Imaging Ligand-Driven PPAR Activities Using Single-Chain Bioluminescent Probes

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 P1-P4. 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, P3 and P4 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.