Development of evolutionary technologies to reprogram protein-protein interactions

项目摘要 蛋白质-蛋白质相互作用(PPI)的改变可导致生物信号的失调。PPI至关重要 许多疾病状态的驱动因素，并日益被认为是重要的治疗目标。 然而，传统疗法很难针对PPI进行治疗，因此通常认为 “无法下药”，因为传统的小分子发现策略不太适合识别分子 具有合适的性质来干扰PPI。当确定与疾病相关的PPI目标时，将其翻译为 将信息转化为有效的分子探针可能需要数年时间，开发一种临床有用的治疗方法可以 几十年后，更糟糕的是，在大多数情况下，这些努力完全失败了。与开发相关的成本 治疗性线索限制了对彻底验证的目标的追求。更快、更便宜、更多 有效的PPI抑制剂发现流程将使研究人员能够快速识别直接用于PPI抑制剂的探针 在相关模型系统中扰动PPI以评估PPI作为治疗靶点的有效性 产生用于临床开发的候选抑制剂。虽然基础生物学和翻译生物学的几乎所有领域 研究受益于21世纪在基因测序、质谱学、 和其他诊断方法，药物发现仍然在很大程度上依赖于20世纪的方法，这些方法已经被证明是 令人沮丧的缓慢和在关键方面不成功-我们提出的技术旨在解决这些问题。 我们假设，持续进化技术将允许我们快速进化PPI抑制剂 胞浆蛋白靶标的范围。具体地说，我们将追求两项关键进展，以实现这一广泛目标。 在目标1中，我们将证明非连续选择之后是噬菌体辅助的连续进化 (PACE)使我们能够使用文库方法快速进化出各种蛋白质的蛋白质结合子。 这种方法将消除速度上的一个关键瓶颈，优化初始选择的严格性，并带来 更接近自动化即插即用，允许更广泛地采用这项技术。在目标2中，我们 将构建和验证PACE兼容的生物传感器，将特定PPI的破坏与噬菌体适合性联系起来 允许我们直接选择PPI抑制功能。我们的目标是演示生成和验证 在不到1个月的时间内为给定的目标提供高效的PPI抑制剂。我们将验证这些演进平台 利用三种已被充分研究的致癌蛋白质-蛋白质相互作用一直是小分子药物的重点 几十年的发展：MDM2-p53，KRAS-RAF和Myc-Max。虽然这是一个崇高的目标，但 长期以来，进化一直被认为是解决这个问题的一种有希望的解决方案；我们希望通过合并 创新的生物传感器设计和不断的进化，我们可以释放实验室进化的全部潜力。 如果成功，这些平台有可能彻底改变药物发现范式，并加速 新型PPI抑制剂的发现。

Project Summary Alterations in protein-protein interactions (PPI) can result in dysregulated biological signaling. PPIs are critical drivers of a plethora of disease states and are increasingly recognized as important therapeutic targets. However, PPIs have been difficult to target with traditional therapeutics, and are often considered “undruggable”, because traditional small molecule discovery strategies are not well suited to identify molecules with suitable properties to disrupt PPIs. When a disease-associated PPI target is identified, translating that information into a validated molecular probe can take years, development of a clinically useful therapeutic can take decades, and even worse, in most cases these efforts fail entirely. The costs associated with developing therapeutic leads limits pursuits towards only thoroughly validated targets. A faster, less expensive, and more efficacious pipeline for PPI inhibitor discovery would allow researchers to quickly identify probes for directly perturbing PPIs in relevant model systems to assess the validity of the PPI as a therapeutic target and to generate candidate inhibitors for clinical development. While almost all areas of basic and translational biology research have benefitted from 21st century technological advances in genetic sequencing, mass spectrometry, and other diagnostics, drug discovery still largely relies on 20th century methods, which have proven to be frustratingly slow and unsuccessful in critical ways—our proposed technology aims to solve these problems. We hypothesize that continuous evolution techniques will allow us to rapidly evolve PPI inhibitors for a wide range of cytosolic protein targets. Specifically, we will pursue two key advancements to realize this broad goal. In Aim 1, we will demonstrate that non-continuous selection followed by phage-assisted continuous evolution (PACE) allows us to rapidly evolve protein binders for diverse proteins using a library-of-libraries approach. This approach will eliminate a crucial bottleneck in PACE, optimization of initial selection stringency, and bring PACE much closer to automated plug-and-play allowing for broader adoption of this technique. In Aim 2, we will construct and validate a PACE compatible biosensor linking disruption of a specific PPI to phage fitness allowing for us to directly select for PPI inhibitor function. Our goal is to demonstrate generation and validation of high potency PPI inhibitors for a given target in under 1 month. We will validate these evolution platforms using three well-studied oncogenic protein-protein interactions that have been the focus of small molecule drug development for decades: MDM2-p53, KRAS-RAF, and Myc-MAX. While this is a lofty goal, the power of evolution has long been recognized as a promising solution to this problem; we are hopeful that by merging innovative biosensor designs and continuous evolution, we can unlock the full potential of laboratory evolution. If successful, these platforms have the potential to revolutionize the drug discovery paradigm and accelerate the discovery of novel PPI inhibitors.