Molecular interaction of a protease inhibitor, leupeptin, with human serum albumin: Insights from calorimetry, spectroscopy, microscopy, and computational approaches

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Leupeptin (LPN) possesses anti-inflammatory and inhibitory effects on periodontitis and gingivitis, and it also inhibits human coronavirus and SARS-CoV-2 in cells. The characterisation of its interaction with human serum albumin (HSA), the key transport protein in the blood circulation of the human body, contributes essential information to the drug's pharmacokinetic properties. Various techniques, such as isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, atomic force microscopy (AFM), molecular docking, and molecular dynamics simulation (MDS), were employed to characterise the LPN-HSA interaction. The ITC result revealed a moderate binding affinity in the LPN-HSA interaction with a binding constant (Ka) value of 4.27 ± 0.27 × 106 M−1. The thermodynamic analysis indicated that LPN binding to HSA was spontaneous, resulting from a negative Gibbs free energy change (∆G° = − 37.91 ± 0.04 kJ mol−1) and involving an exothermic reaction due to a combination of a negative enthalpy change (∆H° = − 11.4 ± 0.9 kJ mol−1) and a positive entropy change (∆S° = 88.82 ± 3.4 J mol−1K−1). The HSA secondary structure remained unaltered upon the addition of LPN, as evident from the far-UV CD results. AFM results showed HSA morphological changes induced by LPN binding due to the occurrence of aggregates in the LPN-HSA complex. Molecular docking results predicted that LPN preferably binds to site III of HSA, involving hydrogen bonds, electrostatic, van der Waals forces, and hydrophobic forces. The MDS demonstrated a slight deviation and fluctuation, which supported the overall stability of the LPN-HSA system. Thus, these findings provide additional information on the pharmacokinetics of this drug and contribute to designing new drugs for clinical use, particularly those related to inflammatory diseases.