Gaining Insights Into the Formation of the Cathepsin B Inhibitor II–Human Serum Albumin Complex: A Multidisciplinary Approach

ABSTRACT Cathepsin B inhibitor II (CBI), a cysteine protease inhibitor, is an anti‐inflammatory and anticancer agent. It targets cathepsin B, an enzyme implicated in the development of diseases such as periodontitis, neurological disorders, and cancer progression. Understanding CBI's interaction with human serum albumin (HSA), the main blood transport protein, is essential for predicting its distribution and bioavailability. Multiple experimental and computational approaches have elucidated this interaction. Isothermal titration calorimetry revealed spontaneous, exothermic binding with moderate affinity ( K a = 6.13 ± 0.10 × 10 5 M −1 ). Circular dichroism showed minimal changes in HSA secondary structure, while atomic force microscopy demonstrated surface aggregation, supporting complex formation. Competitive displacement assays and molecular docking suggested Sudlow's Site I (subdomain IIA) as the preferred binding site, with the lowest docking energy of −34.64 kJ⋅mol −1 . The interaction was stabilized via H‐bonds, hydrophobic, and van der Waals forces, and the bound state of CBI and HSA was preserved during 100‐ns simulation. Esterase activity assays demonstrated that CBI affected HSA's esterase‐like function, reflected by a concurrent decrease in K m and increase in k cat . These findings offer detailed insights into CBI–HSA interactions, enhancing understanding of its behavior in the bloodstream and guiding potential applications in human health.