Molecular interaction of abemaciclib with human serum albumin: Insights from spectroscopy, microscopy, and computational approaches.

Understanding drug-protein interactions at the biointerface is essential for predicting pharmacokinetics and guiding drug delivery strategies. Here, we systematically investigated the binding mechanism of abemaciclib, a selective CDK4/6 inhibitor, with human serum albumin (HSA) using a combination of multi-spectroscopic techniques, atomic force microscopy (AFM), molecular docking, molecular dynamics (MD) simulation, and site-directed mutagenesis. Fluorescence quenching and UV-Vis analyses revealed that abemaciclib interacts with HSA via a static mechanism, forming a 1:1 stoichiometric complex with moderate affinity (KA ∼ 105 M-1). Thermodynamic parameters (ΔH° < 0, ΔS° < 0) indicated that hydrogen bonding and van der Waals forces are the primary driving interactions. Competition experiments and molecular docking identified Sudlow site III (subdomain IB) as the preferred binding pocket, with Arg186 playing a key role, as confirmed by site-directed mutagenesis. Circular dichroism demonstrated minimal alterations in secondary structure, while three-dimensional fluorescence and atomic force microscopy suggested localized hydrophobicity and protein aggregation upon drug binding. Molecular dynamics simulations further validated the stability and dynamic features of the HSA-abemaciclib complex. Collectively, these findings elucidate the structural and thermodynamic determinants of albumin-drug interactions and provide mechanistic insights into the pharmacokinetics of abemaciclib. This work highlights the significance of albumin binding at the molecular interface, offering implications for dose optimization and albumin-based drug delivery strategies.