Hsu Hsiao-Tieh, Trantow Brian M, Waymouth Robert M, Wender Paul A
Department of Chemistry and ‡Department of Chemical and Systems Biology, Stanford University , Stanford, California 94305, United States.
Bioconjug Chem. 2016 Feb 17;27(2):376-82. doi: 10.1021/acs.bioconjchem.5b00469. Epub 2015 Sep 25.
The development of abiological catalysts that can function in biological systems is an emerging subject of importance with significant ramifications in synthetic chemistry and the life sciences. Herein we report a biocompatible ruthenium complex Cp(MQA)Ru(C3H5)PF6(-) 2 (Cp = cyclopentadienyl, MQA = 4-methoxyquinoline-2-carboxylate) and a general analytical method for evaluating its performance in real time based on a luciferase reporter system amenable to high throughput screening in cells and by extension to evaluation in luciferase transgenic animals. Precatalyst 2 activates alloc-protected aminoluciferin 4b, a bioluminescence pro-probe, and releases the active luminophore, aminoluciferin (4a), in the presence of luciferase-transfected cells. The formation and enzymatic turnover of 4a, an overall process selected because it emulates pro-drug activation and drug turnover by an intracellular target, is evaluated in real time by photon counting as 4a is converted by intracellular luciferase to oxyaminoluciferin and light. Interestingly, while the catalytic conversion (activation) of 4b to 4a in water produces multiple products, the presence of biological nucleophiles such as thiols prevents byproduct formation and provides almost exclusively luminophore 4a. Our studies show that precatalyst 2 activates 4b extracellularly, exhibits low toxicity at concentrations relevant to catalysis, and is comparably effective in two different cell lines. This proof of concept study shows that precatalyst 2 is a promising lead for bioorthogonal catalytic activation of pro-probes and, by analogy, similarly activatable pro-drugs. More generally, this study provides an analytical method to measure abiological catalytic activation of pro-probes and, by analogy with our earlier studies on pro-Taxol, similarly activatable pro-drugs in real time using a coupled biological catalyst that mediates a bioluminescent readout, providing tools for the study of imaging signal amplification and of targeted therapy.
能够在生物系统中发挥作用的非生物催化剂的开发是一个新兴的重要课题，在合成化学和生命科学中具有重大影响。在此，我们报道了一种生物相容性钌配合物Cp(MQA)Ru(C3H5)PF6(-) 2（Cp = 环戊二烯基，MQA = 4-甲氧基喹啉-2-羧酸盐）以及一种基于荧光素酶报告系统实时评估其性能的通用分析方法，该系统适用于细胞中的高通量筛选，并可扩展到荧光素酶转基因动物中的评估。前催化剂2在荧光素酶转染的细胞存在下激活烯丙基保护的氨基荧光素4b（一种生物发光前体探针），并释放出活性发光体氨基荧光素（4a）。4a的形成和酶促周转（这一整体过程因其模拟了前药激活和细胞内靶点的药物周转而被选中）通过光子计数实时评估，因为4a被细胞内荧光素酶转化为氧代氨基荧光素并发出光。有趣的是，虽然4b在水中催化转化为4a会产生多种产物，但生物亲核试剂如硫醇的存在可防止副产物形成，并几乎只提供发光体4a。我们的研究表明，前催化剂2在细胞外激活4b，在与催化相关的浓度下表现出低毒性，并且在两种不同的细胞系中效果相当。这项概念验证研究表明，前催化剂2是用于前体探针生物正交催化激活以及类似地可激活前药的有前景的先导物。更一般地说，本研究提供了一种分析方法，用于测量前体探针的非生物催化激活，并通过与我们早期关于前体紫杉醇的研究类比，使用介导生物发光读出的耦合生物催化剂实时测量类似地可激活的前药，为成像信号放大和靶向治疗的研究提供工具。
