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

所有动物细胞都含有多种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有的蛋白质补体，以确保代谢区隔。维持细胞器的特性并确保它们之间物质的准确运输是所有细胞功能的基础。蛋白质进入（内吞作用）和流出（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。在不同阶段，运输包括将蛋白质货物包裹在小泡或小管中，这些小泡或小管作为载体，沿着路径精确地定位到下一个中转站。在此过程中，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。关于参与囊泡运输步骤的关键蛋白（小gtpase的Arf家族）的身份，功能甚至分子结构，有大量的信息。Arf蛋白存在两种状态（开和关），并通过其他具有区室特异性的蛋白质在这两种状态之间切换。我们建议开发两种目前可用的小型化学抑制剂，它们靶向Arf1及其激活剂的复合体，以阻止其激活。这种方法利用了一种叫做界面抑制剂的新型化学抑制剂。大多数现有的化学物质以酶的“活性位点”为目标，或抑制参与反应的成分的结合。相反，界面抑制剂在其功能周期中将蛋白质复合物捕获在“死角”点，使其不能再在细胞中发挥作用。不幸的是，经典的Arf1界面抑制剂会同时对多种途径造成广泛的破坏，因为它不仅针对一种配对（即一个Arf1 GTPase及其在一个室中的特定激活剂），而且针对许多这样的配对，从而阻止多种含Arf1复合物的激活。尽管如此，这种抑制剂的使用已经导致成千上万的出版物给出了更具体抑制剂的潜力的一些想法。我们现在提议合成现有界面抑制剂的精炼版本，以产生更具体的作用的化学物质。通过这种方式，将产生可用于区分蛋白质运输中arf1介导的不同步骤的工具。Arf1及其激活剂和经典抑制剂的分子结构已经已知。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、细胞生物学家和生化学家，已经产生了大量的数据，包括现有化合物之间关键差异的表征，这些特征为我们未来的修饰提供了信息。我们合成的分子的特异性将通过自动细胞成像和基于细胞的生化分析进行测试。我们在这一领域拥有丰富的专业知识，所有的技术都已到位。这项工作具有巨大的潜力，因为Arf1的特定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相关，而且与其他细胞器（如反高尔基网络（TGN）和核内体）相关的运输途径。这一点也很重要，因为我们所针对的分子群调节了一个小G蛋白超家族，这个超家族参与了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一系列受调节活动方面证明是有价值的。

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.