Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

项目摘要 原位控制生物分子的能力对实验过程至关重要。蛋白质尤其是 了解它们很重要，因为它们是细胞功能、细胞信号和活体过程的中心 (例如新陈代谢、组织发育和免疫功能)。对小分子的需求还没有得到满足 “可调”和可逆调节成像相容蛋白标签表达的方法研究 蛋白质功能(S)。这样的标签将允许多模式成像，包括荧光和活体正电子 发射断层扫描(PET)，使研究人员能够检测并控制细胞和 使用小分子配体的动物。例如，标签的荧光成像可以指导正确的 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除时间，以及PET 对标签的成像可以指导调节体内细胞运输的蛋白质。一种特定的 体内应用的例子是在开发嵌合抗原受体(CAR)T细胞的过程中 实体肿瘤的治疗，通过成像了解体内的生物分布、疗效和毒性 这将是至关重要的，而且重要的是，控制汽车的细胞表面表达可能会产生很大的影响 该药物的生物分布、疗效和非肿瘤毒性。小分子抗生素的化学衍生物 甲氧苄氨嘧啶(TMP)已被我们的团队和其他人开发成多模式成像探针。这个 这项提案的目的是为成像兼容的蛋白质调节标签设定一个标准，该标签可以广泛地 领养的。我们提出了靶向嵌合小分子(PROTACs)的蛋白质水解法。 Coli二氢叶酸还原酶(EDHFR)标记的高亲和力融合蛋白。我们的概念验证分子 将TMP和泊马度胺(POM)共价连接，泊马度胺是E3连接酶Cereblon的配体。一种先导化合物，TMP- POM 7c有力地调节各种蛋白质，从YFP和荧光素酶等光学报告蛋白到 转录因子和治疗性膜结合蛋白，如CARS，在原代人类T细胞中。 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 了解连接子长度和组成以及药代动力学特性的影响，以便为 可广泛应用于生物医学科学的可分发原型(S)的基础工作。这种方法 通过将小分子蛋白质对多功能蛋白质标签的调节统一起来，代表了技术上的飞跃 使用荧光成像探针和PET放射性示踪剂进行活体成像。

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.