Selective chemical intervention in membrane trafficking - designing interfacial inhibitors specific to Arf1/Arf-GEF complexes

所有的动物细胞都含有多个被称为细胞器的膜结合结构。每种类型的细胞器都含有一种特征性的蛋白质补充物，以确保代谢区室化。维持细胞器的身份并确保它们之间的物质准确运输是所有细胞功能的基础。蛋白质进入（内吞作用）和离开（胞吐作用）细胞的运输涉及它们通过一系列细胞器的调节运动，这些细胞器包括所谓的内吞/分泌途径。在不同的阶段，运输涉及将蛋白质货物包裹在小泡或小管内，这些小泡或小管充当载体，以准确地靶向沿着途径的下一个中转站。在这个过程中，必须保持接收和发送细胞器的同一性。在这个项目中，我们建议开发特定的化学工具来详细剖析分泌途径的运输步骤。有大量关于参与囊泡转运步骤的关键蛋白（小GTP酶的Arf家族）的身份、功能甚至分子结构的信息。Arf蛋白存在于两种状态（开和关），并通过其他蛋白质在这些状态之间切换，这些蛋白质是特定的。我们建议开发两种目前可用的小化学抑制剂，其靶向Arf 1与其激活剂的复合物以防止其激活。该方法利用了一种称为界面抑制剂的新型化学抑制剂。大多数现有的化学品都是针对酶的“活性位点”或抑制反应中所涉及的成分的结合。相反，界面抑制剂在其功能周期期间将蛋白质复合物捕获在“死端”点，使得它们不再能够在细胞中发挥作用。不幸的是，Arf 1的经典界面抑制剂同时引起多个途径的广泛破坏，因为它不仅靶向一个配对（即一个Arf 1 GTdR及其特异性激活剂在一个隔室上），而且靶向许多这样的配对，从而防止多个含Arf 1复合物的激活。尽管如此，这种抑制剂的使用已经导致了数以千计的出版物，这些出版物给出了更特异性抑制剂的潜力的一些想法。我们现在正在提议合成现有界面抑制剂的改进版本，以产生在其作用中更具体的化学物质。通过这种方式，将生成可用于区分蛋白质转运中不同Arf 1介导步骤的工具。Arf 1及其激活剂和经典抑制剂的分子结构是已知的。这使我们能够模拟其他抑制剂和其他密切相关的蛋白质配对。我们的跨学科团队包括化学家，分子建模师，细胞生物学家和生物化学家，并且已经产生了大量的数据，包括现有化合物之间的关键差异的表征，这些差异为我们未来的修改提供了信息。我们合成的分子的特异性将使用自动化细胞成像和基于细胞的生化分析进行测试。我们在这方面拥有丰富的专业知识，所有的技术都已到位。拟议的工作具有巨大的潜力，因为Arf 1的特异性抑制剂将为科学界提供一套新的工具，用于探测不仅与高尔基体堆叠相关的贩运途径，而且与其他细胞器，如transGolgi网络（TGN）和内体。这一点也很重要，因为我们靶向的一组分子调节着一个小G蛋白超家族，该家族参与几乎所有的细胞过程，包括生长、分裂、代谢和信号传导。因此，从长远来看，我们从这个项目中学到的方法和经验教训可能在分析对健康细胞至关重要的各种受监管活动中证明是有价值的。

All animal cells contain multiple membrane-bound structures known as organelles. Each type of organelle contains a characteristic complement of proteins that ensures metabolic compartmentation. Maintaining the identity of organelles and ensuring accurate transport of material between them underlies all cellular function. Transport of proteins into (endocytosis) and out of (exocytosis) the cell involves their regulated movement through a series of organelles that comprise the so called endocytic/secretory pathway. At various stages, transport involves encasing the protein cargo within small vesicles or tubules that act as carriers to be accurately targeted to the next staging post along the pathway. During this process, the identity of the receiving and dispatching organelles must be maintained. In this project, we propose to develop specific chemical tools to dissect transport steps of the secretory pathway in fine detail. There is a large body of information regarding the identity, function and even molecular structure of key proteins (the Arf family of small GTPases) involved in vesicle transport steps. Arf proteins exist in two states (on and off) and are switched between these states by other proteins that are compartment-specific. We are proposing to develop two currently available small chemical inhibitors that target a complex of Arf1 with its activator to prevent its activation. The approach makes use of a new category of chemical inhibitors called interfacial inhibitors. Most existing chemicals target the 'active site' of an enzyme or inhibit binding of components involved in reactions. In contrast, interfacial inhibitors trap complexes of proteins at a 'dead-end' point during their cycle of function such that they can no longer perform their role in the cell. Unfortunately, the classical interfacial inhibitor of Arf1 causes widespread disruption to multiple pathways simultaneously because it targets not just one pairing (i.e. one Arf1 GTPase and its specific activator on one compartment) but many such pairings, thereby preventing the activation of multiple Arf1-containing complexes. Nonetheless, the use of this inhibitor has led to literally thousands of publications giving some idea of the potential of more specific inhibitors. We are now proposing to synthesize refined versions of existing interfacial inhibitors to generate chemicals that are a lot more specific in their action. In this way, tools will be generated that can be used to discriminate the different Arf1-mediated steps in protein transport. The molecular architecture of Arf1 with its activator and a classical inhibitor is already known. This allows us to model other inhibitors and other closely related protein pairings. Our interdisciplinary team includes a chemist, molecular modeller, cell biologists and biochemists and has already generated a significant amount of data, including a characterization of key differences between existing chemical compounds that inform our future modification. The specificity of the molecules we synthesize will be tested using automated cell imaging and by using cell-based biochemical assays. We have significant expertise in this area and all of the technology is in place for this project. The proposed work has enormous potential since specific inhibitors of Arf1 will provide the scientific community with a new set of tools with which to probe trafficking pathways associated not only with the Golgi stack, but also with other organelles, such as the trans Golgi network (TGN) and endosomes. It is additionally important because the cohort of molecules we are targeting regulate a superfamily of small G proteins that is involved in almost all cellular processes including growth, division, metabolism and signalling. Thus in the long term, the approaches and lessons we learn from this project may prove valuable in the analysis of a wide range of regulated activities critical for a healthy cell.