BACKGROUND Artemisinin-based combination therapy (ACT) is used as the first-line treatment of uncomplicated malaria caused by the Plasmodium falciparum parasite and chloroquine-resistant Plasmodium vivax parasites. Evidence of resistance to ACT has been reported in Cambodia, and without new and effective anti-malarial agents, malaria burden and mortality will rise. METHODS The used MolPrint 2D fingerprints and the Tanimoto similarity index were used to perform a structural similarity search within the Malaria Box collection to select diverse molecular scaffolds that are different from artesunate. Next, the inhibitory potency against the P. falciparum 3D7 strain (SYBR Green I inhibition assay) and the cytotoxicity against HepG2 cells (MTT and neutral red assays) were evaluated. Then, the speed of action, the combination profile of selected inhibitors with artesunate, and the P. berghei in vivo activity of the best compounds were assessed. RESULTS A set of 11 structurally diverse compounds from the Malaria Box with a similarity threshold of less than 0.05 was selected and compared with artesunate. The in vitro inhibitory activity of each compound confirmed the reported potencies (IC50 values ranging from 0.005 to 1 µM). The cytotoxicity of each selected compound was evaluated and used to calculate the selectivity index (SI values ranging from 15.1 to 6100). Next, both the speed of action and the combination profile of each compound with artesunate was assessed. Acridine, thiazolopyrimidine, quinoxaline, benzimidazole, thiophene, benzodiazepine, isoxazole and pyrimidoindole derivatives showed fast in vitro inhibitory activity of parasite growth, whereas hydrazinobenzimidazole, indenopyridazinone and naphthalenone derivatives were slow-acting in vitro inhibitors. Combinatory profile evaluation indicated that thiazolopyrimidinone and benzodiazepine derivatives have an additive profile, suggesting that the combination of these inhibitors with artesunate is favourable for in vitro inhibitory activity. The remaining compounds showed an antagonistic combinatory profile with artesunate. The collected data indicated that the indenopyridazinone derivative, a bc1 complex inhibitor, had a similar association profile in combination with proguanil when compared to atovaquone combined with proguanil, thereby corroborating the correlation between the molecular target and the combination profile. Lastly, the in vivo activity of the thiazolopyrimidinone and benzodiazepine derivatives were assessed. Both compounds showed oral efficacy at 50 mg/kg in a mouse model of Plasmodium berghei malaria (64% and 40% reduction in parasitaemia on day 5 post-infection, respectively). CONCLUSIONS The findings in this paper shed light on the relationship among the speed of action, molecular target and combinatory profile and identified new hits with in vivo activity as candidates for anti-malarial combination therapy.

中文翻译：

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疟疾盒中所选化合物的抗血浆特性：体外评估，作用速度和药物组合研究。

背景技术基于青蒿素的联合疗法（ACT）被用作对由恶性疟原虫和耐氯喹的间日疟原虫寄生虫引起的单纯性疟疾的一线治疗。柬埔寨已报告有对ACT产生抗药性的证据，如果没有新的有效抗疟药，疟疾负担和死亡率将会上升。方法使用所用的MolPrint 2D指纹图谱和Tanimoto相似性指数，在Malaria Box集合内进行结构相似性搜索，以选择与青蒿琥酯不同的各种分子支架。接下来，评估了对恶性疟原虫3D7菌株的抑制能力（SYBR Green I抑制测定）和对HepG2细胞的细胞毒性（MTT和中性红测定）。然后，行动的速度，评估了所选抑制剂与青蒿琥酯的组合情况以及最佳化合物的伯氏疟原虫的体内活性。结果从疟疾盒中选择了11种结构多样的化合物，其相似度阈值小于0.05，并与青蒿琥酯进行了比较。每种化合物的体外抑制活性均证实了所报道的效力（IC50值范围为0.005至1 µM）。评估每种所选化合物的细胞毒性，并将其用于计算选择性指数（SI值范围为15.1至6100）。接下来，评估了作用速度以及每种化合物与青蒿琥酯的组合曲线。cr啶，噻唑并嘧啶，喹喔啉，苯并咪唑，噻吩，苯并二氮杂卓，异恶唑和嘧啶吲哚衍生物对寄生虫的生长具有快速的体外抑制活性，肼基苯并咪唑，茚并哒嗪酮和萘酮衍生物是体外作用缓慢的抑制剂。组合曲线评估表明，噻唑并吡咯烷二酮和苯并二氮杂derivatives衍生物具有累加曲线，表明这些抑制剂与青蒿琥酯的组合对体外抑制活性有利。其余化合物与青蒿琥酯表现出拮抗的组合性质。收集的数据表明，与阿托伐醌与丙胍联合使用相比，bc1复合抑制剂茚并哒嗪酮衍生物与丙胍联合使用时具有相似的缔合曲线，从而证实了分子靶标与结合曲线之间的相关性。最后，评估了噻唑并吡咯烷二酮和苯并二氮杂卓衍生物的体内活性。两种化合物在伯氏疟原虫疟疾的小鼠模型中均以50 mg / kg的剂量表现出口服功效（感染后第5天的寄生虫血症分别降低了64％和40％）。结论本文的研究结果阐明了作用速度，分子靶标和组合谱之间的关系，并确定了具有体内活性的新命中药物作为抗疟疾联合疗法的候选药物。