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

项目总结/摘要 下面的建议描述了我的目标，使用一个已知的蛋白质配体对作为支架，以创建一个分子工具包组成的小分子降解剂的蛋白质调控和核成像探针。具体地说，这种分子工具包由一个小的蛋白质结构域，E。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）。TMP可以被修饰以作为能够以剂量依赖性和可逆方式调节蛋白质的蛋白水解靶向嵌合（PROTAC）分子。此外，我们的小组已经开发了eDHFR作为正电子发射断层扫描（PET）成像报告蛋白，能够在体内成像工程细胞。总之，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，并定量研究动物和潜在人类患者中这些变化的表型输出。 PROTAC调节是用于控制细胞蛋白活性的主要配体介导的敲低方法。配体介导的策略允许研究人员以时间精确度调节敲除强度。我已经证明，eDHFR-TMP PROTAC系统可以在24小时内以纳摩尔浓度诱导OVCAR 8细胞中95%的eDHFR标记蛋白降解。我还证明了这一过程是可逆的，并通过蛋白水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区室中的不同蛋白质。 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敲除感兴趣的蛋白质，可以在临床相关模型和潜在的人类治疗中进行定量分析。为了推进这些分子工具的体内使用，我将使用生物发光成像在小鼠OVCAR 8异种移植肿瘤模型中建立eDHFR-TMP PROTAC的靶向降解动力学。我假设eDHFR-荧光素酶的蛋白质水平响应将滞后于TMP PROTAC在体内的靶标占据。因此，在小鼠中同时给予PROTAC和用已建立的[18 F]氟丙基-甲氧苄啶（[18 F]FPTMP）放射性示踪剂对eDHFR进行PET成像将准确定量PROTAC特异性和动力学活性。总的来说，这项工作是PROTAC和PET技术的一种新应用，这些工具可以一起用于探测体内表达、调节蛋白质活性，并可能被纳入人类免疫疗法中以提高治疗效果。

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.