Delineating the binding site of a series of gold(III) Schiff base chelates on HSA via experimentation and in silico methods

Human serum albumin (HSA) is crucial for drug transport, influencing pharmacokinetics and pharmacological activity. While drug binding to HSA is well-studied, precise identification of primary binding sites remains underexplored. Since unbound drug fractions impact distribution and receptor-site concentration, understanding HSA interactions is essential for evaluating pharmacokinetics and toxicity. This study presents a systematic workflow integrating fluorescence quenching, circular dichroism, and ligand-binding thermodynamics to map ligand binding on HSA. Using patented Schiff base Au (III) chelates, we validate our approach with experimental data, molecular dynamics simulations, and QM/MM TD-DFT methods. Results indicate all three chelates preferentially bind Sudlow's site I, with affinities following AuL3 > AuL1 > AuL1, with logka values ranging from 4.73 to 5.03. Thermodynamic analysis suggests endothermic, hydrophobic-driven binding. Competitive site displacement assays confirm these findings. Overall, our results highlight HSA's potential as a transporter for metal-based therapeutics and demonstrate the efficacy of our workflow in accurately determining drug-binding sites.