Developing a genetic tag for in vivo protein regulation using PROTACs with companion PET imaging

项目摘要/摘要 下面的提案描述了我的目标，即使用一个已知的蛋白质-配体对作为支架，创建一个由小分子降解物组成的分子工具包，用于蛋白质调节和核成像探针。具体地说，这个分子工具盒由一个小的蛋白结构域--大肠杆菌二氢叶酸还原酶(EDHFR)及其小分子抑制剂甲氧苄啶(TMP)组成。TMP可以被修饰为蛋白水解靶向嵌合体(PROTAC)分子，能够以剂量依赖和可逆的方式调节蛋白质。此外，我们的团队还开发了eDHFR作为正电子发射断层扫描(PET)成像报告蛋白，能够在体内对工程细胞进行成像。总而言之，我的方法将使研究人员能够调节任何所需基因的蛋白质活性，并在动物身上和潜在的人类患者中定量研究这些变化的表型输出。 PROTAC调控是配体介导的控制细胞蛋白质活性的主要基因敲除方法。配体介导的策略使研究人员能够以时间精确度调整击倒强度。我已经证明，eDHFR-TMP PROTAC系统可以在24小时内诱导95%的eDHFR标记蛋白在OVCAR8细胞中降解。我还证明了这个过程是可逆的，并通过蛋白质降解介导的降解机制进行。我们正在扩展这项技术，以确定我们是否可以调节多个亚细胞隔间中的不同蛋白质。 最终，这套分子工具将提供一个模块化系统，用于快速诱导和可逆地敲除感兴趣的蛋白质，这些蛋白质可以在临床相关的模型中进行定量分析，并可能在人类疗法中进行分析。为了推进这些分子工具在体内的应用，我将利用生物发光成像技术建立eDHFR-TMP PROTAC在OVCAR8小鼠移植瘤模型中的靶向降解动力学。我推测eDHFR-荧光素酶在体内的蛋白水平反应将落后于TMP PROTAC的靶占有率。因此，用已建立的[18F]氟丙基甲氧嘧啶([18F]FPTMP)放射示踪剂在小鼠体内同时给予PROTAC和eDHFR的PET成像将准确地定量PROTAC的特异性和动力学活性。总体而言，这项工作是PROTAC和PET技术的新应用，这些工具可以用于探测体内表达，调节蛋白质活性，并有可能被纳入人类免疫疗法，以提高治疗效果。

Project Summary/Abstract The following proposal describes my aims to use a known protein-ligand pair as a scaffold to create a molecular tool kit composed of small molecule degraders for protein regulation and nuclear imaging probes. Specifically, this molecular tool kit is comprised of a small protein domain, E. coli dihydrofolate reductase (eDHFR), and its small molecule inhibitor, trimethoprim (TMP). TMP can be modified to function as a proteolysis targeted chimeric (PROTAC) molecule capable of protein regulation in a dose-dependent and reversible fashion. Additionally, our group has developed eDHFR as a positron emission tomography (PET) imaging reporter protein capable of imaging engineered cells in vivo. Taken together, my approach would give researchers the ability to modulate protein activity of any desired gene and quantitatively study the phenotypic outputs of these changes in animals and potentially in human patients. PROTAC regulation is the leading ligand-mediated knockdown approach for controlling cellular protein activity. Ligand-mediated strategies allow researchers to tune knockdown intensity with temporal precision. I have demonstrated that the eDHFR-TMP PROTAC system can induce 95% degradation of eDHFR-tagged protein in OVCAR8 cells at nanomolar concentration within 24 hours. I have also demonstrated that this process is reversible and proceeds through a proteolysis-mediated degradation mechanism. We are expanding this technology to determine if we can regulate diverse proteins in multiple subcellular compartments. Ultimately, this suite of molecular tools will provide a modular system for rapidly inducible and reversible knockdown of proteins of interest that can be analyzed quantitatively in clinically relevant models and potentially in human therapies. To advance these molecular tools for in vivo use, I will establish the kinetics of target degradation of the eDHFR-TMP PROTAC in OVCAR8 xenograft tumor models in mice using bioluminescence imaging. I hypothesize that the protein level response of eDHFR-luciferase will lag behind target-occupancy of TMP PROTAC in vivo. Therefore, simultaneous PROTAC administration and PET imaging of eDHFR with established [18F]fluoropropyl-trimethoprim ([18F]FPTMP) radiotracer in mice will accurately quantify PROTAC specificity and kinetic activity. Overall, this work is a novel application of PROTAC and PET technology, where together, these tools can be used to probe expression in vivo, modulate protein activity, and potentially, be incorporated into human immunotherapies to improve therapeutic outputs.