Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Avenida do Café, Ribeirão Preto, Brazil.
Laboratório de Cristalografia de Proteínas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903 Ribeirão Preto, Brazil; Ludwig Institute for Cancer Research, University of California, School of Medicine, 92093-0669 La Jolla, CA, USA.
Int J Biol Macromol. 2021 Sep 30;187:964-975. doi: 10.1016/j.ijbiomac.2021.07.108. Epub 2021 Jul 24.
Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KHPO, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 °C). rNcGR exhibited a Michaelian behavior (K:0.10 ± 0.02 mM; k:0.076 ± 0.003 s; K:0.006 ± 0.001 mM; k: 0.080 ± 0.003 s). The IC of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 ± 0.2 μM, 11 ± 2 μM, 0.7 ± 0.1 μM, and 0.9 ± 0.2 μM, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition.
刚地弓形虫导致牛类流产造成严重损失。该寄生虫形成了一个复杂的防御氧化还原系统，包括谷胱甘肽还原酶（GR）等酶。亚甲蓝（MB）可降低重组型疟原虫 GR 的活性，并抑制寄生虫在体内和体外的增殖。同样，MB 及其衍生物可抑制刚地弓形虫的增殖，但 MB 的作用机制是否与 GR 功能相关仍不清楚。因此，本研究对刚地弓形虫 GR（NcGR）进行了表征，并确定了其潜在的抑制剂。NcGR 存在于速殖子细胞质中，其结构和序列与同源物相似。我们在 340nm 处检测到 NADPH 吸光度（100mM KHPO，pH7.5，1mM EDTA，离子强度：600mM，25°C）时，验证了 rNcGR（875nM）的体外活性。rNcGR 表现出米氏行为（K：0.10±0.02mM；k：0.076±0.003s；K：0.006±0.001mM；k：0.080±0.003s）。MB、1,9-二甲基亚甲蓝、新亚甲蓝和甲苯胺蓝 O 对 rNcGR 活性的 IC 分别为 2.1±0.2μM、11±2μM、0.7±0.1μM 和 0.9±0.2μM。我们的研究结果表明 NcGR 在刚地弓形虫生物学和抗氧化机制中具有重要意义。此外，本文的数据强烈表明 NcGR 是刚地弓形虫增殖抑制中苯并噻嗪染料的重要靶标。
