Programmable peptide-guided protein degradation

可编程肽引导的蛋白质降解

• 批准号: 10741655
• 负责人: Pranam Chatterjee
• 金额: $ 38.77万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10741655
• 关键词: 2019-nCoV; Active Sites; Address; Algorithms; Amino Acid Sequence; Antibodies; Architecture; Base Sequence; Binding; Biological Assay; CRISPR/Cas technology; Cells; Chemistry; Chimera organism; Chimeric Proteins; Code; Complex; Consumption; DNA; Data Set; Databases; Development; Disease; Employment; Encapsulated; Engineering; Fibronectins; Fluorescence; Formulation; Foundations; Gene Delivery; Generations; Genetic; Goals; Human; In Vitro; Laboratories; Language; Learning; Length; Libraries; Malignant Neoplasms; Masks; Mediating; Messenger RNA; Methodology; Methods; Modality; Modeling; Molecular Conformation; Mutagenesis; Output; Pathogenicity; Peptides; Phosphoric Monoester Hydrolases; Phosphotransferases; Positioning Attribute; Post-Translational Protein Processing; Protac; Protein Engineering; Protein Isoforms; Proteins; Proteome; Protocols documentation; RNA; Rapid screening; Research; Specific qualifier value; Structure; Techniques; Technology; Testing; Therapeutic; Training; Translating; Translations; Ubiquitin-Proteasomal Pathway; Validation; Variant; Work; design; disease phenotype; experimental analysis; gene therapy; grasp; in silico; in vivo; in vivo evaluation; interest; iterative design; lipid nanoparticle; mutant; nanobodies; novel therapeutic intervention; novel therapeutics; pharmacologic; protein aminoacid sequence; protein complex; protein degradation; protein protein interaction; rapid testing; scaffold; screening; small molecule; small molecule inhibitor; tool; ubiquitin-protein ligase

Summary Over 600 human proteins have been recently prioritized as key cancer targets, with nearly half being considered ‘intractable’ by standard small-molecule inhibition approaches, due to target instability and active site accessibility constraints. By redirecting the ubiquitin-proteasomal pathway (UPS) for targeted protein degradation, the proteolysis-targeting chimera (PROTAC) technology provides a potential solution, enabling rapid and continuous target consumption as well as the stronger pharmacological effects than small molecule inhibition. Nonetheless, PROTACs suffer from similar developmental hurdles as small molecules and cannot be easily designed for motif or post-translational modification-specific targeting. To address these hurdles, research efforts have shifted toward gene therapy approaches by introducing the concept of protein-mediated protein degradation. Here, E3 ubiquitin ligases are redirected by replacing their natural substrate binding domains with “off-the-shelf” binding domains, including nanobodies, antibodies, and DARPins, to generate target-specific ubiquibodies. To augment this platform, we recently exploited natural protein-protein interaction information to develop algorithmic pipelines that prioritize target-selective peptides which can be fused to the E3 ubiquitin ligase conjugation domains to induce target protein degradation. In this project, we will augment our current methods to enable the development of these ubiquibodies (uAbs) for any protein, including those deemed ‘intractable’ by small molecule-based means. To do this, we will automate a bipartite algorithmic pipeline that leverages recent advancements in protein language modeling as well as existing co-complex databases to design peptide binders to diverse protein targets, including those with solved co-crystals as well as those with minimal structural information. Specifically, our pipeline will take user-specified target proteins as inputs, and generate prioritized lists of candidate peptide binders as outputs, enabling subsequent generation of uAbs for target degradation. Through library-on-library fluorescence-based assays in human cells and subsequent encapsulation of uAb mRNA in lipid nanoparticles (LNPs), we will develop a scalable method to test and translate our degraders for downstream in vivo validation. In total, this work will generate a robust peptide design tool that will enhance targeted protein degradation efforts and lay the foundation for programmable proteome editing.

总结 最近，超过600种人类蛋白质被优先考虑为关键的癌症靶点，其中近一半是 被标准的小分子抑制方法认为是“难治的”，由于靶标不稳定和活性， 网站访问限制。通过重定向泛素-蛋白酶体途径（UPS）， 蛋白水解靶向嵌合体（PROTAC）技术提供了一种潜在的解决方案， 快速、持续的靶向消耗以及比小分子更强的药理作用 抑制作用尽管如此，PROTAC遭受与小分子类似的发展障碍，并且不能被用于治疗。 易于设计用于基序或翻译后修饰特异性靶向。为了克服这些障碍， 通过引入蛋白质介导的概念，研究工作已经转向基因治疗方法。 蛋白质降解在这里，E3泛素连接酶通过取代它们的天然底物结合而重定向， 结构域与“现成的”结合结构域，包括纳米抗体、抗体和DARPin，以产生 靶特异性泛体。为了增强这个平台，我们最近利用了天然蛋白质-蛋白质相互作用 信息，以开发算法管道，该算法管道优先考虑可以融合到 诱导靶蛋白降解的E3泛素连接酶缀合结构域。在这个项目中，我们将增加 我们目前的方法，使这些泛抗体（uAb）的发展，任何蛋白质，包括那些 通过基于小分子的方法被认为是“难处理的”。为了做到这一点，我们将自动化一个二分算法， 利用蛋白质语言建模的最新进展以及现有的协同复合物的管道 数据库，以设计针对不同蛋白质靶点的肽结合剂，包括那些具有溶解共晶体的蛋白质 和那些结构信息最少的一样。具体来说，我们的管道将用户指定的目标蛋白作为 输入，并生成候选肽结合剂的优先级列表作为输出，使得能够进行后续生成 用于靶降解。通过在人类细胞中进行基于库对库荧光的测定， 随后将uAb mRNA包封在脂质纳米颗粒（LNP）中，我们将开发一种可扩展的方法， 测试和翻译我们的降解剂，用于下游体内验证。总之，这项工作将产生一个强大的 肽设计工具，将增强靶向蛋白质降解的努力，并奠定基础， 可编程蛋白质组编辑。