Interaction of rofecoxib with human serum albumin: Determination of binding constants and the binding site by spectroscopic methods

doi:10.1016/j.jphotochem.2007.06.011

Journal of Photochemistry and Photobiology A: Chemistry

Volume 193, Issues 2–3, 25 January 2008, Pages 81-88

https://doi.org/10.1016/j.jphotochem.2007.06.011 Get rights and content

Abstract

The interaction of rofecoxib with human serum albumin (HSA) under physiological condition was investigated by fluorescence, UV–vis absorbance and Fourier transfer infrared (FT-IR) spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by rofecoxib was the result of the formed complex of HSA–rofecoxib, and the site binding constants (K_a) were 4.840 × 10⁴, 3.450 × 10⁴, and 2.325 × 10⁴ M⁻¹ at 298, 304, and 310 K, respectively. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be −46.90 kJ mol⁻¹ and −67.59 J mol⁻¹ K⁻¹ according to van’t Hoff equation. The spectroscopic measurements and the thermodynamic parameters suggested that van der Waals interaction and hydrogen bonds were the predominant intermolecular forces to stabilize the complex. The distance r = 5.1 nm between donor (Trp²¹⁴) and accepter (rofecoxib) was obtained according to the Förster theory of non-radiative energy transfer. FT-IR spectra and UV–vis absorbance showed that the change of protein secondary structures resulted from the rofecoxib binding to several amino acids on the hydrophobic pocket of HSA. Furthermore, it is observed from the probe of competitive experiments that the binding location of rofecoxib with HSA could be the same as the warfarin site I of HSA, which was also revealed by fluorescence anisotropy.

Introduction

Serum albumin, as the most abundant protein constituent of blood plasma [1], facilitates the disposition and transportation of varieties of exogenous and endogenous ligands. The protein is capable of binding an extraordinarily broad range of pharmaceuticals, including fatty acids, amino acids, steroids, metal ions, etc. It is also responsible for the maintenance of blood pH [2], the drug disposition and efficacy [3], and the contribution of colloid osmotic blood pressure. Much of the clinical and pharmaceutical interest in the serum albumin derives from its effects on the drug pharmacokinetics [4]. HSA is considered to have at least three specific binding sites for high-affinity binding of drugs, sites I, II and III [5], [6] and a single tryptophan (Trp²¹⁴) in the subdomain IIA. X-ray measurements have revealed that ligands binding to HSA were located in hydrophobic cavities in subdomains IIA and IIIA. It is important to study the interaction of the drugs with HSA owing to the interaction of drugs with HSA influence the drugs’ pharmacology and pharmacodynamics.

Rofecoxib(4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone), a prescription COX-2 selective, non-steroidal anti-inflammatory drug (NSAID) was proved by FDA in May 1999 for the relief of signs and symptoms of osteoarthritis, for the management of acute pain in adults, and for the treatment of menstrual symptoms, and was later approved for the relief of signs and symptoms of rheumatoid arthritis in adults and children. As its structure shown in Fig. 1, it has been developed for treating acute pain and chronic inflammatory disorders without gastric side effects associated with the use of COX-1 inhibitors [7], [8], [9].

It is currently approved for the treatment of acute and chronic symptoms of osteoarthritis, rheumatoid arthritis, acute pain and menstrual pain [10]. Furthermore, rofecoxib can also slow the growth of human pancreatic cancer by changing the gene expression that favors cell cyclic arrest [11]. On 30 September 2004, however, Merck & Co., Inc. announced a voluntary withdrawal of rofecoxib (Vioxx) from the U.S. and worldwide market due to the safety concerns of an increasing risk of cardiovascular events (including heart attack and stroke) in patients on rofecoxib [12].

It is widely accepted that the distribution, metabolism, and efficacy of many drugs can be altered based on their affinity to serum albumin. Significantly, the determination and understanding of rofecoxib interacting with serum albumin are important for the therapy and design of the drug [2]. The knowledge of the interaction and binding to HSA is poorly understood but may open new avenues for the design of the most suitable rofecoxib derivatives without side-effects. Investigating the influence of the drug on protein not only provide the pharmacological action of rofecoxib, but also can illuminate its binding mechanisms.

In this paper, we have studied the interaction of rofecoxib with human serum albumin (HSA) at three temperatures (298, 304, and 310 K) under physiological condition. Spectroscopic data were used to quantify the binding constants of rofecoxib to HSA and the action distance which was based on the Förster energy transference (FET). UV–vis and FT-IR spectroscopy revealed that the change of protein secondary structure resulted from the rofecoxib binding to several amino acids on the hydrophobic pocket of HSA. What is more, the interaction of the mainly acting forces and the binding site of the location were characterized by optical spectroscopy.

Section snippets

Materials and solutions

Human serum albumin (HSA, fatty acid free), purchased from Sigma Chemical Company, was used without further purification. Tris–Base had a purity of no less than 99.5%, and NaCl, HCl, etc., were all of analytical purity. HSA was dissolved in Tris–HCl buffer solution (50mM Tris–Base, 100mM NaCl, pH 7.4 ± 0.1). Rofecoxib was obtained from USA of Merck & Co., Inc., prepared by absolute dimethyl sulfoxide (DMSO) to form 5 mM solution, and the DMSO in the solution of HSA was at a concentration of

Fluorescence quenching mechanism and binding constant

The fluorescence of protein was a sensitive method to investigate the conformation of protein when its environment and structure change. Fluorescence quenching can be dynamic, resulting from collisional encounters between the fluorophore and quencher, or static, resulting from the formation of a ground state complex between the fluorophore and quencher [16], which gives information about the changes of the molecular microenvironment in a vicinity of the chromophore molecules and the binding to

Conclusions

The interaction of rofecoxib with HSA under physiological condition was studied by spectral methods. The results indicated that the probable mechanism of rofecoxib interaction with HSA is a static quenching process. The binding process was exothermic, enthalpy driven and spontaneous, as indicated by the thermodynamic parameters analyzed, and the major part of the action force is van der Waals and H-bonds. On the efficiency of energy transfer between the donor and acceptor, the distance between

Acknowledgements

We gratefully acknowledge the financial support of National Natural Science Foundation of China (grant nos. 30570015, 20621502); Natural Science Foundation of Hubei Province; and Research Foundation of Chinese Ministry of Education.

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Interaction of rofecoxib with human serum albumin: Determination of binding constants and the binding site by spectroscopic methods

Abstract

Introduction

Section snippets

Materials and solutions

Fluorescence quenching mechanism and binding constant

Conclusions

Acknowledgements

Virology

Eur. J. Med. Chem.

J. Biol. Chem.

Chem. Phys. Lett.

J. Mol. Struct.

J. Pharm. Biomed. Anal.

Bioorg. Med. Chem.

J. Photochem. Photobiol. A: Chem.

Prog. Biophys. Mol. Biol.

J. Inorg. Biochem.

Biochim. Biophys. Acta

Anal. Biochem.

Biochem. Pharmacol.

J. Boil. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta

J. Biomol. Screen

Chem. Biol. Interact.

Biochim. Biophys. Acta

Angew. Chem. Int. Ed. Engl.

Med. Res. Rev.

Nature

Nat. Struct. Biol.