Systematic stabilization of specific protein-protein interactions

摘要 蛋白质-蛋白质相互作用（PPI）在生物学中普遍存在，并且它们的失调与以下密切相关： 从癌症到神经退行性疾病，再到罕见的遗传疾病。PPI通常形成复杂的网络， 包括高度相互作用“中枢”蛋白。调节单一相互作用的方法将提供对以下方面的深入了解： 这些枢纽的功能。小分子探针和药物先导物集中于阻断PPI;然而， 稳定PPI对于药物发现同样重要，并且可以为化学药物提供更大的选择性。 生物学然而，很少有系统的方法来发现PPI稳定剂的前瞻性。这 一项提案的重点是系统地发现选择性小分子PPI稳定剂，使用枢纽蛋白 14-3-3作为一个模型系统。14-3-3s是七种高度同源的接头蛋白，它们与丝氨酸结合， 客户蛋白上的苏氨酸位点以改变其功能和命运。数百种信号转导蛋白 通路、细胞周期调节、转录调节和蛋白质稳态是14-3-3的客户。鉴于 蛋白质磷酸化的重要性和14-3-3作为磷酸化效应物的普遍性， 令人惊讶地被低估了。我们建议开发一种选择性的，细胞活性的天然稳定剂的工具包， 14-3-3/客户PPI将刺激生物学研究，并可能导致新药。基于结构的多样性， 客户，我们假设我们可以开发客户选择性PPI稳定剂，结合到复合物14-3- 3/客户端界面。这些选择性稳定剂应该放大14-3-3/客户端复合物的天然生物学。我们 将通过三个目标为这种方法提供概念验证：目标1。选择性稳定剂的筛选 14-3-3/磷酸肽客户。我们以前已经发现了二硫键结合的片段，稳定14-3- 3/磷酸肽复合物。我们现在将筛选六个，结构和生物多样性14-3-3/客户端 复合物，使用仅在14-3-3 β同种型上发现的天然C38残基。我们假设客户端序列 在C38附近具有更开放或更灵活的结构将产生更高质量的命中。目标2.优化14-3-3/客户端 细胞活性的稳定剂。我们已经证明了将二硫化物转化为细胞活性物质的能力。 亲电子弹头和调整14-3-3亲电子/客户端稳定剂的选择性。我们将优化C38结合 具有（或不具有）亲电体的片段，目的是在细胞中实现靶选择性PPI稳定化， 14-3-3客户CRAF激酶、雌激素受体β 1（ER β）和转录因子FOXO 1。目标3.设计 基于PROTAC的14-3-3客户端降级器。PROTAC是双功能分子， 在泛素连接酶和目标之间，导致目标的降解。我们将扩大可达目标 通过使用14-3-3作为支架蛋白将内在无序蛋白（IDP）连接到PROTAC技术， 泛素连接酶我们将首先开发用于ER的技术，其中已知PROTAC，然后将我们的 向国内流离失所者学习，包括FOXO 1。这些目标的成功实现将提供广泛适用的方法， 适用于14-3-3网络，并可扩展到其他原生和非原生PPI。

Abstract Protein-protein interactions (PPIs) are ubiquitous in biology, and their dysregulation is closely associated with diseases, from cancer to neurodegeneration to rare genetic disorders. PPIs often form complex networks that include highly interacting ‘hub’ proteins. Methods to modulate single interactions would provide great insight into the functions of these hubs. Small-molecule probes and drug leads have focused on blocking PPIs; however, stabilizing PPIs could be just as important for drug discovery and could provide greater selectivity for chemical biology. However, there are few systematic methodologies to discover PPI stabilizers prospectively. This proposal focuses on the systematic discovery of selective small-molecule PPI stabilizers, using the hub protein 14-3-3 as a model system. 14-3-3s are seven highly homologous adaptor proteins that bind to serine and threonine sites on client proteins to alter their function and fate. Hundreds of proteins in signal transduction pathways, cell-cycle regulation, transcription regulation, and protein homeostasis are clients of 14-3-3. Given the importance of protein phosphorylation and the ubiquity of 14-3-3 as an effector of phosphorylation, it is surprisingly underappreciated. We propose that developing a tool kit of selective, cell-active stabilizers of native 14-3-3/client PPIs will stimulate biological study and may lead to new drugs. Based on the structural diversity of clients, we hypothesize that we can develop client-selective PPI stabilizers that bind to the composite 14-3- 3/client interface. These selective stabilizers should amplify the native biology of the 14-3-3/client complex. We will provide proof-of-concept for this approach through three aims: Aim 1. Screen for selective stabilizers of 14-3-3/phosphopeptide clients. We have previously discovered disulfide-bound fragments that stabilize 14-3- 3/phosphopeptide complexes. We will now screen six, structurally and biologically diverse 14-3-3/client complexes, using a native C38 residue found only on the 14-3-3 isoform. We hypothesize that client sequences with more open or flexible structures near the C38 will yield higher quality hits. Aim 2. Optimize 14-3-3/client stabilizers for cell-based activity. We have demonstrated the ability to convert disulfides to cell-active electrophilic warheads and to tune the selectivity of 14-3-3/client stabilizers. We will optimize C38-bound fragments with (or without) an electrophile with the goal of achieving target-selective PPI stabilization in cells for the 14-3-3 clients CRAF kinase, estrogen receptor  (ER), and the transcription factor FOXO1. Aim 3. Design PROTAC-based degraders of 14-3-3 clients. PROTACs are bifunctional molecules that induce proximity between a ubiquitin ligase and a target, leading to the target’s degradation. We will expand the targets accessible to PROTAC technology by using 14-3-3 as a scaffolding protein to link intrinsically disordered proteins (IDPs) to the ubiquitin ligase. We will first develop the technology for ER, where PROTACs are known, then translate our learning to IDPs, including FOXO1. Successful completion of these aims will provide approaches that are broadly applicable to the 14-3-3 network and are extendable to other native and nonnative PPIs.