Mechanistic Insights Into the Cooperative Interactions of PI3K/mTOR Inhibitor Pictilisib and EGCG With Human Serum Albumin: A Comprehensive Spectroscopic and Computational Study

ABSTRACT Combining PI3K/mTOR inhibitor Pictilisib with green tea polyphenol EGCG holds promise in cancer therapy but may alter pharmacokinetics via human serum albumin (HSA) binding. This study elucidates their cooperative interactions with HSA under physiological conditions using multispectroscopy (steady‐state fluorescence, UV–Vis, circular dichroism [CD], synchronous, and three‐dimensional [3D] fluorescence), competitive binding assays, molecular docking, and molecular dynamics (MD) simulations. Fluorescence quenching revealed coadministration of Pictilisib and EGCG with HSA exhibits higher binding constants (up to 10 6 M −1 ) and stronger hydrophobic interactions compared to binary systems, driven by entropy‐dominated static quenching. Structural characterization by CD, synchronous, and 3D fluorescence spectroscopy demonstrated that combined drug binding induces progressive α‐helix reduction and conformational reorganization in HSA, which alters the microenvironment of Trp/Tyr residues. Molecular docking and site probes show Pictilisib displaces prebound EGCG from Sudlow Site I (IIA), whereas EGCG binds subdomain IB when Pictilisib occupies Site I first, highlighting administration‐order dependency. One‐hundred‐nanosecond MD simulations confirm ternary complexes increase HSA's solvent accessibility (SASA), flexibility (RMSF), and structural expansion (Rg), particularly in Subdomains IIA and IIIA. These results demonstrate that HSA‐mediated EGCG–Pictilisib interactions are sequence dependent, critically influencing drug distribution and bioavailability. This mechanistic insight provides a foundation for optimizing coadministration schedules in combinatorial cancer therapy.