Integrative analysis of the anti-diabetic inhibitor GW9508 binding to human serum albumin: computational, spectroscopic and ITC insights

GW9508 (GW) is a promising anti-diabetic agent with potential to regulate glucose metabolism. This study explored its binding interaction with human serum albumin (HSA), the major blood transport protein, using in silico modeling, isothermal titration calorimetry (ITC), and various spectroscopic techniques. Molecular docking suggested that GW preferentially binds to subdomain IIA (Site I) of HSA, stabilized by hydrogen bonding, hydrophobic interactions, and van der Waals forces. Displacement studies confirmed this binding site. Molecular dynamics simulations supported the stability of the GW-HSA complex. ITC and fluorescence spectroscopy indicated a moderate binding affinity (Ka = 104-105M-1) and a spontaneous, thermodynamically favorable interaction (ΔG° = -31.01 ± 1.28 kJ mol-1). Negative enthalpy (ΔH° = -45.05 ± 0.85 kJ mol-1) and entropy (ΔS° = -47.10 ± 0.57 J mol-1K-1) values suggested contributions from hydrogen bonds and van der Waals forces. Fluorescence quenching followed a static mechanism, supported by a high bimolecular quenching rate constant (kq > 10¹⁰ M-1s-1) and UV-Vis spectral shifts. AFM analysis revealed morphological changes and protein aggregation upon GW binding. 3D fluorescence spectra showed microenvironmental alterations around Trp/Tyr residues, while FT-IR spectroscopy indicated minor changes in HSA's secondary structure. These findings provide insights into the transport and pharmacokinetic behavior of GW in human circulation.