Coiled-coil Technology for Regulating Intracellular Protein-protein Interactions

蛋白质是生物学的主力。蛋白质很少单独工作，它们通过所谓的蛋白质-蛋白质相互作用进行合作。通过这种方式，它们形成了更大的蛋白质集合和网络。反过来，它们为控制所有调节生命的细胞过程提供了框架。潜在的蛋白质-蛋白质相互作用失调可导致疾病。细胞内蛋白质-蛋白质相互作用框架的规模是巨大的；据估计，大约有65万种不同的相互作用。这为干预生物过程(a)更好地了解健康细胞和(b)在亚细胞机制出现问题时开发新的治疗方法提供了相当大的机会。有不同的方法可以做到这一点。我们提出了一种新的方法，利用合成蛋白质模块(i)破坏蛋白质-蛋白质相互作用（ii）劫持内源性细胞机制。合成的化学探针——比如小分子药物——通过与体内的靶蛋白结合而起作用。这可以用来干扰目标蛋白的功能，以帮助了解其生物学作用，并作为药物发现的起点。大多数化学探针与蛋白质中定义明确的口袋结合；这类似于钥匙能插进锁里。相比之下，用于干扰蛋白-蛋白相互作用的探针的设计通常需要探针与相互作用的靶蛋白之间的一种根本不同的关联类型；类似于握球的手。因此，开发针对蛋白质-蛋白质相互作用的有效探针提出了未来化学生物学和药物发现需要满足的新挑战。有两种新兴的方法有望实现这一目标，我们建议在本次拨款申请中将它们结合起来。第一种是合成生物学方法。它使用一种被称为“从头开始”的合成蛋白质作为支架，从头开始构建新的蛋白质相互作用。这是很有吸引力的，因为天然的螺旋状蛋白质表现出一系列蛋白质识别特性，我们可以设计具有不同结构的新螺旋状蛋白质，从而扩大它们的潜力。第二种方法是有针对性地破坏细胞蛋白质；从本质上讲，这是一种“搜索-摧毁”策略，利用细胞自身的废物处理机制来阻断蛋白质功能或清除导致疾病的有害蛋白质。拟议的研究将开发新的方法(i)破坏特定的蛋白质-蛋白质相互作用和（ii）针对某些感兴趣的蛋白质进行降解。通过这种方式，我们将调节细胞中的特定过程。为了做到这一点，我们不会使用传统的小分子作为探针来干预潜在的蛋白质-蛋白质相互作用。相反，我们将使用合成线圈，并调整它们来识别目标蛋白质。此外，对于第二次应用，卷曲的线圈将被进一步修饰，以将目标蛋白连接到细胞的降解途径。我们的目标是提供可用于研究生物功能和开发治疗疾病的新药的方法和试剂。这项工作必然是跨学科的。因此，我们汇集了一个由计算和实验化学家、生物化学家和细胞生物学家组成的团队来解决这个问题，我们与一家生物技术公司合作，及时和相关地翻译工作。

Proteins are the workhorses of biology. Proteins rarely work alone, and they cooperative via so called protein-protein interactions. In this way, they form larger assemblies and networks of proteins. In turn, these provide frameworks for controlling all cellular processes that regulate life. Dysregulation of the underlying protein-protein interactions can result in disease. The scale of this framework of protein-protein interactions within a cell is enormous; it has been estimated to be around 650,000 different interactions. These present considerable opportunities for intervening in biological processes (a) to understand healthy cells better, and (b) to develop new therapeutics when subcellular mechanisms go wrong. There are different ways to do this. We propose a new approach that employs synthetic protein modules (i) to disrupt protein-protein interactions and (ii) to hijack endogenous cell machineries. Synthetic chemical probes-such as small-molecule drugs-function by binding to a protein target within the body. This can be used to interfere with the target protein's function to help understand its biological role and as a starting point for drug discovery. Most chemical probes bind to well-defined pockets in proteins; this is analogous to a key fitting into a lock. By contrast, the design of probes to interfere with protein-protein interactions generally requires a fundamentally different type of association between the probe and one of the interacting target proteins; analogous to a hand gripping a ball. Thus, the development of effective probes that target protein-protein interactions raises new challenges that need to be met in future chemical biology and drug discovery. Two emerging approaches are promising for this, and we propose to combine them in this grant application.The first is a synthetic-biology approach. This uses synthetic proteins called de novo coiled coils as scaffolds for building new protein-protein interactions from scratch. This is attractive because natural coiled-coil proteins exhibit an array of protein-recognition properties and we can design de novo coiled-coils with diverse structures thereby expanding their potential. The second involves the targeted destruction of cellular proteins; in essence, this is a search-and-destroy strategy that co-opts the cell's own waste-disposal machineries so as to block protein function or remove harmful proteins that cause disease. The proposed research will develop new methodologies (i) to disrupt specified protein-protein interactions and (ii) to target certain proteins of interest for degradation. In this way, we will regulate specified processes in cells. To do this, we will not use conventional small molecules as the probes for intervening in the underlying protein-protein interactions. Rather, we will employ the synthetic coiled coils and adapt these to recognise the target proteins. In addition, for the second application, the coiled coils will be modified further to link the target protein to the cell's degradation pathways. Our aim is to deliver methods and reagents that will be of use to others in studying biological function and for developing new drugs to treat disease.This work is necessarily interdisciplinary. Therefore, we bring together a team of computational and experimental chemists, biochemists and cell biologists to tackle it, and we partner with a biotech company to translate the work in timely and relevant manner.