■棘阿米巴感染是一个严重的公共卫生问题,需要开发有效和安全的抗棘阿米巴化学疗法。聚(ADP-核糖)聚合酶(PARP)控制着大量的生物过程,比如DNA损伤修复,蛋白质降解和凋亡。多种PARP靶向化合物已被批准用于癌症治疗。然而,对PARP抑制剂治疗棘阿米巴的再利用知之甚少。
■在本研究中,我们试图通过进行抗棘阿米巴功效测定来填补这些知识空白,细胞生物学实验,生物信息学,和转录组学分析。
■使用棘阿米巴聚(ADP-核糖)聚合酶(PARPs)的同源模型,已批准药物的分子对接揭示了三种潜在的抑制化合物:奥拉帕尼,venadaparib和AZ9482。特别是,venadaparib表现出优异的对接得分(-13.71)和良好的预测结合自由能(-89.28kcal/mol),其次是AZ9482,其显示-13.20的对接得分和-92.13kcal/mol的结合自由能。值得注意的是,venadaparib中带正电荷的环丙胺在结合袋中建立了盐桥(通过E535)和氢键(通过N531)。为了比较,AZ9482被周围的芳族残基(包括H625、Y652、Y659和Y670)很好地堆叠。在对滋养体生存能力的评估中,AZ9482通过抑制棘阿米巴PARP活性表现出剂量和时间依赖性的抗滋养体作用,不同于奥拉帕利和韦纳帕利。膜联蛋白V-异硫氰酸荧光素/碘化丙啶凋亡测定显示AZ9482诱导滋养体坏死细胞死亡而不是凋亡。对AZ9482处理的棘阿米巴滋养体进行的转录组学分析显示了差异调节的蛋白质和基因的图谱,并发现AZ9482迅速上调滋养体的大量DNA损伤修复途径,有趣地下调了几个毒力基因。分析与DNA损伤修复途径相关的基因表达和嘌呤/嘧啶(AP)位点的速率表明AZ9482处理后棘阿米巴滋养体的DNA损伤功效和修复调节。
■集体,这些发现突出了AZ9482作为一种结构独特的PARP抑制剂,为推进抗棘阿米巴药物研究提供了有希望的原型。
UNASSIGNED: Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood.
UNASSIGNED: In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses.
UNASSIGNED: Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of
trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba
trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in
trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba
trophozoites following AZ9482 treatment.
UNASSIGNED: Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.