Exploring Fundamental Strategies For Imaging Hydrolytic Enzymes

探索水解酶成像的基本策略

• 批准号: RGPIN-2022-05367
• 负责人: Phenix, Christopher
• 金额: $ 2.62万
• 依托单位: University of Saskatchewan
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=748047
• 关键词: Exploring; Fundamental; Strategies; Imaging; Hydrolytic

The focus of my research program is to apply fundamental bio-organic chemistry approaches, enzymology and rationale probe design to develop innovative chemical strategies for imaging lysosomal enzymes: most notably (CTB) and glucocerebrosidase (GCase). To improve MI of lysosomal proteases, in vitro and in vivo, we have prepared a series of novel prodrug inspired peptide derivatives designed to be substrate-based probes for CTB. However, a substrate-based approach requires the accumulation of the reporter signal, fluorescent or radioactive, within the local area of enzyme activity to improve detection. To test this approach, we prepared substrates bearing various aminoquinoline (AMQ) reporters that would become ionized and accumulate into the acidic environment of the lysosomes after CTB release. Weakly basic, lipophilic amines, like AMQs, are well known lysosomotropic molecules that spontaneously accumulate to high levels inside of cells. Indeed, peptides derivatized with PABA, a common prodrug linker and AMQs proved to be excellent substrates of CTB. However, most AMQs have no inherent fluorescent properties and cannot be easily labeled with 18F for positron emission tomography (PET) imaging studies in vivo. To address these limitations and explore lysosomal trapping to improve imaging of protease enzymes, we synthesized a series of AMQ derivatives designed to be highly fluorescent and lysosomotropic to enable cell imaging studies and to guide PET tracer discovery. We discovered novel AMQ derivatives that are highly fluorescent, have tunable lipophilicity, pKa, and accumulate inside the lysosomes of living cells through the lysosomotropic effect. Surprisingly, one derivative had near-infrared fluorescence emission and accumulated in the lysosomes of breast cancer cells, generating high resolution images using confocal microscopy. Further, several derivatives were fluorinated, using approaches compatible with 18F radiochemistry while also maintaining strong fluorescence. We will continue to investigate the photochemical properties of these novel AMQs (relative quantum yields, modes of fluorescence), physiochemical properties (pKa, logD) and biological properties (lysosomal trapping, toxicity). This will guide the development of new derivatives with improved imaging performance. To demonstrate the utility of these novel AMQs as powerful new reporter groups, we will conjugate the best derivatives onto irreversible inhibitors of GCase and into peptide conjugates to generate fluorogenic substrate-based probes for detecting CTB. In parallel, we will use the most efficient and selective substrates, bearing fluorinated AMQs, as potential PET tracers for imaging CTB in vivo. Importantly, the same prodrug strategies and lysosomotropic reporter groups developed for CTB can be easily adapted for imaging other proteases as well as other enzyme classes including glucosidases such as GCase thus having broad long term impact.

我的研究计划的重点是应用基本的生物有机化学方法，酶学和基本原理探针设计，以开发成像溶酶体酶的创新化学策略：最显着的是（CTB）和葡萄糖脑苷脂酶（GCase）。为了在体外和体内改善溶酶体蛋白酶的MI，我们制备了一系列新型前药启发的肽衍生物，旨在成为CTB的基于底物的探针。然而，基于底物的方法需要在酶活性的局部区域内积累荧光或放射性的报告信号以改善检测。 为了测试这种方法，我们制备了携带各种氨基喹啉（AMQ）报告分子的底物，这些报告分子在CTB释放后会被电离并积累到溶酶体的酸性环境中。弱碱性亲脂性胺，如AMQ，是众所周知的亲溶酶体分子，其在细胞内自发积累到高水平。事实上，用PABA（一种常见的前药接头）和AMQ衍生的肽被证明是CTB的优良底物。然而，大多数AMQ没有固有的荧光特性，并且不能容易地用18F标记用于体内正电子发射断层扫描（PET）成像研究。为了解决这些限制并探索溶酶体捕获以改善蛋白酶的成像，我们合成了一系列AMQ衍生物，其被设计为具有高度荧光性和亲溶酶体性，以使细胞成像研究成为可能并指导PET示踪剂的发现。我们发现了新的AMQ衍生物，其具有高荧光性，具有可调的亲脂性，pKa，并通过亲溶酶体效应在活细胞的溶酶体内积累。令人惊讶的是，一种衍生物具有近红外荧光发射，并在乳腺癌细胞的溶酶体中积累，使用共聚焦显微镜产生高分辨率图像。此外，几种衍生物被氟化，使用与18F放射化学相容的方法，同时还保持强荧光。 我们将继续研究这些新型AMQ的光化学性质（相对量子产率，荧光模式），理化性质（pKa，logD）和生物学性质（溶酶体捕获，毒性）。这将指导具有改进的成像性能的新衍生物的开发。为了证明这些新型AMQ作为强大的新报告基团的实用性，我们将最好的衍生物缀合到GCase的不可逆抑制剂上并缀合到肽缀合物中以产生用于检测CTB的基于荧光底物的探针。同时，我们将使用最有效和选择性的基板，轴承氟化AMQ，作为潜在的PET示踪剂成像CTB在体内。重要的是，为CTB开发的相同前药策略和亲溶酶体报告基团可以容易地适用于成像其他蛋白酶以及其他酶类别，包括葡萄糖醛酸酶如GCase，因此具有广泛的长期影响。