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

使用 PROTAC 和伴随 PET 成像开发用于体内蛋白质调节的遗传标签

基本信息

批准号：
10535045
负责人：
Jean Marie Etersque
金额：
$ 4.68万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10535045
关键词：
3-methyladenine Affinity Animals Binding Biological Assay Biology Bioluminescence Cell model Cells Chemicals Companions Complex Coupling Degradation Pathway Dihydrofolate Reductase Dose Drug Delivery Systems Drug Formulations Drug Kinetics Engineering Escherichia coli Excretory function Family member Firefly Luciferases Flow Cytometry Genes Genetic Goals Guanosine Triphosphate Phosphohydrolases Homologous Gene Hour Human Hydroxychloroquine Image Imaging Techniques Immunotherapy Interleukin-2 Investments Kinetics Knock-out Lead Length Libraries Ligands Luciferases Lymphocyte Lymphocyte-Specific p56LCK Tyrosine Protein Kinase Malignant neoplasm of ovary Measures Mediating Metabolism Modeling Molecular Mus Output Patients Peptides Pharmaceutical Preparations Pharmacodynamics Phenotype Positron-Emission Tomography Process Property Protac Proteins Proteolysis RNA Interference Radiolabeled Radiopharmaceuticals Regulation Reporter Reporter Genes Research Research Personnel Rodent Model Series Signal Transduction Specificity Sum Synthesis Chemistry System Technology Tertiary Protein Structure Testing Therapeutic Tissue Model Tissue imaging Trimethoprim Ubiquitination Validation Western Blotting Work absorption analog bioluminescence imaging cancer cell career cell growth regulation clinically relevant design enzyme substrate epoxomicin experimental study first-in-human imaging probe imaging study improved in vitro Assay in vitro activity in vivo in vivo monitoring inhibitor interest kidney cell knock-down molecular imaging multicatalytic endopeptidase complex mutant nanomolar novel nuclear imaging pomalidomide portability protein degradation protein expression quantitative imaging radiotracer response scaffold skills small molecule small molecule inhibitor technology development tissue culture tool tool development tumor xenograft ubiquitin-protein ligase uptake virtual

项目摘要

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.

项目概要/摘要以下提案描述了我的目标，即使用已知的蛋白质-配体对作为支架来创建由用于蛋白质调节和核成像探针的小分子降解剂组成的分子工具包。具体来说，该分子工具包由小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 可以修饰为蛋白水解靶向嵌合 (PROTAC) 分子，能够以剂量依赖性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，能够对工程细胞进行体内成像。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，并定量研究这些变化在动物和人类患者中的表型输出。 PROTAC 调节是用于控制细胞蛋白质活性的主要配体介导的敲低方法。配体介导的策略使研究人员能够以时间精度调整敲低强度。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 OVCAR8 细胞中 eDHFR 标记蛋白降解 95%。我还证明了这个过程是可逆的，并且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地敲低感兴趣的蛋白质，可以在临床相关模型中进行定量分析，并可能在人类疗法中进行定量分析。为了推进这些分子工具在体内的应用，我将使用生物发光成像在小鼠的 OVCAR8 异种移植肿瘤模型中建立 eDHFR-TMP PROTAC 的目标降解动力学。我假设 eDHFR-荧光素酶的蛋白质水平响应将落后于体内 TMP PROTAC 的目标占据。因此，在小鼠体内同时使用已建立的[18F]氟丙基甲氧苄氨嘧啶（[18F]FPTMP）放射性示踪剂对eDHFR进行PROTAC给药和PET成像将准确量化PROTAC特异性和动力学活性。总体而言，这项工作是 PROTAC 和 PET 技术的新颖应用，这些工具一起可用于探测体内表达、调节蛋白质活性，并有可能纳入人类免疫疗法中以提高治疗效果。