Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

使用甲氧苄啶 PROTAC 调节 eDHFR 标记蛋白

• 批准号: 10714294
• 负责人: Mark A Sellmyer
• 金额: $ 59.49万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 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 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10714294
• 关键词: Adopted; Affect; Affinity; Amides; Animals; Antibiotics; Antigens; Benchmarking; Binding; Binding Proteins; Biodistribution; Biological Assay; Biological Models; Bone Marrow; CAR T cell therapy; Cancer Model; Cell physiology; Cell surface; Cells; Chemicals; Chimeric Proteins; Clinical; Companions; Coupled; Coupling; Cytokine Receptors; Cytolysis; Cytoplasm; Degradation Pathway; Development; Dihydrofolate Reductase; Dose; Drug Kinetics; Drug Targeting; Engineering; Enzymes; Escherichia coli; Experimental Models; Flow Cytometry; Fluorescence; Future; Half-Life; Human; Ikaros protein; Image; Imides; In Situ; In Vitro; Lead; Length; Ligands; Link; Luciferases; Malignant neoplasm of ovary; Measures; Mediating; Membrane; Membrane Proteins; Metabolism; Microscopy; Monitor; Multimodal Imaging; Optical reporter; Organism; Patients; Pharmaceutical Preparations; Positron-Emission Tomography; Process; Property; Protac; Proteins; RUNX1 gene; Radioisotopes; Regulation; Research Personnel; Research Project Grants; Rodent; Rodent Model; Science; Serum; Signal Transduction; Solid Neoplasm; System; T-Lymphocyte; Technology; Testing; Therapeutic; Time; Tissues; Toxic effect; Translations; Trimethoprim; Validation; Western Blotting; cell killing; chimeric antigen receptor; chimeric antigen receptor T cells; experimental study; fibroblast-activating factor; fluorescence imaging; human imaging; imaging probe; immune function; immunoregulation; improved; in vivo; in vivo imaging; inhibitor; interest; intraperitoneal; knock-down; multicatalytic endopeptidase complex; pomalidomide; protein degradation; protein expression; protein function; protein protein interaction; prototype; quantitative imaging; radiotracer; small molecule; tool; trafficking; transcription factor; tumor; ubiquitin-protein ligase

Project Abstract The ability to control biomolecules in situ is critical to the experimental process. Proteins are especially important to understand as they are central to cellular function, cell signaling, and living organism processes (e.g. metabolism, tissue development, and immune function). There is an unmet need for a small molecule approach to “tunably” and reversibly regulate expression of an imaging-compatible protein tag to investigate protein function(s). Such a tag would allow multi-modal imaging, including fluorescence and in vivo positron emission tomography (PET), allowing investigators to detect, then control, the tagged protein in cells and animals using small molecule ligands. For example, fluorescence imaging of the tag could guide the proper timing for protein knock-down based on subcellular localization or protein-protein interactions in vitro, and PET imaging of the tag could guide the regulation of proteins modulating cellular trafficking in vivo. A specific example of the in vivo application is in the process of developing chimeric antigen receptor (CAR) T-cell therapies for solid tumors, where understanding in vivo biodistribution, efficacy, and toxicity using imaging would be crucial, and importantly, controlling the cell surface expression of the CAR may have a large impact on that biodistribution, efficacy, and off-tumor toxicity. Chemical derivatives of the small molecule antibiotic trimethoprim (TMP) have been developed into multi-modality imaging probes by our group and others. The objective of this proposal is to set a standard for imaging-compatible protein regulation tags that can be widely adopted. We propose proteolysis targeting chimeric small molecules (PROTACs) based on TMP that target E. coli dihydrofolate reductase (eDHFR) tagged fusion proteins with high affinity. Our proof-of-concept molecules covalently link TMP and pomalidomide (POM), a ligand for the E3 ligase Cereblon. A lead compound, TMP- POM 7c robustly regulates diverse proteins, from optical reporter proteins, such as YFP and luciferase, to transcription factors and therapeutic membrane-bound proteins, such as CARs, in primary human T-cells. Optimization, characterization, and application of these compounds is needed, especially in terms of understanding the impact of linker length and composition, as well as pharmacokinetic properties, to lay the groundwork for a distributable prototype(s) that can be applied broadly in biomedical science. This approach represents a technological leap forward by uniting small molecule protein regulation of a versatile protein tag with fluorescence imaging probes and PET radiotracers for in vivo imaging.

项目摘要 控制生物分子原位的能力对于实验过程至关重要。蛋白质尤其是 重要的是要理解它们是细胞功能，细胞信号传导和生物过程的核心 （例如，代谢，组织发育和免疫功能）。对小分子的需求未满足 使用成像兼容蛋白质标签的“调节”和可逆地调节表达的方法 蛋白质功能。这样的标签将允许多模式成像，包括荧光和体内正电子 发射断层扫描（PET），允许研究人员检测，然后控制细胞中标记的蛋白质和 动物使用小分子配体。例如，标签的荧光成像可以指导正确 基于亚细胞定位或蛋白质 - 蛋白质相互作用的蛋白质敲低时间，PET 标签的成像可以指导调节体内细胞运输的蛋白质。特定的 体内应用的示例是在开发嵌合抗原受体（CAR）T细胞的过程中 实体瘤的疗法，其中了解体内生物分布，效率和使用成像的毒性 至关重要，重要的是，控制汽车的细胞表面表达可能会有很大的冲击 在生物分布，有效性和肿瘤毒性上。小分子抗生素的化学衍生物 甲氧苄啶（TMP）已被我们的小组和其他人发展为多模式成像问题。 该建议的目的是为与成像兼容的蛋白质调节标签设定标准，该标签可以广泛 采用。我们提出靶向基于靶向E的TMP的嵌合小分子（Protac）的蛋白水解。 大肠杆菌二氢叶酸还原酶（EDHFR）标记具有高亲和力的融合蛋白。我们的概念验证分子 共价连接TMP和Pomalidomide（POM），这是E3连接酶Cereblon的配体。铅化合物TMP- POM 7C可靠地调节不同的蛋白质，从光学报告蛋白（例如YFP和荧光素酶）到 原代人T细胞中的转录因子和治疗性膜结合的蛋白，例如汽车。 需要这些化合物的优化，表征和应用，尤其是 了解接头长度和组成以及药代动力学特性的影响 可分布原型的基础工作，可以广泛应用于生物医学科学。这种方法 通过团结多功能蛋白标签的小分子蛋白调节来代表技术飞跃 带有荧光成像探针和PET放射性示例，用于体内成像。