Accepted_test

Photodynamic therapy (PDT) is a non-invasive treatment that uses photosensitizers (PSs) to produce reactive oxygen species upon light activation, inducing controlled cell death and enhancing immune responses. Despite their potential, PSs face challenges such as aggregation. To address these issues, human serum albumin (HSA) can be used as a carrier to improve PS solubility and distribution. Understanding PS-HSA interactions is crucial, but traditional methods like fluorescence techniques often provide ambiguous results.

This study employs an integrative approach combining laser-induced EPR spectroscopy (LaserIMD) with advanced computational techniques, including molecular docking and molecular dynamics simulations, to investigate the structural heterogeneity and binding dynamics of PSs with HSA. LaserIMD EPR spectroscopy provides precise measurements of distances between spin labels at Cys34 of HSA and PSs, capturing full distance distributions. This method overcomes the limitations of other techniques by discriminating between different binding sites and providing clear structural information.

Molecular docking, particularly blind docking, is used to predict potential binding sites on HSA, followed by filtering and clustering based on experimental data. Focused docking and molecular dynamics simulations further refine and validate the identified binding sites. The study reveals that PS binding often involves multiple sites on HSA, not just the standard ones, and that substituent charge and the presence of metal ions influence binding site localization.

The integrative approach provides a comprehensive, experimentally validated map of PS binding sites on HSA, enhancing our understanding of PS-HSA interactions. This methodology is applicable to other biomolecular systems, offering a powerful tool for optimizing PSs for PDT.