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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.

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Secondary Perkinsus marinus growth-inhibition screen (IC50).Biological triplicate cultures were exposed to an 8 -point dose-response curve (10 µM to 0.156 µM). The effect of the drugs on P. marinus proliferation was evaluated as above.
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pone-0111051-g003: Secondary Perkinsus marinus growth-inhibition screen (IC50).Biological triplicate cultures were exposed to an 8 -point dose-response curve (10 µM to 0.156 µM). The effect of the drugs on P. marinus proliferation was evaluated as above.

Mentions: Three drug-like and three probe- like of the 13 compounds with the highest inhibitory effect on P. marinus (Table 2) were randomly selected for calculating the IC50 in an 8-point dose-response curve (10 µM to 0.156 µM). We found that the IC50 varied between 1.05 µM for MV66602 and 5.35 µM for MMV665941; for MV006522 the IC50 was 35.6 µM a high concentration or leaving the compound longer time would have resulted in a fitted sigmoidal curve. In this study the IC50 for the selected compounds (Figure 3) was in the lower µM range and much lower than for the compounds tested in our previous study [13], still it was higher than the corresponding P. falciparum IC50 values (Table 2); consequently, without knowing the mechanism of action of the compounds, we cannot rule out off-target effects due to non-specific cytotoxic agents, including detergent effects, multi-targeting and oxidative effects. The nature of the assays (Plasmodium falciparum relies on infected erythrocytes and the P. marinus screen is performed in the absence of the host cells) and culture medium can also account for the differences in the IC50 values. With a direct life cycle, P. marinus trophozoites are phagocytized by the oyster hemocytes [37], [38] where they resist oxidative killing [39]. Interestingly, MMV666021 has been involved in the inhibition of glutathione-S-transferase (GST) activity of prostaglandin D2 synthase (PGDS) [40]. GST are involved in parasite survival by protecting them against oxidative stress from the host or from products derived from their own metabolism [41], and in P. falciparum it has been associated with chloroquine-resistance [42]. We grow Perkinsus in a host cell-free culture medium; hence, if the MMV666021 is indeed affecting the oxidative stress, it is most likely dealing with the ROS derived from the parasites' own metabolism. Perkinsus marinus trophozoites have an expanded transporter repertoire, which is useful not only for transporting nutrients but also for secreting extracellular products (ECP) intended to inactivate the host defense and to break down host tissues [43], [44]. Protease activity variations significantly decrease the migration of hemocytes [44] and have been associated with differences in the average cell size and growth rate [45]. MMV665807 is believed to target transmembrane serine proteases. Interestingly, the P. marinus genome encodes multiple putative serine protease genes (e.g. XM_002788359, XM_002786609, XM_002766692); numerous studies have identified serine protease activities in the spent medium of cultured P. marinus and P. mediterraneus[46], [47] and mutations in the promoter region of serine protease inhibitors (SPIs) in C. virginica which confer resistance to Dermo disease [48], [49]. The parasite proteases could be the target of MMV665807; however, to prove this hypothesis would require further experiments outside the scope of this study.


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

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

Secondary Perkinsus marinus growth-inhibition screen (IC50).Biological triplicate cultures were exposed to an 8 -point dose-response curve (10 µM to 0.156 µM). The effect of the drugs on P. marinus proliferation was evaluated as above.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111051-g003: Secondary Perkinsus marinus growth-inhibition screen (IC50).Biological triplicate cultures were exposed to an 8 -point dose-response curve (10 µM to 0.156 µM). The effect of the drugs on P. marinus proliferation was evaluated as above.
Mentions: Three drug-like and three probe- like of the 13 compounds with the highest inhibitory effect on P. marinus (Table 2) were randomly selected for calculating the IC50 in an 8-point dose-response curve (10 µM to 0.156 µM). We found that the IC50 varied between 1.05 µM for MV66602 and 5.35 µM for MMV665941; for MV006522 the IC50 was 35.6 µM a high concentration or leaving the compound longer time would have resulted in a fitted sigmoidal curve. In this study the IC50 for the selected compounds (Figure 3) was in the lower µM range and much lower than for the compounds tested in our previous study [13], still it was higher than the corresponding P. falciparum IC50 values (Table 2); consequently, without knowing the mechanism of action of the compounds, we cannot rule out off-target effects due to non-specific cytotoxic agents, including detergent effects, multi-targeting and oxidative effects. The nature of the assays (Plasmodium falciparum relies on infected erythrocytes and the P. marinus screen is performed in the absence of the host cells) and culture medium can also account for the differences in the IC50 values. With a direct life cycle, P. marinus trophozoites are phagocytized by the oyster hemocytes [37], [38] where they resist oxidative killing [39]. Interestingly, MMV666021 has been involved in the inhibition of glutathione-S-transferase (GST) activity of prostaglandin D2 synthase (PGDS) [40]. GST are involved in parasite survival by protecting them against oxidative stress from the host or from products derived from their own metabolism [41], and in P. falciparum it has been associated with chloroquine-resistance [42]. We grow Perkinsus in a host cell-free culture medium; hence, if the MMV666021 is indeed affecting the oxidative stress, it is most likely dealing with the ROS derived from the parasites' own metabolism. Perkinsus marinus trophozoites have an expanded transporter repertoire, which is useful not only for transporting nutrients but also for secreting extracellular products (ECP) intended to inactivate the host defense and to break down host tissues [43], [44]. Protease activity variations significantly decrease the migration of hemocytes [44] and have been associated with differences in the average cell size and growth rate [45]. MMV665807 is believed to target transmembrane serine proteases. Interestingly, the P. marinus genome encodes multiple putative serine protease genes (e.g. XM_002788359, XM_002786609, XM_002766692); numerous studies have identified serine protease activities in the spent medium of cultured P. marinus and P. mediterraneus[46], [47] and mutations in the promoter region of serine protease inhibitors (SPIs) in C. virginica which confer resistance to Dermo disease [48], [49]. The parasite proteases could be the target of MMV665807; however, to prove this hypothesis would require further experiments outside the scope of this study.

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