The combined use of BRAF and MEK inhibitors has transformed the management of BRAFV600-mutated melanoma, yet the pharmacokinetic interplay between cobimetinib (COB) and vemurafenib (VEM) remains incompletely understood. Here, we investigated the binding interactions of COB and VEM with human serum albumin (HSA) using a multidisciplinary approach combining fluorescence spectroscopy, isothermal titration calorimetry, circular dichroism, and molecular simulations. Both inhibitors form stable complexes with HSA, predominantly at Sudlow’s site II, driven by different interactions pattern. Thermodynamic and kinetic analyses revealed distinct binding behaviors: COB binding is entropy-driven (ΔH = +5.88 ± 0.32 kJ/mol; ΔG = −24.19 kJ/mol), with a dissociation constant (Kd) of 58.2 μM and a residence time of 1.45 s, indicating rapid and dynamic engagement. In contrast, VEM displays a more enthalpy-favored profile (ΔH = −22.05 ± 0.31 kJ/mol), stronger binding affinity (Kd≈ 4.8 μM), and a longer residence time of 18.5 s. Stoichiometry for both ligands is approximately 1:1, as determined by ITC. Structural analyses revealed subtle conformational alterations in HSA upon ligand binding, while enzymatic assays demonstrated that both COB and VEM competitively inhibit HSA’s esterase-like activity. These findings highlight distinct binding kinetics and functional consequences for each drug, offering critical insights into their pharmacokinetic behavior during combination therapy and providing a foundation for optimizing systemic exposure and therapeutic efficacy.
