEJs co-stained with the discriminatory viability dyes FDA and PI (Fig. 7A). The second methodology, using the HFB as a more objective method for determining NEJ viability [25], confirmed this concentration-dependent effect and established an LD50 of 17.7 μM for 7-keto-sempervirol against F. hepatica NEJs (Fig. 7B). When compared to the mammalian HepG2 cell line (S1 Fig.), 7-keto-sempervirol displayed an anti-NEJ therapeutic index of 4.5. To identify whether 7-keto-sempervirol also affected the surface integrity of F. hepatica adults, similar to S. mansoni (Fig. 4), SEM analyses were performed on adult liver flukes co-cultured with this diterpenoid (Fig. 8). Here, in comparison to control flukes incubated with 1% (v/v) DMSO (Fig. 8A-C), prolonged (48 hr) exposure to 7-keto-sempervirol (50 μM) induced substantial spine shortening and spine loss that was apparent on both dorsal and ventral sides of the organism (Fig. 8D-F).


The diterpenoid 7-keto-sempervirol, derived from Lycium chinense, displays anthelmintic activity against both Schistosoma mansoni and Fasciola hepatica.

Edwards J, Brown M, Peak E, Bartholomew B, Nash RJ, Hoffmann KF - PLoS Negl Trop Dis (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358835&req=5

pntd.0003604.g007: 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.
Mentions: To assess 7-keto-sempervirol’s activity on F. hepatica NEJs, two complementary methodologies were employed (Fig. 7). The first methodology, using well-established motility and phenotypic metrics [31,32], indicated that 7-keto-sempervirol induced a negative concentration-dependent effect on NEJ movement and viability (Fig. 7A). This finding was supported by fluorescent microscopy of NEJs co-stained with the discriminatory viability dyes FDA and PI (Fig. 7A). The second methodology, using the HFB as a more objective method for determining NEJ viability [25], confirmed this concentration-dependent effect and established an LD50 of 17.7 μM for 7-keto-sempervirol against F. hepatica NEJs (Fig. 7B). When compared to the mammalian HepG2 cell line (S1 Fig.), 7-keto-sempervirol displayed an anti-NEJ therapeutic index of 4.5. To identify whether 7-keto-sempervirol also affected the surface integrity of F. hepatica adults, similar to S. mansoni (Fig. 4), SEM analyses were performed on adult liver flukes co-cultured with this diterpenoid (Fig. 8). Here, in comparison to control flukes incubated with 1% (v/v) DMSO (Fig. 8A-C), prolonged (48 hr) exposure to 7-keto-sempervirol (50 μM) induced substantial spine shortening and spine loss that was apparent on both dorsal and ventral sides of the organism (Fig. 8D-F).

Bottom Line: This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology.As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM).Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss. 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biological, Environmental and Rural Sciences (IBERS), Aberystwyth University, Aberystwyth, United Kingdom.

ABSTRACT

Background: Two platyhelminths of biomedical and commercial significance are Schistosoma mansoni (blood fluke) and Fasciola hepatica (liver fluke). These related trematodes are responsible for the chronic neglected tropical diseases schistosomiasis and fascioliasis, respectively. As no vaccine is currently available for anti-flukicidal immunoprophylaxis, current treatment is mediated by mono-chemical chemotherapy in the form of mass drug administration (MDA) (praziquantel for schistosomiasis) or drenching (triclabendazole for fascioliasis) programmes. This overreliance on single chemotherapeutic classes has dramatically limited the number of novel chemical entities entering anthelmintic drug discovery pipelines, raising significant concerns for the future of sustainable blood and liver fluke control.

Methodology/ principle findings: Here we demonstrate that 7-keto-sempervirol, a diterpenoid isolated from Lycium chinense, has dual anthelmintic activity against related S. mansoni and F. hepatica trematodes. Using a microtiter plate-based helminth fluorescent bioassay (HFB), this activity is specific (Therapeutic index = 4.2, when compared to HepG2 cell lines) and moderately potent (LD50 = 19.1 μM) against S. mansoni schistosomula cultured in vitro. This anti-schistosomula effect translates into activity against both adult male and female schistosomes cultured in vitro where 7-keto-sempervirol negatively affects motility/behaviour, surface architecture (inducing tegumental holes, tubercle swelling and spine loss/shortening), oviposition rates and egg morphology. As assessed by the HFB and microscopic phenotypic scoring matrices, 7-keto-sempervirol also effectively kills in vitro cultured F. hepatica newly excysted juveniles (NEJs, LD50 = 17.7 μM). Scanning electron microscopy (SEM) evaluation of adult F. hepatica liver flukes co-cultured in vitro with 7-keto-sempervirol additionally demonstrates phenotypic abnormalities including breaches in tegumental integrity and spine loss.

Conclusions/ significance: 7-keto-sempervirol negatively affects the viability and phenotype of two related pathogenic trematodes responsible for significant human and animal infectious diseases. This plant-derived, natural product is also active against both larval and adult developmental forms. As such, the data collectively indicate that 7-keto-sempervirol is an important starting point for anthelmintic drug development. Medicinal chemistry optimisation of more potent 7-keto-sempervirol analogues could lead to the identification of novel chemical entities useful for future combinatorial or replacement anthelmintic control.

Show MeSH
Related in: MedlinePlus