Developing COX-2-targeted fluorescent probes for HClO/ONOO⁻ detection in cancer cells: an integrated experimental and computational study

Overexpression of cyclooxygenase-2 (COX-2) and associated reactive species, specifically hypochlorous acid (HClO) and peroxynitrite (ONOO⁻), in the tumor microenvironment are critical hallmarks for early cancer diagnosis. In this work, we designed and synthesized two fluorescent probes,  S1  and  S2 , for the targeted imaging of HClO and ONOO⁻, respectively. Both probes integrate a celecoxib ligand for COX-2 targeting with a naphthalimide fluorophore. The probes operate via a photoinduced electron transfer (PET) mechanism, producing a distinct fluorescence turn-on response with excellent linearity against analyte concentration ( R 2  = 0.9902 for  S1  with HClO;  R 2  = 0.9897 for  S2  with ONOO⁻). This mechanism was theoretically validated through DFT calculations. In vitro cellular imaging demonstrated that both probes exhibit low cytotoxicity and achieve superior tumor cell selectivity, showing significantly higher fluorescence intensity in COX-2 overexpressing MCF-7 cancer cells than in normal HUVEC cells. The molecular basis for targeting was rigorously investigated using molecular docking and 100 ns molecular dynamics simulations. MM/PBSA calculations revealed favorable binding enthalpies for both  S1  (−80.05 ± 7.19 kcal/mol) and  S2  (−83.76 ± 3.88 kcal/mol) with COX-2, confirming stable and specific complex formation. This integrated experimental and computational strategy yields highly sensitive and selective probes, positioning  S1  and  S2  as promising tools for studying cancer-related biomarkers at the cellular level. Graphical