Ipatasertib binding to human serum albumin: An integrated multi-technique perspective

Ipatasertib (IPA), a pan-AKT inhibitor under clinical investigation for triple-negative breast cancer, may have its pharmacological behavior influenced by plasma-protein binding, which affects systemic transport and the free circulating drug fraction. Human serum albumin (HSA), the major carrier protein in blood, is therefore expected to contribute to IPA distribution in vivo. In this study, the interaction between IPA and HSA was investigated through a combined experimental and computational approach. Ultraviolet-visible spectroscopy, steady-state fluorescence spectroscopy, synchronous and three-dimensional fluorescence spectroscopy, circular dichroism, site-marker displacement assays, and isothermal titration calorimetry were used to define the binding behavior, thermodynamic profile, and structural consequences of complex formation, while molecular modeling and enhanced sampling simulations provided a molecular-level interpretation of the interaction and its kinetic stability. ITC revealed a micromolar binding affinity (Kd = 145 ± 27 μM) and a thermodynamically spontaneous interaction (ΔG = −21.9 ± 0.6 kJ/mol), with favorable contributions from both enthalpy (ΔH = −12.7 ± 2.9 kJ/mol) and entropy (-TΔS = −9.2 ± 3.5 kJ/mol), together with an apparent stoichiometry of n = 1.23 ± 0.09. Spectroscopic data support complex formation and indicate limited global structural perturbation of HSA, accompanied by ligand-dependent local environmental changes. Competitive displacement experiments and molecular modeling consistently identify Sudlow site II as the preferential binding region of IPA. Enhanced sampling simulations further indicate a short residence time, consistent with a reversible binding process. These findings define a quantitative physicochemical framework for interpreting albumin-mediated transport of IPA and its possible impact on systemic availability.