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Abstract：
Background: Lumefantrine (LM), piperaquine (PQ), and amodiaquine (AQ), the long-acting components of the artemisinin-based combination therapies (ACTs), are a cornerstone of malaria treatment in Africa. Studies have shown that PQ, AQ, and LM resistance may arise independently of predicted modes of action. Protein kinases have emerged as mediators of drug action and efficacy in malaria parasites; however, the link between top druggable Plasmodium kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant Plasmodium berghei parasites, we have evaluated the association of choline kinase (CK), pantothenate kinase 1 (PANK1), diacylglycerol kinase (DAGK), and phosphatidylinositol-4 kinase (PI4Kβ), and calcium-dependent protein kinase 1 (CDPK1) with LM, PQ, and AQ resistance in Plasmodium berghei ANKA. Methods: We used in silico bioinformatics tools to identify ligand-binding motifs, active sites, and sequence conservation across the different parasites. We then used PCR and sequencing analysis to probe for single nucleotide polymorphisms (SNPs) within the predicted functional motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1. Using qPCR analysis, we finally measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages. Results: We reveal sequence conservation and unique ligand-binding motifs in the CK, PANK1, DAGK, PI4Kβ, and CDPK1 across malaria species. DAGK, PANK1, and PI4Kβ possessed nonsynonymous mutations; surprisingly, the mutations only occurred in the AQr parasites. PANK1 acquired Asn394His while DAGK contained K270R and K292R mutations. PI4Kβ had Asp366Asn, Ser1367Arg, Tyr1394Asn and Asp1423Asn. We show downregulation of PANK1, DAGK, and PI4Kβ in the trophozoites but upregulation at the schizonts stages in the AQr parasites. Conclusions: The selective acquisition of the mutations and the differential gene expression in AQ-resistant parasites may signify proteins under AQ pressure. The role of the mutations in the resistant parasites and the impact on drug responses require further investigations in malaria parasites.
摘要：
背景：Lumefantrine（LM），哌喹（PQ），和阿莫地喹（AQ），以青蒿素为基础的联合疗法（ACTs）的长效成分，是非洲疟疾治疗的基石。研究表明PQ,AQ,和LM阻力可能独立于预测的作用模式而出现。蛋白激酶已成为疟疾寄生虫中药物作用和功效的介体；然而，顶级可药用疟原虫激酶与LM之间的联系，PQ,AQ抗性仍不清楚。使用LM，PQ,或者抗AQ的伯氏疟原虫寄生虫，我们已经评估了胆碱激酶（CK），泛酸激酶1（PANK1），二酰基甘油激酶（DAGK），和磷脂酰肌醇-4激酶（PI4Kβ），和钙依赖性蛋白激酶1（CDPK1）与LM，PQ,伯氏疟原虫ANKA的AQ抗性。方法：我们使用电子生物信息学工具来鉴定配体结合基序，活跃的网站，以及不同寄生虫的序列保守。然后，我们使用PCR和测序分析来探测CK中预测的功能基序内的单核苷酸多态性（SNP）。PANK1,DAGK,PI4Kβ,和CDPK1。使用qPCR分析，我们最终测量了PANK1，DAGK，和PI4Kβ在滋养体和裂殖体阶段。结果：我们揭示了CK中的序列保守性和独特的配体结合基序，PANK1,DAGK,PI4Kβ,和CDPK1跨疟疾物种。DAGK,PANK1和PI4Kβ具有非同义突变；令人惊讶的是，突变仅发生在AQr寄生虫中。PANK1获得Asn394His,而DAGK含有K270R和K292R突变。PI4Kβ有Asp366Asn，Ser1367Arg,Tyr1394Asn和Asp1423Asn。我们显示了PANK1、DAGK、和PI4Kβ在滋养体中，但在AQr寄生虫的分裂阶段上调。结论：AQ抗性寄生虫中突变的选择性获得和差异基因表达可能表明AQ压力下的蛋白质。突变在抗药性寄生虫中的作用以及对药物反应的影响需要在疟疾寄生虫中进行进一步研究。
