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 原位控制生物分子的能力对实验过程至关重要。蛋白质尤其 重要的是要了解，因为它们是细胞功能，细胞信号传导和活生物体过程的中心 (e.g.代谢、组织发育和免疫功能）。人们对小分子的需求尚未得到满足， 一种“可调”和可逆调节成像相容蛋白标签表达的方法， 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成像。 发射断层扫描（PET），使研究人员能够检测，然后控制，细胞中的标记蛋白质， 使用小分子配体的动物。例如，标签的荧光成像可以引导适当的 基于体外亚细胞定位或蛋白质-蛋白质相互作用的蛋白质敲低时机，以及PET 标记物的成像可以指导调节体内细胞运输的蛋白质的调节。特定 体内应用的实例是在开发嵌合抗原受体（CAR）T细胞的过程中 实体瘤治疗，其中使用成像技术了解体内生物分布、疗效和毒性 将是至关重要的，重要的是，控制CAR的细胞表面表达可能会产生很大的影响， 生物分布、功效和肿瘤外毒性。小分子抗生素的化学衍生物 甲氧苄氨嘧啶（TMP）已被本小组和其他人开发成多模态成像探针。的 该建议的目的是为成像兼容的蛋白质调节标签设定标准， 领养的我们提出了基于靶向大肠杆菌的TMP的靶向嵌合小分子（PROTAC）的蛋白水解。 大肠杆菌二氢叶酸还原酶（eDHFR）标记的融合蛋白。我们的概念验证分子 共价连接TMP和泊马度胺（POM），一种E3连接酶Cereblon的配体。一种先导化合物，TMP- POM 7 c稳健地调节多种蛋白质，从光学报告蛋白，如YFP和荧光素酶， 在原代人T细胞中，本发明涉及转录因子和治疗性膜结合蛋白，如汽车。 需要对这些化合物进行优化、表征和应用，特别是在以下方面： 了解连接体长度和组成的影响，以及药代动力学特性， 为可广泛应用于生物医学科学的分布式原型奠定了基础。这种方法 通过将多功能蛋白质标签的小分子蛋白质调节结合起来， 使用荧光成像探针和PET放射性示踪剂进行体内成像。