Probing the binding sites of antibiotic drugs doxorubicin and N-(trifluoroacetyl) doxorubicin with human and bovine serum albumins

Abstract

We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K(DOX-BSA) = 7.8 (± 0.7) × 10(3) M(-1), K(FDOX-BSA) = 4.8 (± 0.5)× 10(3) M(-1) and K(DOX-HSA) = 1.1 (± 0.3)× 10(4) M(-1), K(FDOX-HSA) = 8.3 (± 0.6)× 10(3) M(-1). The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47-44% (drug-complex) and 57% (free HSA) to 51-40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro.

Figure 1. Chemical structures of doxorubicin, N-(trifluoroacetyl) doxorubicin and BSA and HSA with tryptophan residues in green color.

Figure 2. FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 7.4) for free BSA (0.25 mM) and its drug complexes with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).

Figure 3. FTIR spectra in the region of 1800–600 cm⁻¹ of hydrated films (pH 7.4) for free HSA (0.25 mM) and its drug complexes with difference spectra (diff.) (bottom two curves) obtained at different drug concentrations (indicated on the figure).

Figure 4. Second derivative resolution enhancement and curve-fitted amide I region (1700–1600 cm⁻¹) for free BSA and HSA (0.25 mM) and their drug complexes with 0.5 mM drug concentration.

Figure 5. Fluorescence emission spectra of drug-BSA systems in 10 mM Tris-HCl buffer pH 7.4 at 25°C presented for (A) drug–BSA: (a) free BSA (10 µM), (b–o) with drug 1, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µM; (B) drug– HSA: (a) free HSA (10 µM), (b–o) drug at 1, 5, 7.5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µM.

Inset: F _0/(F ₀–F) vs 1/[drug] for A’ (DOX-BSA), B’ (FDOX-BSA), C’ (DOX-HSA) and D’ (FDOX-HSA).

Figure 6. Stern-Volmer plots of fluorescence quenching constant (Kq) for the drug-BSA and drug-HSA complexes at different drug concentrations (A) DOX-BSA and (B) FDOX-BSA (C) DOX-HSA and (D) FDOX-HSA.

Figure 7. The plot of Log (F₀–F/F) as a function of Log (drug concentration) and the number of bound drug molecules per protein (n).

Figure 8. Best docked conformations of drug–BSA and drug–HSA complexes.

(A, A’) for DOX complexed to BSA, (B, B’) for FDOX complexed to BSA, (C, C’) for DOX complexed to HSA, (D, D’) for FDOX complexed to HSA.