Shedding light on the molecular interaction between the Hepatitis B virus inhibitor, clevudine, and human serum albumin: Thermodynamic, spectroscopic, microscopic, and in silico analyses

Abstract

The molecular interaction between clevudine (CLV), a hepatitis B virus inhibitor, and human serum albumin (HSA), the major transporter in plasma, was investigated via fluorescence, UV–vis absorption, circular dichroism (CD), atomic force microscopy (AFM), and molecular modelling approaches. A steady reduction in the Stern-Volmer quenching constant (Ksv) with increasing temperature endorsed the static mode of the protein fluorescence quenching upon CLV addition, thus confirming CLV-HSA complex formation. This finding was supported by alteration in the UV–vis absorption spectrum of HSA upon CLV addition, higher value (>10 10 M−1 s−1) of the bimolecular quenching rate constant (kq), along with swelling of the HSA molecule in the presence of CLV, as revealed by AFM. Fluorescence quenching titration established a weak binding affinity from the values of the binding constant (Ka=8.65-11.20×103 M−1) at various temperatures (290, 300, 310 and 315 K) for the CLV-HSA system. According to the thermodynamic data (ΔS = +51.89 J mol−1 K−1 and ΔH = −7.69 KJmol−1) of the binding reaction, hydrophobic interactions, hydrogen bonds, and van der Waals forces stabilized the CLV-HSA complex. Lesser changes in both secondary and tertiary structures of HSA upon CLV binding were ratified from the far-UV and the near-UV CD spectral results. In addition, the microenvironment around HSA’s Trp and Tyr residues was also altered, as deduced from three-dimensional fluorescence spectra. The protein showed some resistance against thermal stress in the presence of CLV. The binding site of CLV in subdomain IIA (Site I) of HSA was identified by competitive displacement and molecular docking findings.