A Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi
A Novel Antifungal Compound Z-705 Specifically Inhibits Protein Kinase C of Filamentous Fungi
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新型抗真菌化合物 Z-705 特异性抑制丝状真菌的蛋白激酶 C
DOI：
10.1128/aem.02923-18
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发表时间：
2019
影响因子：
4.4
通讯作者：
Keietsu Abe
中科院分区：
文献类型：
--
作者：
Asume Sugahara;Akira Yoshimi;Fumio Shoji;Tomonori Fujioka;Kiyoshi Kawai;Hideaki Umeyama;Katsuichiro Komatsu;Masaru Enomoto;Shigefumi Kuwahara;Daisuke Hagiwara;Takuya Katayama;Hiroyuki Horiuchi;Ken Miyazawa;Mayumi Nakayama;Keietsu Abe
细胞壁完整性信号传导（CWIS）途径参与真菌细胞壁的生物发生。该通路由传感器蛋白、蛋白激酶C（PKC）和丝裂原激活蛋白激酶（MAPK）通路组成，控制许多细胞壁相关基因的转录。 PKC 在该通路中发挥着关键作用；模式丝状真菌构巢曲霉中的 PkcA 缺陷和稻瘟病菌 Magnaporthe grisea 中的 MgPkc1p 缺陷是致命的。这表明丝状真菌中的 PKC 是抗真菌药物的潜在靶点。在本研究中，为了寻找 MgPkc1p 抑制剂，我们通过三维 (3D) 结构模型进行了硅筛选，并在琼脂平板上进行了稻瘟病菌的生长抑制测试。我们从大约 800,000 种候选化合物中选择了 Z-705，并评估了其对酿酒酵母细胞中表达的嵌合 PKC 的抑制活性，其中天然酿酒酵母 PKC 的激酶结构域被丝状真菌 PKC 的激酶结构域取代。对酿酒酵母中编码 PKC 下游因子的 MLP1 进行转录分析，并对 PKC 下游激活的丝裂原激活蛋白激酶 (MAPK) Mpk1p 进行磷酸化分析，结果表明 Z-705 特异性抑制丝状真菌的 PKC。此外，Z-705的抑制活性与众所周知的PKC抑制剂星形孢菌素相似。有趣的是，Z-705 抑制 M 细胞壁应激诱导的黑化。稻瘟病。我们讨论了 PKC 和黑色素生物合成之间的关系。 重要性 通过硅筛选鉴定出丝状真菌蛋白激酶 C (PKC) 的候选抑制剂 Z-705。在酿酒酵母中构建了评估真菌 PKC 抑制剂效果的筛选系统。使用该系统，我们发现与酿酒酵母 PKC 相比，Z-705 对丝状真菌 PKC 具有高度选择性。对稻瘟菌 Magnaporthe grisea 中 AGS1mRNA 水平的分析表明，Z-705 具有与十字孢菌素相当的 PKC 抑制作用，AGS1mRNA 水平由 Mps1p 丝裂原激活蛋白激酶 (MAPK) 通过 PKC 调节。米卡芬净诱导 M 菌丝黑化。稻瘟病菌，以及这种黑化，这是稻瘟病菌致病性所必需的。稻瘟病菌被 Z-705 和十字孢菌素的 PKC 抑制作用所抑制。 4HNR、3HNR 和 SCD1 的 mRNA 水平对于 M 的黑化至关重要。稻瘟病菌，均被两种 PKC 抑制剂抑制。
The cell wall integrity signaling (CWIS) pathway is involved in fungal cell wall biogenesis. This pathway is composed of sensor proteins, protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) pathway, and it controls the transcription of many cell wall-related genes. PKC plays a pivotal role in this pathway; deficiencies in PkcA in the model filamentous fungus Aspergillus nidulans and in MgPkc1p in the rice blast fungus Magnaporthe grisea are lethal. This suggests that PKC in filamentous fungi is a potential target for antifungal agents. In the present study, to search for MgPkc1p inhibitors, we carried outin silicoscreening by three-dimensional (3D) structural modeling and performed growth inhibition tests for M. grisea on agar plates. From approximately 800,000 candidate compounds, we selected Z-705 and evaluated its inhibitory activity against chimeric PKC expressed in Saccharomyces cerevisiae cells in which the kinase domain of native S. cerevisiae PKC was replaced with those of PKCs of filamentous fungi. Transcriptional analysis ofMLP1, which encodes a downstream factor of PKC in S. cerevisiae, and phosphorylation analysis of the mitogen-activated protein kinase (MAPK) Mpk1p, which is activated downstream of PKC, revealed that Z-705 specifically inhibited PKCs of filamentous fungi. Moreover, the inhibitory activity of Z-705 was similar to that of a well-known PKC inhibitor, staurosporine. Interestingly, Z-705 inhibited melanization induced by cell wall stress inM. grisea. We discuss the relationships between PKC and melanin biosynthesis.IMPORTANCEA candidate inhibitor of filamentous fungal protein kinase C (PKC), Z-705, was identified byin silicoscreening. A screening system to evaluate the effects of fungal PKC inhibitors was constructed in Saccharomyces cerevisiae. Using this system, we found that Z-705 is highly selective for filamentous fungal PKC in comparison with S. cerevisiae PKC. Analysis of theAGS1mRNA level, which is regulated by Mps1p mitogen-activated protein kinase (MAPK) via PKC, in the rice blast fungus Magnaporthe grisea revealed that Z-705 had a PKC inhibitory effect comparable to that of staurosporine. Micafungin induced hyphal melanization inM. grisea, and this melanization, which is required for pathogenicity ofM. grisea, was inhibited by PKC inhibition by both Z-705 and staurosporine. The mRNA levels of4HNR,3HNR, andSCD1, which are essential for melanization inM. grisea, were suppressed by both PKC inhibitors.