First‐principles simulations of the fluorescence modulation of a <scp>COX</scp>‐2‐specific fluorogenic probe upon protein dimerization for cancer discrimination

Cyclooxygenase-2 (COX-2) plays a crucial role in inflammation and has been implicated in cancer development. Understanding the behavior of COX-2 in different cellular contexts is essential for developing targeted therapeutic strategies. In this study, we investigate the fluorescence spectrum of a fluorogenic probe, NANQ-IMC6, when bound to the active site of human COX-2 in both its monomeric and homodimeric forms. We employ a multiscale first-principles simulation protocol that combines ground state MM-MD simulations with multiple excited state adiabatic QM/MM Born-Oppenheimer MD simulations based on linear response TD-DFT, which allows to account for protein heterogeneity effects on excited-state properties. Emission is then estimated from polarizable embedding TD-DFT QM/MMPol calculations. Our findings indicate that the emission shift arises from dimerization of the highly overexpressed COX-2 in cancer tissues, in contrast to the monomer structure present in inflammatory lesions and in normal cells with constitutive COX-2. This spectral shift is linked to changes in specific protein-probe interactions upon dimerization due to changes in the environment, whereas steric effects related to modulation of the NANQ geometry by the protein scaffold are found to be minor. This research paves the way for detailed investigations on the impact of environment structural transitions on the spectral properties of fluorogenic probes. Moreover, the fact that COX-2 exists as homodimer just in cancer tissues, but as monomer elsewhere, gives novel hints for therapeutical avenues to fight cancer and contributes to the development of drugs targeted to COX-2 dimer in cancer, but without affecting constitutive COX-2, thus minimizing off-target effects.