Imaging enzyme activity by Overhauser-enhanced MRI

摘要 绘制体内酶的活性图谱，直接在其自然环境中进行，可能会提供独特的见解 一种特定的酶在各种疾病的病理生理学中的作用，但仍然具有极大的挑战性。 最近，Pi和他的同事首次展示了电子顺磁共振(EPR)的能力 使用碱性磷酸酶(ALP)的氮氧化物自由基底物成像酶活性。然而，在 氮氧化物片段的快速生物还原阻碍了体内应用。该项目旨在开发 第一个对酶活性敏感的生物稳定顺磁探针。该设计是基于三芳基甲基 ()自由基支架，以其在生物环境中的非凡稳定性、窄线宽和高 Overhauser增强磁共振成像(OMRI)的极化性能。为实现这一目标 我们将依靠我们在功能探针化学方面的最新进展，这些探针允许多功能 使用EPR成像和/或OMRI进行映射。为了证明这个概念，我们将在特定目标1(SA1)下合成 光谱经酶修饰的碱性磷酸酶顺磁性底物 去磷酸化。此外，合成的探针还将继承自由基对组织的内在敏感性 PO2，从而使它们成为酶促碱性磷酸酶和pO2的双功能探针。在SA2的情况下，我们将在体外进行 双碱性磷酸酶和氧分压OMRI序列的优化。根据SA3，作为这一新概念的首次应用 在活体中，我们将使用Omri成像碱性磷酸酶(ALP)活性与组织氧合。 HeLa皮下侧翼肿瘤模型的肿瘤微环境癌症研究最近 经历了从看似明显的肿瘤细胞靶点到关键支持系统的范式转变 癌症，肿瘤微环境(TME)。组织中的PO2和酶的活性是重要的 生物标记物。成像结果将使用独立的技术进行验证，即PO2的羟基石 以及ALP的光学成像。该项目重点介绍了使用OMRI作为证据的ALP和PO2双重映射 概念，但探针支架的设计是为了方便地应用于许多其他重要的酶 未来。这一提议的成功将为体内酶学提供一种独特的工具，使其能够绘制出活性图谱 并在空间上将这种活性与组织的PO2相关联。这种独特的工具将允许出类拔萃 在药物筛选、治疗优化、预测对特定治疗的反应或 研究特定酶在生理学和生理学中的作用。

ABSTRACT Mapping the activity of an enzyme in vivo, directly within its natural environment, may provide unique insights into the role of a particular enzymes in pathophysiology of various diseases but remains extremely challenging. Recently, PI and colleagues demonstrated for the first time the ability of electron paramagnetic resonance (EPR) to image enzymatic activity using a nitroxide radical substrate of alkaline phosphatase (ALP). However, the in vivo application is hampered by the fast bioreducation of the nitroxide fragment. This project aims to develop the first biostable paramagnetic probes sensitive to enzymatic activity. The design is based on the triarylmethyl (TAM) radical scaffold known for its exceptional stability in biological milieu, narrow linewidth and high performance in polarization for Overhauser enhanced magnetic resonance imaging (OMRI). To achieve this goal we will rely on our recent advances in the chemistry of TAM functional probes that allow for multifunctional mapping using EPR imaging and/or OMRI. To prove the concept, under specific aim 1 (SA1) we will synthesize TAM-based paramagnetic substrates of alkaline phosphatase whose spectrum is modified upon enzymatic dephosphorylation. In addition, the synthesized probes will inherit the intrinsic sensitivity of TAM radical to tissue pO2, therefore making them dual function enzymatic ALP & pO2 probes. Under SA2 we will perform in vitro optimization of the OMRI sequences for dual ALP & pO2. Under SA3, as a first application of this new concept in vivo, we will image alkaline phosphatase (ALP) activity concurrently with tissue oxygenation using OMRI in the tumor microenvironment of a HeLa subcutaneous flank tumor model. Cancer research has recently experienced a paradigm shift from the seemingly obvious target of tumor cells towards key support systems of cancer, the tumor microenvironment (TME). Tissue pO2 and the activities of enzymes are among the important TME biomarkers. The imaging results will be validated using independent techniques, namely Oxylite for pO2 and optical imaging for ALP. The project focuses on the dual ALP & pO2 mapping using OMRI as a proof of concept but the probe scaffold has been designed for easy application to many other important enzymes in the future. The success of this proposal will provide a unique tool for in vivo enzymology allowing to map the activity of an enzyme and to correlate spacially this activity with tissue pO2. This unique tools will allows for outstanding applications in drug screening, therapy optimization, predication of the response to a particular treatment or to study the role of a particular enzyme in physiology and physiopathology.