Evaluation of the Interactions between Human Serum Albumin (HSA) and Non-Steroidal Anti-Inflammatory (NSAIDs) Drugs by Multiwavelength Molecular Fluorescence, Structural and Computational Analysis

Abstract

The interaction between drugs and transport proteins, such as albumins, is a key factor in drug bioavailability. One of the techniques commonly used for the evaluation of the drug-protein complex formation is fluorescence. This work studies the interaction of human serum albumin (HSA) with four non-steroidal anti-inflammatory drugs (NSAIDs)-ibuprofen, flurbiprofen, naproxen, and diflunisal-by monitoring the fluorescence quenching when the drug-albumin complex is formed. Two approaches-the double logarithm Stern-Volmer equation and the STAR program-are used to evaluate the binding parameters. The results are analyzed considering the binding properties, determined by using other complementary techniques and the available structural information of albumin complexes with NSAID-related compounds. Finally, this combined analysis has been synergistically used to interpret the binding of flurbiprofen to HSA.

Keywords: drug-protein interactions; fluorescence multiwavelength data treatment; fluorescence quenching; human serum albumin; molecular modeling; non-steroidal anti-inflammatory drugs.

Figure 1

Comparison of fluorescence spectra in the study of diflunisal-HSA: (a) Emission; (b) synchronous Δλ 15 nm; (c) synchronous Δλ 60 nm modes at 25 °C.

Figure 2

Fluorescence spectrum for diflunisal-HSA at: (a) Emission; (b) synchronous Δλ = 15 nm; (c) synchronous Δλ = 60 nm modes. The sixteen additions of diflunisal performed to cover the ratio 0.7:13 with respect to the initial concentration of HSA in the cell (4.5 μM).

Figure 3

Chemical structures of the NSAIDs and related compounds included in the analysis of X-ray crystallographic structures.

Figure 4

Representation of the main clusters observed for (S)-ibuprofen, (S)-naproxen, diflunisal, and NSAID-related compounds in the X-ray crystallographic structures of their complexes with albumin from different organisms. The protein backbone of selected albumins (PDB entries 2BXG, 6OCI, 4OT2, 4LUH, 6OCL, and 5OSW; see also Table 6) are shown as a light gray cartoon. The only tryptophan residue (Trp241) present in human serum albumin is shown as gray spheres. Ligands bound to a common binding site are shown in different colors.

Figure 5

The predicted pose of diflunisal bound to sites IIIA, IIA, IIA-IIB, and IIC. The crystallographic poses of diflunisal (bound at sites IIIA, IIA, and IIA-IIB; PDB entry 2BXE), and naproxen (bound at site IIC; PDB entry 4OR0) are shown as sticks (C atoms in green and dark blue, respectively). The docked pose of diflunisal is shown with C atoms as orange sticks. The protein backbone is displayed as a gray cartoon. Selected interactions with HSA residues are represented as dashed lines.

Figure 6

The predicted pose of (S)-flurbiprofen bound to sites IIIA, IIA, IIA-IIB, and IIC. The crystallographic poses of diflunisal (bound at sites IIIA, IIA, and IIA-IIB; PDB entry 2BXE), and (S)-naproxen (bound at site IIC; PDB entry 4OR0) are shown as sticks (C atoms in green and dark blue, respectively). The docked pose of (S)-flurbiprofen is shown with C atoms as orange sticks. The protein backbone is displayed as a gray cartoon. Selected interactions with HSA residues are represented as dashed lines.

Figure 7

The predicted pose of (R)-flurbiprofen bound to sites IIIA, IIA, IIA-IIB, and IIC. The crystallographic poses of diflunisal (bound at sites IIIA, IIA, and IIA-IIB; PDB entry 2BXE) and (S)-naproxen (bound at site IIC; PDB entry 4OR0) are shown as sticks (C atoms in green and dark blue, respectively). The docked pose of (R)-flurbiprofen is shown with C atoms as orange sticks. The protein backbone is displayed as a gray cartoon. Selected interactions with HSA residues are represented as dashed lines.

Figure 8

Representation of the binding mode of (S)-naproxen, indomethacin, 3,5-diiodosalicylic acid, and diclofenac (these ligands are shown as sticks with C atoms in dark blue, orange, yellow, and cyan, respectively) to binding site IIC in albumin (taken from X-ray structures 4OR0, 2BXK, 2BXM, 2BXQ, 4JK4, 4LUH, 5OSW, and 4Z69). The orientation of the side chain of Trp214, which is found in two distinct conformations, is highlighted as sticks. The protein backbone is displayed as a gray cartoon.