Drug Screening of Nitroreductase Modulators Using an Atomic-Engineered Activatable Fluorescent Probe for Tumor Imaging.

Nitroreductase (NTR), a hypoxia-upregulated enzyme, is a promising biomarker for solid tumors, driving demand for effective biosensors. Cyanine dyes are prominent fluorophores widely used in fluorescence imaging, tumor phototheranostics, and image-guided surgery. Guided by atomic engineering through strategic heteroatom substitution (C, O, and S), we designed four NTR-activated probes. Optical characterization identified HCy-NS-4 as the lead candidate, exhibiting near-infrared emission (λem = 712 nm) and over 18-fold fluorescence enhancement upon activation. The probe enabled precise imaging of hypoxic NTR and distinguished malignant from nonmalignant cells, while in vivo and in vitro studies supported the use of the probe in intraoperative tumor navigation. Additionally, using HCy-NS-4 as a screening tool, we identified luteolin as a potential NTR modulator that exhibited concentration-dependent suppression of NTR-associated fluorescence activation and significant antitumor activity in vivo. Collectively, this continuum─from atomic-scale molecular design to novel NTR inhibitor discovery─provides an innovative model for drug screening.