Limits...
Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay.

Alemán Resto Y, Fernández Robledo JA - PLoS ONE (2014)

Bottom Line: Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites.The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species.The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

View Article: PubMed Central - PubMed

Affiliation: Research Experiences for Undergraduates (REU) NSF Program - 2013 - Bigelow Laboratory for Ocean Sciences, Boothbay, Maine, United States of America.

ABSTRACT
"Dermo" disease caused by the protozoan parasite Perkinsus marinus (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. Perkinsus spp. are also a concern worldwide because there are limited approaches to intervention against the disease. Based on the phylogenetic affinity between the Perkinsozoa and Apicomplexa, we exposed Perkinsus trophozoites to the Medicines for Malaria Venture Malaria Box, an open access compound library comprised of 200 drug-like and 200 probe-like compounds that are highly active against the erythrocyte stage of Plasmodium falciparum. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites. Six compounds with IC50 in the µM range were used to compare the degree of susceptibility in vitro of eight P. marinus strains from the USA and five Perkinsus species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species. Both Perkinsus marinus isolates and Perkinsus spp. presented different patterns of response to the panel of compounds tested, supporting the concept of strain/species variability. Here, we expanded the range of compounds available for inhibiting Perkinsus proliferation in vitro and characterized Perkinsus phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

Show MeSH

Related in: MedlinePlus

Percentage of inhibition of Perkinsus marinus using the MMV Malaria Box.Biological triplicate cultures were grown in sterile 96-well plates (100 µl; 2.0×106 cells/ml) and cells were exposed to the MMV Malaria Box (20 µM). The effect of the drugs on P. marinus proliferation was evaluated using the ATPlite assay at day 4 post-exposure to the selected drugs. Readings for each concentration were normalized to the control wells with each solvent (100% activity). A total of 122 (67.0%) compounds resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (Figure 2).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4206467&req=5

pone-0111051-g001: Percentage of inhibition of Perkinsus marinus using the MMV Malaria Box.Biological triplicate cultures were grown in sterile 96-well plates (100 µl; 2.0×106 cells/ml) and cells were exposed to the MMV Malaria Box (20 µM). The effect of the drugs on P. marinus proliferation was evaluated using the ATPlite assay at day 4 post-exposure to the selected drugs. Readings for each concentration were normalized to the control wells with each solvent (100% activity). A total of 122 (67.0%) compounds resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (Figure 2).

Mentions: In this study, we screened the MMV Malaria Box for compounds that might inhibit P. marinus proliferation in vitro, an approach that has been successfully used to identify compounds against other protozoan parasites [19]–[21]. In our previous study, the effect of the drugs on P. marinus proliferation was evaluated at days 2, 4, and 8 post-exposure; however, it was at day 4 post-exposure when the inhibitory effect(s) of most drugs tested became apparent [13]. Consequently, for the MMV Malaria Box screening we measured cell viability at day 4 post-exposure. We found that 46% of the compounds active against the P. falciparum erythrocyte life stage were also active against P. marinus trophozoites (Table S2). A total of 58 compounds (31.8%) resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (Figure 1). The repertory of available anti- Perkinsus drugs has gradually increased over the past two decades thanks to the establishment of the culture methodologies for Perkinsus spp. [28]–[30] (Figure 2A). Still, prior to this study, the number of available compounds against Perkinsus spp. was very limited (Figure 2B) compared to compounds against protozoan parasites of medical and veterinary relevance [31]–[34]. Previous screenings for compounds inhibiting Perkinsus proliferation have been based on the strong line of evidence for the presence in Perkinsus, like those in apicomplexan parasites, of pathways linked to a relic plastid [12], [13], [35], [36]. Here we have shown that the MMV Malaria Box offers a promising alternative way of finding compounds effective against Perkinsus spp.


Identification of MMV Malaria Box inhibitors of Perkinsus marinus using an ATP-based bioluminescence assay.

