Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

使用甲氧苄啶 PROTAC 调节 eDHFR 标记蛋白

基本信息

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
项目类别：
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

项目摘要

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于实验过程尤其重要。理解它们很重要，因为它们是细胞功能、细胞信号传导和生物体过程的核心（例如新陈代谢、组织发育和免疫功能）。方法“可调谐”并可逆地调节成像兼容蛋白标签的表达以进行研究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)，使研究人员能够检测并控制细胞中的标记蛋白例如，使用小分子配体的动物，标签的荧光成像可以指导正确的选择。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 标签的成像可以指导调节体内特定细胞运输的蛋白质。体内应用的例子是嵌合抗原受体（CAR）T细胞的开发过程实体瘤疗法，利用成像了解体内生物分布、功效和毒性将是至关重要的，而且重要的是，控制 CAR 的细胞表面表达可能会产生很大的影响小分子抗生素的生物分布、功效和非肿瘤毒性。我们组和其他人已将甲氧苄啶（TMP）开发成多模态成像探针。该提案的目的是为成像兼容的蛋白质调控标签制定一个标准，该标签可以广泛应用我们提出基于针对大肠杆菌的 TMP 的蛋白水解靶向嵌合小分子 (PROTAC)。大肠杆菌二氢叶酸还原酶 (eDHFR) 标记的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 和泊马度胺 (POM)，后者是 E3 连接酶 Cereblon 的先导化合物 TMP- 的配体。 POM 7c 强有力地调节多种蛋白质，从光学报告蛋白（如 YFP 和荧光素酶）到原代人类 T 细胞中的转录因子和治疗性膜结合蛋白，例如 CAR。需要对这些化合物进行优化、表征和应用，特别是在了解接头长度和组成的影响以及药代动力学特性，以奠定为可广泛应用于生物医学科学的可分发原型奠定了基础。通过将小分子蛋白调节与多功能蛋白标签结合起来，代表了技术的飞跃具有用于体内成像的荧光成像探针和 PET 放射性示踪剂。