Binding thermodynamics of (E)-2-((2-hydroxybenzylidene)amino)-5-methylbenzonitrile and (E)-2-((2-hydroxybenzylidene)amino)-5-methylbenzonitrile cobalt(ii) with HSA: an experimental and molecular dynamic study

Abstract

The interaction of small-molecule ligands with human serum albumin (HSA) critically influences drug bioavailability and pharmacokinetics. This study examines the binding characteristics of the free ligand HL and its cobalt(II) complex, CoL, using spectroscopic techniques, molecular docking, and molecular dynamics (MD) simulations. Fluorescence quenching assays confirm a dose-dependent interaction via a dynamic quenching mechanism, with HL exhibiting a 1.8-fold higher binding affinity at 310 K (log K_a = 5.13) than CoL (log K_a = 4.90), indicating reduced protein interaction upon metal complexation. Thermodynamic analyses reveal ΔH and ΔS values for HL and CoL to be 16.34, 10.36 kJ mol⁻¹ and 0.15, 0.13 J K⁻¹ mol⁻¹, respectively, indicating an entropy-driven binding process, with HL demonstrating greater entropic contribution. Circular dichroism spectroscopy (UV-CD) shows minimal secondary structure perturbation, though CoL induces a slight reduction in α-helicity. Near-UV CD shifts and in silico docking confirm preferential binding at Sudlow's site I, near Trp-214, supporting fluorescence data. Furthermore, site displacement assays suggest possible multi-site binding for both CoL and HL. Experimental data was well supported by MD simulations over a 100 ns trajectory. These findings suggest HL's stronger HSA affinity could influence its bioavailability, whereas CoL exhibits reduced binding due to steric and electronic effects of metal coordination. This study enhances understanding of protein–ligand interactions, informing rational drug design strategies for albumin-bound therapeutics.