Alemán Resto Y, Fernández Robledo JA - PLoS ONE (2014)

Percentage of inhibition of Perkinsus marinus using the MMV Malaria Box.Biological triplicate cultures were grown in sterile 96-well plates (100 µl; 2.0×106 cells/ml) and cells were exposed to the MMV Malaria Box (20 µM). The effect of the drugs on P. marinus proliferation was evaluated using the ATPlite assay at day 4 post-exposure to the selected drugs. Readings for each concentration were normalized to the control wells with each solvent (100% activity). A total of 122 (67.0%) compounds resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (Figure 2).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206467&req=5

pone-0111051-g001: Percentage of inhibition of Perkinsus marinus using the MMV Malaria Box.Biological triplicate cultures were grown in sterile 96-well plates (100 µl; 2.0×106 cells/ml) and cells were exposed to the MMV Malaria Box (20 µM). The effect of the drugs on P. marinus proliferation was evaluated using the ATPlite assay at day 4 post-exposure to the selected drugs. Readings for each concentration were normalized to the control wells with each solvent (100% activity). A total of 122 (67.0%) compounds resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (Figure 2).
Mentions: In this study, we screened the MMV Malaria Box for compounds that might inhibit P. marinus proliferation in vitro, an approach that has been successfully used to identify compounds against other protozoan parasites [19]–[21]. In our previous study, the effect of the drugs on P. marinus proliferation was evaluated at days 2, 4, and 8 post-exposure; however, it was at day 4 post-exposure when the inhibitory effect(s) of most drugs tested became apparent [13]. Consequently, for the MMV Malaria Box screening we measured cell viability at day 4 post-exposure. We found that 46% of the compounds active against the P. falciparum erythrocyte life stage were also active against P. marinus trophozoites (Table S2). A total of 58 compounds (31.8%) resulted in at least 50% inhibition; from these compounds, 13 (7.1%) resulted in at least 90% inhibition (Figure 1). The repertory of available anti- Perkinsus drugs has gradually increased over the past two decades thanks to the establishment of the culture methodologies for Perkinsus spp. [28]–[30] (Figure 2A). Still, prior to this study, the number of available compounds against Perkinsus spp. was very limited (Figure 2B) compared to compounds against protozoan parasites of medical and veterinary relevance [31]–[34]. Previous screenings for compounds inhibiting Perkinsus proliferation have been based on the strong line of evidence for the presence in Perkinsus, like those in apicomplexan parasites, of pathways linked to a relic plastid [12], [13], [35], [36]. Here we have shown that the MMV Malaria Box offers a promising alternative way of finding compounds effective against Perkinsus spp.

Bottom Line: Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites.The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species.The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

View Article: PubMed Central - PubMed

Affiliation: Research Experiences for Undergraduates (REU) NSF Program - 2013 - Bigelow Laboratory for Ocean Sciences, Boothbay, Maine, United States of America.

ABSTRACT
"Dermo" disease caused by the protozoan parasite Perkinsus marinus (Perkinsozoa) is one of the main obstacles to the restoration of oyster populations in the USA. Perkinsus spp. are also a concern worldwide because there are limited approaches to intervention against the disease. Based on the phylogenetic affinity between the Perkinsozoa and Apicomplexa, we exposed Perkinsus trophozoites to the Medicines for Malaria Venture Malaria Box, an open access compound library comprised of 200 drug-like and 200 probe-like compounds that are highly active against the erythrocyte stage of Plasmodium falciparum. Using a final concentration of 20 µM, we found that 4 days after exposure 46% of the compounds were active against P. marinus trophozoites. Six compounds with IC50 in the µM range were used to compare the degree of susceptibility in vitro of eight P. marinus strains from the USA and five Perkinsus species from around the world. The three compounds, MMV666021, MMV665807 and MMV666102, displayed a uniform effect across Perkinsus strains and species. Both Perkinsus marinus isolates and Perkinsus spp. presented different patterns of response to the panel of compounds tested, supporting the concept of strain/species variability. Here, we expanded the range of compounds available for inhibiting Perkinsus proliferation in vitro and characterized Perkinsus phenotypes based on their resistance to six compounds. We also discuss the implications of these findings in the context of oyster management. The Perkinsus system offers the potential for investigating the mechanism of action of the compounds of interest.

Show MeSH
Related in: MedlinePlus