Systematic stabilization of specific protein-protein interactions

特定蛋白质-蛋白质相互作用的系统稳定

• 批准号: 10703416
• 负责人: Michelle Arkin
• 金额: $ 31.67万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10703416
• 关键词: Adaptor Signaling Protein; Address; Binding; Binding Proteins; Binding Sites; Biological; Biological Models; Biological Process; Biology; Cell Cycle; Cell Cycle Progression; Cell Cycle Regulation Pathway; Cells; Cellular Assay; Chemicals; Client; Complex; Development; Disease; Disulfides; Estrogen Receptor alpha; Estrogen Receptors; FOXO1A gene; Genetic Transcription; Glues; Goals; Homodimerization; Human; Individual; Knowledge; Learning; Length; Link; Malignant Neoplasms; Methodology; Methods; Modification; Molecular; Molecular Conformation; Multiprotein Complexes; Natural Products; Nature; Nerve Degeneration; Outcome; Peptides; Pharmaceutical Preparations; Phosphopeptides; Phosphorylation; Phosphotransferases; Physiological Processes; Play; Positioning Attribute; Protac; Protein Isoforms; Proteins; Proteome; Role; Scaffolding Protein; Serine; Signal Transduction; Signal Transduction Pathway; Site; Specificity; Structure; Surface; System; Technology; Testing; Therapeutic; Threonine; Transcriptional Regulation; Translating; design; drug discovery; drug-like compound; enzyme activity; estrogen-related receptor; flexibility; fusicoccin; immune modulating agents; inhibitor; insight; iterative design; non-Native; novel; novel therapeutics; prospective; protein complex; protein protein interaction; proteostasis; raf Kinases; rare genetic disorder; scaffold; screening; small molecule; synthetic protein; tool; transcription factor; ubiquitin ligase

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.

抽象的 蛋白质 - 蛋白质相互作用（PPI）在生物学上无处不在，它们的失调与 从癌症到神经退行性的疾病再到罕见的遗传疾病。 PPI经常形成复杂的网络 包括高度相互作用的“集线器”蛋白。调节单个互动的方法将为您提供深入的了解 这些枢纽的功能。小分子问题和药物铅已重点是阻止PPI。然而， 稳定PPI对于药物发现同样重要，并且可以为化学品提供更大的选择性 生物学。但是，很少有系统的方法可以预期发现PPI稳定器。这 提案的重点是使用集线器蛋白的选择性小分子PPI稳定器的系统发现 14-3-3作为模型系统。 14-3-3s是七个高度同源的衔接蛋白，与串行结合和 客户蛋白上的苏氨酸位点以改变其功能和命运。信号转导中数百种蛋白质 途径，细胞周期调节，转录调节和蛋白质稳态为14-3-3的客户。鉴于 蛋白质磷酸化和无处不在14-3-3作为磷酸化作用的重要性，它是 令人惊讶的是被低估了。我们建议开发一个天然的选择性，细胞活性稳定器的工具包 14-3-3/客户PPI将刺激生物学研究，并可能导致新药。基于结构多样性 客户，我们假设我们可以开发客户选择性的PPI稳定剂，这些稳定器与复合材料14-3-- 3/客户端接口。这些选择性稳定器应放大14-3-3/客户端复合物的天然生物学。我们 将通过三个目的为这种方法提供概念验证：目标1。选择性稳定器的屏幕 14-3-3/磷酸肽客户。我们以前发现了二硫键结合的片段，可稳定14-3-- 3/磷酸肽复合物。现在，我们将在结构和生物学上筛选六个，14-3-3/客户 复合物，使用仅在14-3-3同工型上发现的天然C38居住地。我们假设该客户端序列 在C38附近，更开放或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 基于细胞活性的稳定器。我们已经证明了将二硫化物转化为细胞活性的能力 亲电弹头，以调整14-3-3/客户稳定器的选择性。我们将优化C38限制 具有（或没有）电力的碎片，目的是实现细胞中目标选择性PPI稳定的目的 14-3-3个客户CRAF激酶，雌激素受体（ER）和转录因子FOXO1。目标3。设计 14-3-3客户的基于Protac的降级器。 Protac是影响接近性的双功能分子 在泛素连接酶和靶标之间，导致靶标降解。我们将扩展目标可访问的目标 通过使用14-3-3用作脚手架蛋白来将原始无序蛋白（IDP）连接到Protac Technology 泛素连接酶。我们将首先开发Er技术，那里是Protac是已知的，然后翻译我们的 学习包括FoxO1在内的IDP。这些目标的成功完成将提供广泛的方法 适用于14-3-3网络，扩展到其他本地和非本地PPI。

项目成果

Reversible Dual-Covalent Molecular Locking of the 14-3-3/ERRγ Protein-Protein Interaction as a Molecular Glue Drug Discovery Approach.

• DOI: 10.1021/jacs.2c12781
• 发表时间: 2023-03-29
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Somsen, Bente A.;Schellekens, Rick J. C.;Verhoef, Carlo J. A.;Arkin, Michelle R.;Ottmann, Christian;Cossar, Peter J.;Brunsveld, Luc
• 通讯作者: Brunsveld, Luc

Structure-Based Optimization of Covalent, Small-Molecule Stabilizers of the 14-3-3σ/ERα Protein-Protein Interaction from Nonselective Fragments.

• DOI: 10.1021/jacs.3c05161
• 发表时间: 2023-09-20
• 期刊: JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
• 影响因子: 15
• 作者: Konstantinidou, Markella;Visser, Emira J.;Vandenboorn, Edmee;Chen, Sheng;Jaishankar, Priyadarshini;Overmans, Maurits;Dutta, Shubhankar;Neitz, R. Jeffrey;Renslo, Adam R.;Ottmann, Christian;Brunsveld, Luc;Arkin, Michelle R.
• 通讯作者: Arkin, Michelle R.

From Tethered to Freestanding Stabilizers of 14-3-3 Protein-Protein Interactions through Fragment Linking.

通过片段连接从 14-3-3 蛋白质-蛋白质相互作用的束缚稳定剂到独立稳定剂。

• DOI: 10.1002/anie.202308004
• 发表时间: 2023
• 期刊: Angewandte Chemie (International ed. in English)
• 作者: Visser,EmiraJ;Jaishankar,Priyadarshini;Sijbesma,Eline;Pennings,MarloesAM;Vandenboorn,EdmeeMF;Guillory,Xavier;Neitz,RJeffrey;Morrow,John;Dutta,Shubhankar;Renslo,AdamR;Brunsveld,Luc;Arkin,MichelleR;Ottmann,Christian
• 通讯作者: Ottmann,Christian