Identification of MMV Malaria Box Inhibitors of Perkinsus marinus Using an ATP-Based Bioluminescence Assay

“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.

丁莫（Dermo）病由原生动物寄生虫帕金虫（Perkinsus marinus）（隶属于帕金虫门（Perkinsozoa））引发，是美国牡蛎种群恢复面临的核心障碍之一。帕金虫属（Perkinsus spp.）在全球范围内均受到广泛关注，因为目前针对该类疾病的干预手段极为有限。基于帕金虫门与顶复门（Apicomplexa）之间的系统发育亲缘关系，我们将帕金虫滋养体（Perkinsus trophozoites）暴露于疟疾药物创新伙伴（Medicines for Malaria Venture）疟疾化合物库——这是一个开放获取的化合物文库，包含200种类药物化合物与200种探针类化合物，对恶性疟原虫（Plasmodium falciparum）的红细胞阶段具有强效抗疟活性。我们采用20 μM的终浓度开展实验，结果显示：化合物暴露4天后，有46%的受试化合物可有效抑制帕金虫滋养体的增殖。我们选取了6个半数抑制浓度（IC50）处于微摩尔级别的化合物，用于对比分析8株美国本土帕金虫（P. marinus）菌株以及全球范围内5种帕金虫属物种的体外易感性差异。其中MMV666021、MMV665807与MMV666102这3种化合物，在所有测试的帕金虫菌株与物种中均表现出稳定一致的作用效果。帕金虫（P. marinus）分离株与其他帕金虫属物种对该受试化合物组合呈现出截然不同的响应模式，这印证了帕金虫菌株/物种间存在易感性差异的学术观点。本研究不仅拓宽了体外可用于抑制帕金虫增殖的化合物谱系，还基于6种受试化合物的抗性特征，对帕金虫的表型进行了系统表征。此外，我们还探讨了上述研究结果在牡蛎种群管理中的应用意义。帕金虫模型为探究目标化合物的作用机制提供了极具潜力的研究途径。