This work presents a comprehensive investigation into the structural and energetic consequences of KI binding to HSA, with an emphasis on long-range perturbations and potential effects on the protein’s enzymatic behavior. The study focuses not only on quantifying binding affinities, but also on probing how the presence of kinase inhibitors may alter the conformational landscape and functional dynamics of the albumin molecule.
To this end, we adopt a combined experimental and computational strategy. On the experimental side, we employ a suite of biophysical and biochemical tools, including intrinsic fluorescence quenching to evaluate binding interactions and determine thermodynamic parameters; UV–visible and circular dichroism spectroscopy to monitor conformational changes upon ligand binding; and isothermal titration calorimetry (ITC) to assess enthalpic and entropic contributions to the binding process. Importantly, we also investigate how KI binding affects the esterase-like activity of albumin, which serves as a functional readout of conformational and active-site accessibility changes. These measurements are essential to understand whether drug-induced structural perturbations extend beyond the primary binding sites, potentially compromising HSA’s transport or catalytic roles.
Complementing the experimental data, we apply state-of-the-art computational techniques relying on HPC infrastructures. These tools allow us to assess binding energetics, explore conformational flexibility, and investigate the residence time and kinetic stability of albumin–drug complexes. Rather than focusing solely on local interactions at the drug-binding sites, we aim to characterize their systemic consequences across the protein scaffold, with attention to emergent allosteric-like effects.
Rather than considering albumin as a static reservoir for drug storage, we propose a view of HSA as a flexible, dynamic, and potentially responsive protein scaffold. In this perspective, drug binding becomes not merely a passive event, but a possible modulator of protein function. This has direct implications for therapeutic design: if KI–HSA interactions alter albumin’s ability to carry endogenous ligands (e.g., fatty acids, bilirubin, hormones) or modulate its esterase-like activity, this could result in unforeseen drug–drug interactions, reduced bioavailability, or altered toxicity profiles.
While the computational tools employed in this study are accessible and widely validated, their interpretation within a systemic and protein-functional context provides new opportunities for insight. Indeed, we argue that combining standard modeling approaches with experimental activity profiling can reveal otherwise hidden dimensions of drug–protein interactions, offering a more nuanced view of pharmacokinetic and pharmacodynamic behavior.
This work represents an early yet essential step toward re-evaluating the pharmacological role of serum proteins in drug efficacy. The integrated methodology we propose is broadly applicable, and may be adapted to study other classes of drugs with high serum protein affinity. Moreover, understanding how binding affects both protein structure and downstream functionality may eventually support the rational design of novel kinase inhibitors with improved therapeutic indices and reduced off-target systemic effects.