Structural Insights into the Competitive Binding of Diclofenac and Naproxen by Equine Serum Albumin

Abstract

The binding modes to equine serum albumin (ESA) of two nonsteroidal anti-inflammatory drugs (NSAIDs), diclofenac (Dic) and naproxen (Nps), were studied by X-ray crystallography and isothermal titration calorimetry. On the basis of the crystal structure of ESA/Dic determined to a resolution of 1.92 Å and the structure of the previously described ESA/Nps complex (2.42 Å), it was found that both NSAIDs bind within drug site 2 (DS2) of ESA and both occupy secondary binding sites in separate cavities of domain II (Nps) and domain III (Dic). The two structures of the ternary complex ESA/Dic/Nps, obtained by competitive cocrystallization (2.19 Å) and through a displacement experiment (2.35 Å), were determined to investigate possible competition of these widely used pharmaceutical drugs in binding to ESA. In these complexes Nps occupies the DS2 pocket common for both drugs, whereas the other distinct binding sites of Dic and Nps remain unaffected. These results suggest that combined application of both drugs may result in increased concentration of free diclofenac in plasma.

Structural investigations of stereoselective profen binding by equine and leporine serum albumins

“…The four determined crystal structures of ESA‐IBU (at 2.54 Å), ESA‐SUP (2.5 Å), LSA‐KET (1.9 Å), and LSA‐SUP (2.35 Å) significantly expand the structural data on chiral profen binding by albumins, which were limited to the structures of albumins (BSA, ESA, and LSA) with NPS, and the structures of HSA with NPS and IBU . The common binding location in albumin molecule for all four profens is DS2 in subdomain IIIA (Figure ), which is the primary and high‐affinity site for NPS . All aforementioned complexes show the binding mode of ( S )‐profens, despite that racemic mixtures of drugs were used for crystallization.…”

“…Based on our thermodynamic analysis of the profen binding by albumins, NPS is the most tightly bound profen to ESA with K a = 7.73 × 10 6 M −1 . This agrees with the ITC data obtained for HSA and BSA .…”

“…However, Lys414 of HSA presented slightly different conformation creating the third hydrogen bond with IBU (instead of only two hydrogen bonds observed in ESA). In our previous studies, 34,53 DS2 was also identified as the NPS binding site in ESA, BSA, and LSA. The conformation of all four drugs in DS2 is preserved with only minor differences in the angle of the carboxylic groups and aromatic moieties (Figure 4).…”

“…16 The common binding location in albumin molecule for all four profens is DS2 in subdomain IIIA (Figure 2), which is the primary and high-affinity site for NPS. 53 All aforementioned complexes show the binding mode of (S)-profens, despite that racemic mixtures of drugs were used for crystallization. Conformation of KET, SUP, and IBU within the DS2 in all four determined structures is very similar (Figure 3).…”

Binding thermodynamics of Diclofenac and Naproxen with human and bovine serum albumins: A calorimetric and spectroscopic study

“…It is a 67 kDa globular monomeric, hydrophilic, and non-glycosylated protein. It is arranged in a globular heart-shaped conformation and is composed of three α-helical domains (I, II and III), each with two subdomains (IA, IB, IIA, IIB, IIIA, IIIB) and several folded disulfide bridged loops with up to five different potential binding sites [8][9][10][11]. Two so-called Sudlow sites (site I and site II) are major binding regions within subdomains IIA and IIIA that allow the protein to bind a variety of hydrophobic, heterocyclic and anionic compounds including 70% of man-made drugs [12][13][14][15][16][17].…”