Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, Chofu, Tokyo 182-8585, Japan.
School of Life Science and Technology, Tokyo Institute of Technology, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Bioconjug Chem. 2024 Sep 18;35(9):1391-1401. doi: 10.1021/acs.bioconjchem.4c00303. Epub 2024 Aug 15.
Bioluminescence (BL) generated by luciferase-coelenterazine (CTZ) reactions is broadly employed as an optical readout in bioassays and in vivo molecular imaging. In this study, we demonstrate a systematic approach to elucidate the luciferase-CTZ binding chemistry with a full set of regioisomeric CTZ analogs, where all the functional groups were regiochemically modified. When the chemical structures were categorized into Groups 1-6, the even-numbered Groups (2, 4, and 6) of the CTZ analogs are found to be exceptionally bright with NanoLuc enzyme. A CTZ analogue was the brightest with NanoLuc and the reason was deciphered by a computational analysis of the binding modes. We also report that (i) the regioisomeric CTZ analogs collectively create unique intensity patterns according to each marine luciferase, (ii) the quantitative structure-activity relationship analysis revealed the roles of respective functional groups of CTZ analogs, and (iii) the regioisomeric CTZ analogs also exert red shifts of the BL spectra and color variation: that is, the λ values are near 500 nm with NanoLuc, near 530 nm with ALuc16, and near 570 nm with RLuc86SG. The advantages of the regioisomeric CTZ analogs were finally demonstrated using (i) a dual-luciferase system with -specific NanoLuc and native CTZ-specific ALuc16, (ii) an estrogen activatable single-chain BL probe by imaging, and (iii) BL imaging of live mice bearing tumors expressing NanoLuc and RLuc8.6SG. This study is the first systematic approach to elucidate the regiochemistry in BL imaging studies. This study provides new insights into how CTZ analogs regiochemically work in BL reporter systems and guides the specific applications to molecular imaging.
荧光素-腔肠素（CTZ）反应产生的生物发光被广泛应用于生物测定和体内分子成像的光学读出。在这项研究中，我们展示了一种系统的方法来阐明荧光素-CTZ 结合化学，使用了一整套区域异构体 CTZ 类似物，其中所有的功能基团都进行了区域化学修饰。当化学结构被分类为第 1-6 组时，发现 CTZ 类似物的偶数组（第 2、4 和 6 组）与 NanoLuc 酶结合时异常明亮。CTZ 类似物 与 NanoLuc 结合最亮，原因通过对结合模式的计算分析得到了解释。我们还报告说，（i）区域异构体 CTZ 类似物根据每种海洋荧光素共同产生独特的强度模式，（ii）定量构效关系分析揭示了 CTZ 类似物各自功能基团的作用，以及（iii）区域异构体 CTZ 类似物还会使生物发光光谱发生红移和颜色变化：即 λ 值在与 NanoLuc 结合时接近 500nm，与 ALuc16 结合时接近 530nm，与 RLuc86SG 结合时接近 570nm。区域异构体 CTZ 类似物的优势最终通过以下方式得到证明：（i）使用具有特异性 NanoLuc 和天然 CTZ 特异性 ALuc16 的双荧光素酶系统，（ii）通过成像的雌激素激活单链生物发光探针，以及（iii）表达 NanoLuc 和 RLuc8.6SG 的荷瘤活鼠的生物发光成像。这项研究是首次系统地阐明生物发光成像研究中的区域化学。这项研究为 CTZ 类似物如何在生物发光报告系统中进行区域化学提供了新的见解，并指导了特定的分子成像应用。
