Exploring Fundamental Strategies For Imaging Hydrolytic Enzymes

我的研究项目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来开发成像溶酶体酶的创新化学策略：最著名的是（CTB）和葡萄糖脑苷酶（GCase）。为了提高体内和体外溶酶体蛋白酶的MI，我们制备了一系列新的前药启发肽衍生物，设计为基于底物的CTB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报告信号以提高检测。为了测试这种方法，我们制备了含有各种氨基喹啉（AMQ）报告因子的底物，这些底物在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AMQs，是众所周知的溶酶体性分子，在细胞内自发积累到高水平。事实上，由PABA衍生的肽，一种常见的前药连接物和amq被证明是CTB的优秀底物。然而，大多数amq没有固有的荧光特性，不能很容易地用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，我们合成了一系列具有高荧光性和溶酶体性的AMQ衍生物，以实现细胞成像研究并指导PET示踪剂的发现。我们发现了新的AMQ衍生物，它们具有高荧光性，具有可调的亲脂性，pKa，并通过溶酶体性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，并积聚在乳腺癌细胞的溶酶体中，使用共聚焦显微镜产生高分辨率图像。此外，使用与18F放射化学相容的方法对若干衍生物进行了氟化处理，同时保持了强荧光。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），物理化学性质（pKa, logD）和生物学性质（溶酶体捕获，毒性）。这将指导具有改进成像性能的新衍生物的开发。为了证明这些新的amq作为强大的新报告基团的效用，我们将把最好的衍生物偶联到不可逆的GCase抑制剂和肽偶联物上，以产生基于荧光底物的检测CTB的探针。同时，我们将使用最有效和选择性的底物，含氟amq，作为体内CTB成像的潜在PET示踪剂。重要的是，同样的前药策略和为CTB开发的溶酶性报告群可以很容易地适用于其他蛋白酶以及其他酶类（包括葡萄糖苷酶如GCase）的成像，因此具有广泛的长期影响。

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.