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

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.

Figure 1. 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).

Figure 2. Drug discovery against Dermo disease.

(A) Time line for the discovery of drugs against Dermo disease, starting when the etiological agent was described until this study. Most of the discoveries did happen after the development of the culture methodologies for Perkinsus spp. in 1993 and most studies have been carried out in in vitro cultures. (B) Percentage of the compounds active against Perkinsus based on their chemical nature. (C) Percentage of available compounds against Dermo tested in Perkinsus marinus and Perkinsus olseni.

Figure 3. 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.