The binuclear cyclopalladated complex CP2 is targeting ubiquinol-cytochrome c reductase (complex III) of Leishmania amazonensis

Angela Maria Arenas Velásquez , Irwin Alexander Patino Linares , Lawrence D. Gaspers , Paula J. Bartlett , Jecika M. Velasques , Adelino V.G. Netto , Andrew P. Thomas , Marcia A.S. Graminha
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Abstract

Leishmaniasis is a neglected disease that remains with a limited number of drugs available for chemotherapy and has an increased drug resistance that affects treatment outcomes. Metal-based drugs such as cyclopalladated complex [Pd(dmba)(μ-N3)]2 (CP2), a Leishmania topoisomerase IB inhibitor involved in calcium dysregulation and mitochondrial dysfunction of the parasite, had been an alternative to outline the appearance of chemoresistance. To identify new molecular targets and point out possible resistance mechanisms, a CP2-resistant Leishmania amazonensis (LaR) was selected by stepwise exposure to increasing drug pressure until a line capable of growth in 13.3 μM CP2. LaR IC50 value was 52.4 μM (4-fold higher than L. amazonensis–wild type, La). LaR promastigotes were cross-resistant to other DNA topoisomerase I inhibitors (camptothecin) and more susceptible to anti-leishmanial drugs pentamidine and miltefosine. A protective effect on cell viability was observed by pretreating the parasite with Ca2+ channel blockers followed by CP2 in La but not in LaR. Analyses of the cell viability of La and LaR using electron transport chain (ETC) inhibitors demonstrated that La is more sensitive than LaR. The studies of mitochondrial oxygen consumption demonstrated that LaR is less susceptible to complex III (ubiquinol-cytochrome c reductase – CcR) inhibitor, antimycin A (AA). CcR activities of La and LaR were equal for both strains in the absence of CP2 and significantly decreased, 69 % for La and 51 % for LaR, in the presence of CP2. This resistance is attributed to overexpression of CcR, confirmed by the RT-qPCR. CcR inhibition by CP2 leads the parasite to increase the reactive oxygen species (ROS) production, principally in La. Therefore, in this work, we suggested that CcR is the main target of CP2 in the mitochondria, acting to inhibit mitochondria respiratory, and the LaR mutant has increased activity of CcR, which reduces the formation of ROS.

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双核环palladated复合物CP2靶向亚马逊利什曼原虫的泛醇-细胞色素c还原酶(复合物III)。
利什曼病是一种被忽视的疾病,用于化疗的药物数量仍然有限,而且耐药性增加,影响治疗结果。金属基药物,如环钯化络合物[Pd(dmba)(μ-N3)]2 (CP2),一种利什曼原虫拓扑异构酶IB抑制剂,参与寄生虫钙失调和线粒体功能障碍,已成为概述化学耐药外观的替代方案。为了寻找新的分子靶点并指出可能的耐药机制,通过逐步增加药物压力,直到能够在13.3 μM的CP2中生长,选择了耐CP2的亚马逊利什曼原虫(Leishmania amazonensis, LaR)。LaR的IC50值为52.4 μM,是野生型L. amazonensis的4倍。LaR promastigotes对其他DNA拓扑异构酶I抑制剂(喜树碱)有交叉耐药,对抗利什曼原虫药物喷他脒和米替福辛更敏感。用Ca2+通道阻滞剂和CP2在La(而不是LaR)中预处理寄生虫,观察到对细胞活力的保护作用。利用电子传递链(ETC)抑制剂对La和LaR的细胞活力分析表明,La比LaR更敏感。线粒体耗氧量的研究表明,LaR对复合物III(泛醇-细胞色素c还原酶- CcR)抑制剂抗霉素A (AA)的敏感性较低。在CP2不存在的情况下,La和LaR的CcR活性相等,而在CP2存在的情况下,La和LaR的CcR活性显著降低,分别为69%和51%。RT-qPCR证实,这种抗性归因于CcR的过表达。CP2抑制CcR导致寄生虫增加活性氧(ROS)的产生,主要是在La。因此,在本工作中,我们提出CcR是CP2在线粒体中的主要靶点,起到抑制线粒体呼吸的作用,而LaR突变体增加了CcR的活性,从而减少了ROS的形成。
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来源期刊
CiteScore
7.90
自引率
7.50%
发文量
31
审稿时长
48 days
期刊介绍: The International Journal for Parasitology – Drugs and Drug Resistance is one of a series of specialist, open access journals launched by the International Journal for Parasitology. It publishes the results of original research in the area of anti-parasite drug identification, development and evaluation, and parasite drug resistance. The journal also covers research into natural products as anti-parasitic agents, and bioactive parasite products. Studies can be aimed at unicellular or multicellular parasites of human or veterinary importance.
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