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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes
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Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes

机译：通过靶向蛋白质-蛋白质复合物，基于结构的Sec7结构域的Arf激活抑制剂的发现

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摘要

Small molecules that produce nonfunctional protein-protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin-Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein-protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.
机译：产生非功能性蛋白质-蛋白质复合物的小分子是竞争性抑制剂对蛋白质功能的抑制的替代方法。在这里，我们的目标是通过鸟嘌呤核苷酸交换因子ARNO的Sec7催化域激活小GTP结合蛋白Arf1（膜运输的主要调节剂）。 Arf1-GDP / ARNO复合物的晶体结构引发交换反应，通过对Arf1 / ARNO界面附近的柔性腔进行计算机筛选，发现了抑制剂LM11。使用荧光动力学和各向异性，核磁共振波谱和诱变，我们表明LM11遵循非竞争机制的行为，其中抑制剂既针对Alf1-GDP和Arf1-GDP / ARNO复合物，又产生非功能性的Alf-GDP / ARNO复合物，其亲和力为类似于本地综合体。另外，LM11识别Arf / Sec7界面附近的Arf和ARNO的特征，这使人联想到范式界面抑制剂布雷菲德菌素A。然后我们证明LM11是一种细胞活性抑制剂，可损害高尔基地区依赖Arf的运输结构。 。此外，LM11抑制Madin-Darby犬肾（MDCK）细胞的ARNO依赖性迁移，表明ARNO是细胞中LM11的靶标。值得注意的是，LM11在体外抑制Arf1的激活，但不抑制Arf6的激活，这表明在细胞迁移中，ARNO在Arf1和Arf6激活之间可能存在协同作用。我们的设计方法表明，蛋白质-蛋白质复合物中的柔性区域为可药用位点提供了开发研究和抑制信号通路的新型工具的潜力。

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