Developing Autophagy-Targeting Chimeras and Optimizing Cell Penetration of Large-Molecule Therapeutics

开发自噬靶向嵌合体并优化大分子治疗的细胞渗透

• 批准号: 10558145
• 负责人: Joshua A Kritzer
• 金额: $ 57.9万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10558145
• 关键词: Address; Adopted; Algorithm Design; Area; Autophagocytosis; Binding; Biological Assay; CRISPR screen; Cells; Cellular biology; Chimera organism; Cytosol; DNA Damage; Data; Degradation Pathway; Disease; Endosomes; Genetic; Human; Kinetics; Learning; Malignant Neoplasms; Measures; Mediating; Methods; Modeling; Molecular Evolution; Penetration; Protac; Protein Family; Proteins; Series; Structure; Testing; Therapeutic; Time; Work; cell type; chemotherapy; design; drug development; drug discovery; inhibition of autophagy; inhibitor; innovation; novel; protein degradation; protein protein interaction; research and development; screening; small molecule inhibitor; stapled peptide

The Kritzer lab focuses on inhibiting protein-protein interactions involved in autophagy. Autophagy is a protein degradation pathway that is active in all human cells, and inhibiting autophagy shows promise as a therapy for late-stage cancers especially in combination with DNA-damaging chemotherapies. Autophagy research and drug development currently rely on compounds that inhibit autophagy indirectly. Better, more specific inhibitors of autophagy would be broadly adopted. A large amount of genetics and cell biology work supports that inhibiting the LC3/GABARAP family of proteins can block autophagy selectively. In one series of projects, we will develop novel stapled peptide and small molecule inhibitors of LC3/GABARAP and evaluate them in models of late-stage cancers and other diseases. Because of our expertise in compounds that bind LC3/GABARAP proteins, we also propose to evaluate related compounds as autophagy-targeting chimeras (AUTACs). These compounds could be used to selectively degrade any proteins in the cell, similar to proteolysis-targeting chimeras (PROTACs) but potentially more versatile and easier to develop. Based on strong preliminary data that validate the AUTAC concept, we will develop novel AUTACs and demonstrate their ability to degrade endogenous proteins, unlocking a broad new area for drug development in targeted protein degradation. Over the course of developing stapled peptides as autophagy modulators, the Kritzer lab encountered a common problem in the field: how to measure the amount that actually reaches the cytosol. In an independent series of projects, the Kritzer lab has developed novel assays that quantitate the cytosolic penetration of large-molecule therapeutics. In this proposal, we describe new opportunities to address challenging problems in drug development for large-molecule therapeutics. We describe new methods to measure penetration to different cellular compartments in any cell type, including primary cells. We also describe a molecular evolution approach to develop a new “turn-on” assay that measures the real-time kinetics of cytosolic penetration. We describe pooled CRISPR screens to reveal the cellular components that mediate endosomal escape. Finally, we describe a novel screening platform that will allow us to screen thousands to millions of molecules at a time for those that are most cell-penetrant; the new screen will be incorporated into a design-test-learn cycle to produce data-driven design algorithms for cytosol-penetrant molecules. All together, these data will represent a huge leap in our understanding of structure-penetration relationships for several classes of large-molecule therapeutics.

Kritzer 实验室专注于抑制自噬中涉及的蛋白质-蛋白质相互作用。自噬是一种蛋白质 降解途径在所有人类细胞中都很活跃，并且抑制自噬显示出作为治疗的希望 晚期癌症，尤其是与 DNA 损伤性化疗相结合时。自噬研究与 目前药物开发依赖于间接抑制自噬的化合物。更好、更特异的抑制剂 自噬将被广泛采用。大量的遗传学和细胞生物学工作支持这一点 抑制 LC3/GABARAP 家族蛋白可以选择性地阻断自噬。在一系列项目中，我们 将开发新型钉合肽和 LC3/GABARAP 小分子抑制剂，并在 晚期癌症和其他疾病的模型。 由于我们在结合 LC3/GABARAP 蛋白的化合物方面拥有专业知识，我们还建议评估相关的 化合物作为自噬靶向嵌合体（AUTAC）。这些化合物可用于选择性地 降解细胞中的任何蛋白质，类似于蛋白水解靶向嵌合体 (PROTAC)，但可能更多 用途广泛且更易于开发。基于验证 AUTAC 概念的强有力的初步数据，我们将 开发新型 AUTAC 并证明其降解内源蛋白质的能力，解锁 靶向蛋白质降解药物开发的广阔新领域。 在开发作为自噬调节剂的钉合肽的过程中，Kritzer 实验室遇到了 该领域的常见问题：如何测量实际到达细胞质的量。在一个 Kritzer 实验室开发了一系列独立的项目，开发了定量细胞溶质的新颖测定方法。 大分子疗法的渗透。在本提案中，我们描述了解决问题的新机会 大分子治疗药物开发中的挑战性问题。我们描述了新的方法 测量对任何细胞类型（包括原代细胞）中不同细胞区室的渗透。我们也 描述了一种分子进化方法来开发一种新的“开启”测定法，该测定法可以实时测量 细胞质渗透动力学。我们描述了混合 CRISPR 筛选以揭示细胞成分 介导内体逃逸。最后，我们描述了一个新颖的筛选平台，使我们能够筛选 对于那些最具细胞渗透性的分子，一次有数千到数百万个分子；新屏幕将是 纳入设计-测试-学习周期，为细胞质渗透剂生成数据驱动的设计算法 分子。总而言之，这些数据将代表我们对结构穿透理解的巨大飞跃 几类大分子疗法的关系。