Kim Sung-Bae, Furuta Tadaomi, Kamiya Genta, Maki Shojiro A, Orioka Mariko, Watanabe Ryuji, Hiruta Yuki, Thangudu Suresh, Natarajan Arutselvan, Paulmurugan Ramasamy
Environmental Management Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 16-1 Onogawa, Tsukuba 305-8569, Japan.
School of Life Science and Technology, Institute of Science Tokyo, B-62 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
ACS Omega. 2025 Jul 24;10(30):33850-33861. doi: 10.1021/acsomega.5c04665. eCollection 2025 Aug 5.
Nuclear receptors (NRs) play pivotal roles in functionally diverse cell signaling cascades, regulating metabolism and homeostasis. This study introduces a broadly applicable molecular imaging platform for NR activities based on four rationally designed single-chain bioluminescent probes named -. As all the ligand binding domains (LBDs) of NRs are highly conserved, the probe portfolio was exemplified using the LBD of peroxisome proliferator-activated receptor γ (PPARγ-LBD), i.e., the LBD was centrally positioned and fused with luciferases and/or fluorescent proteins based on the schemes of the protein-fragment complementation assay (PCA), molecular strain (MS) probe, circular permutation (CP) probe, and bioluminescence resonance energy transfer (BRET) system. In the developing process, we identified the optimal dissection sites for the marine luciferase RLuc for PCA systems and validated them in in vitro and in vivo studies in response to various ligands. Among the probes, and series probes exhibited strong BL intensities in response to a PPARγ agonist with signal-to-background ratios of maximally 14-fold. The animal study using furimazine (FMZ) substrate analogs such as Ad-FMZ showed that the probes can sustain agonist-dependent BL signals for up to 24 h in animal models. Considering the biological importance of NRs, the molecular imaging platform with the portfolio of probes developed in this study can contribute to interrogate many NR-related cell signaling pathways by replacing the PPARγ-LBD with LBDs from other NRs in the probes.
核受体（NRs）在功能多样的细胞信号级联反应中发挥关键作用，调节代谢和体内平衡。本研究基于四种合理设计的单链生物发光探针（命名为-），引入了一种广泛适用的用于NR活性的分子成像平台。由于NRs的所有配体结合结构域（LBDs）高度保守，该探针组合以过氧化物酶体增殖物激活受体γ的LBD（PPARγ-LBD）为例进行说明，即基于蛋白质片段互补分析（PCA）、分子应变（MS）探针、环状排列（CP）探针和生物发光共振能量转移（BRET）系统的方案，将LBD置于中心位置并与荧光素酶和/或荧光蛋白融合。在开发过程中，我们确定了用于PCA系统的海洋荧光素酶RLuc的最佳切割位点，并在体外和体内研究中针对各种配体对其进行了验证。在这些探针中，和系列探针在响应PPARγ激动剂时表现出强烈的生物发光强度，信号与背景比最大为14倍。使用诸如Ad-FMZ等呋虫嗪（FMZ）底物类似物的动物研究表明，这些探针在动物模型中可维持激动剂依赖性生物发光信号长达24小时。考虑到NRs的生物学重要性，本研究开发的具有探针组合的分子成像平台可通过将探针中的PPARγ-LBD替换为其他NRs的LBD，有助于探究许多与NR相关的细胞信号通路。
