Chepngetich Jean, Muriithi Brenda, Gachie Beatrice, Thiong'o Kevin, Jepkorir Mercy, Gathirwa Jeremiah, Kimani Francis, Mwitari Peter, Kiboi Daniel
Department of Molecular Biology and Biotechnology, Pan African University Institute for Basic Sciences, Technology and Innovation, Nairobi, 62000, 00200, Kenya.
Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, Nairobi, 54840, 00200, Kenya.
Open Res Afr. 2023 Oct 19;5:28. doi: 10.12688/openresafrica.13436.1. eCollection 2022.
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 kinases with LM, PQ, and AQ resistance remains unclear. Using LM, PQ, or AQ-resistant 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 ANKA.
We used 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 measured the mRNA amount of PANK1, DAGK, and PI4Kβ at trophozoites and schizonts stages.
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.
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 their impact on drug responses require investigations using reverse genetics techniques in malaria parasites.
双氢青蒿素哌喹（LM）、磷酸哌喹（PQ）和阿莫地喹（AQ）是青蒿素联合疗法（ACTs）的长效成分，是非洲疟疾治疗的基石。研究表明，PQ、AQ和LM耐药性可能独立于预测的作用模式出现。蛋白激酶已成为疟原虫药物作用和疗效的介质；然而，可靶向的主要激酶与LM、PQ和AQ耐药性之间的联系仍不清楚。我们使用对LM、PQ或AQ耐药的疟原虫，评估了胆碱激酶（CK）、泛酸激酶1（PANK1）、二酰基甘油激酶（DAGK）、磷脂酰肌醇-4激酶（PI4Kβ）和钙依赖性蛋白激酶1（CDPK1）与ANKA株疟原虫对LM、PQ和AQ耐药性的关联。
我们使用生物信息学工具来识别不同疟原虫之间的配体结合基序、活性位点和序列保守性。然后，我们使用聚合酶链反应（PCR）和测序分析来探测CK、PANK1、DAGK、PI4Kβ和CDPK1中预测功能基序内的单核苷酸多态性（SNP）。使用定量聚合酶链反应（qPCR）分析，我们测量了滋养体和裂殖体阶段PANK1、DAGK和PI4Kβ的mRNA量。
我们揭示了不同疟原虫物种中CK、PANK1、DAGK、PI4Kβ和CDPK1的序列保守性和独特的配体结合基序。DAGK、PANK1和PI4Kβ存在非同义突变；令人惊讶的是，这些突变仅发生在对AQ耐药的疟原虫中。PANK1获得了Asn394His突变，而DAGK包含K270R和K292R突变。PI4Kβ有Asp366Asn、Ser1367Arg、Tyr1394Asn和Asp1423Asn突变。我们发现，在对AQ耐药的疟原虫中，滋养体阶段PANK1、DAGK和PI4Kβ表达下调，但在裂殖体阶段表达上调。
对AQ耐药的疟原虫中选择性获得的突变和差异基因表达可能表明这些蛋白质受到AQ压力的影响。这些突变在耐药疟原虫中的作用及其对药物反应的影响需要使用疟原虫反向遗传学技术进行研究。
