Original article
Locating the binding sites of anticancer tamoxifen and its metabolites 4-hydroxytamoxifen and endoxifen on bovine serum albumin

https://doi.org/10.1016/j.ejmech.2011.07.005Get rights and content

Abstract

The breast anticancer drug tamoxifen and its metabolites bind serum albumins. We located the binding sites of tamoxifen, 4-hydroxytamoxifen and endoxifen on bovine serum albumin (BSA). FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to characterize the drug binding mode, binding constant and the effect of drug binding on BSA stability and conformation. Structural analysis showed that tamoxifen and its metabolites bind BSA via hydrophobic and hydrophilic interactions with overall binding constants of Ktam–BSA = 1.96 (±0.2) × 104 M−1, K4-hydroxytam–BSA = 1.80 (±0.4) × 104 M−1 and Kendox–BSA = 8.01 (±0.8) × 103 M−1. The number of bound drug molecules per protein is 1.7 (tamoxifen), 1.4 (4-hydroxitamoxifen) and 1.13 (endoxifen). The participation of several amino acid residues in drug–protein complexes is stabilized by extended hydrogen bonding network with the free binding energy of −13.47 (tamoxifen), −13.79 (4-hydroxtamoxifen) and −12.72 kcal/mol (endoxifen). The order of binding is 4-hydroxy-tamoxen > tamoxifen > endoxifen. BSA conformation was altered by a major reduction of α-helix from 63% (free BSA) to 41% with tamoxifen, to 39% with 4-hydroxytamoxifen, and to 47% with endoxifen. In addition, an increase in turn and random coil structures was found, suggesting partial protein unfolding. These results suggest that serum albumins might act as carrier proteins for tamoxifen and its metabolites in delivering them to target tissues.

Graphical abstract

Highlights

► The binding sites of antitumor tamoxifen and its metabolites are located on BSA. ► Tamoxifen formed stronger complexes than 4-hydroxytamoxifen and endoxifen. ► The complexation of tamoxifen and its metabolites induced a partial protein unfolding. ► BSA can transport tamoxifen and its metabolites in vitro.

Introduction

Tamoxifen [trans-1-(4-β-dimethylaminoethoxyphenyl)-1,2-diphenylbut-1-ene] (Scheme 1) is the most commonly used drug for the treatment of estrogen receptor α (ERα)-positive breast cancer in pre- and post-menopausal women [1], [2]. Several clinical studies have shown that tamoxifen reduces the incidence of ERα-positive breast cancer by 30–50% [3]. It is also used for the treatment of male breast cancer [4]. Despite the availability and use of aromatase inhibitors for breast cancer treatment in recent years, tamoxifen is the drug of choice for breast cancer treatment, because it is cost effective, life saving and devoid of major adverse side effects in the majority of patients [1], [2], [5], [6].

Tamoxifen is an antiestrogen and it acts on the ERα in target tissues. The female sex hormone, estradiol plays an important role in the induction and progression of breast cancer by its binding to the ERα and provoking a conformational state of the receptor for facile binding to coactivator proteins and recognition of the estrogen response element (ERE), that is present in the promoter/enhancer elements of estrogen-responsive genes [7], [8]. In breast cancer, ERα binding to the ERE triggers the expression of a cascade of genes involved in cell cycle regulation and cell proliferation. Tamoxifen exerts its action as a breast cancer drug/chemoprevention agent by antagonizing the action of estradiol, by its binding to the ligand binding domain of ERα and provoking a conformational state of the protein that is incapable of binding to the ERE [9]. However, tamoxifen is capable of triggering the effects of ERα in a tissue-specific manner in conjunction with coactivator proteins. Thus tamoxifen causes endometrial cancers in some patients [10].

Tamoxifen undergoes extensive metabolism in the human body, and several metabolites have been detected in human serum [11], [12 ], [13]. CYP2D6 and CYP3A4/5 enzymes, respectively, convert tamoxifen to 4-hydroxytamoxifen and N-desmethyltamoxifen. These metabolites are further converted to endoxifen (Scheme 1) by the action of CYP2D6 and CYP3A4/5 [14], [15], [16]. 4-Hydroxytamoxifen and endoxifen are potent antiestrogens in vitro and have been used to understand the mechanism of action of tamoxifen using breast cancer cell culture models [17], [18]. In serum, more than 98% of tamoxifen is bound to albumin, however, the nature of tamoxifen interaction with serum albumins is not yet clear. Chen and Hage [19] used indirect chromatographic approaches to determine the binding constants of tamoxifen–HSA interaction. Since bovine serum is extensively used in cell culture experiments, we wished to determine the binding affinity of tamoxifen and its metabolites with BSA.

Serum albumins are the major soluble protein constituents of the circulatory system and have many physiological functions [20]. The most important property of this group of proteins is that they serve as transporters for a variety of compounds such as drugs and fatty acids. BSA (Scheme 2) has been one of the most extensively studied of this group of proteins, particularly because of its structural homology with human serum albumin (HSA). The BSA molecule is made up of three homologous domains (I, II, III) that are divided into nine loops (L1–L9) by 17 disulfide bonds. The loops in each domain are made up of a sequence of large–small–large loops forming a triplet. Each domain in turn is the product of two subdomains (IA, IB, etc.). X-ray crystallographic data show that the albumin structure is predominantly α-helical with the remaining polypeptide, occurring in turns and in extended or flexible regions between subdomains with no β-sheets [21], [22], [23], [24]. BSA has two tryptophan residues that possess intrinsic fluorescence [25]. Trp-134 and Trp-212 are located in the first and second domain, respectively. Trp-212 is located within a hydrophobic binding pocket of the protein and Trp-134 is located on the surface of the molecule. While there are marked similarities between BSA and HSA in their compositions, HSA has only one tryptophan residue, Trp-214, while BSA contains two tryptophans Trp-212 and Trp-134 as fluorophores, capable of fluorescence quenching.

Fluorescence quenching is considered as a technique for measuring binding affinity between ligands and proteins. Fluorescence quenching is the decrease of the quantum yield of fluorescence from a fluorophore, induced by a variety of molecular interactions with quencher molecule(s) [25], [26]. Therefore, it is of interest to use quenching of the intrinsic tryptophan fluorescence of BSA as a tool to study the interaction of polyphenols with BSA in an attempt to characterize the nature of tamoxifen/metabolite–protein complex formation.

We report the results of spectroscopic analysis and docking studies of BSA complexes with tamoxifen, 4-hydroxytamoxifen, and endoxifen. Structural information regarding drug binding mode, and the effect of drugs on protein stability and secondary structure has been determined in this study.

Section snippets

Materials

BSA fraction V, tamoxifen and 4-hydroxytamoxifen were from Sigma Chemical Company. The synthesis of endoxifen was conducted at the Chemical Synthesis Core Facility by Fauq et al. [27]. Other chemicals were of reagent grade and used without further purification.

Preparation of stock solutions

Bovine serum albumin was dissolved in 10 mM Tris–HCl buffer (pH 7.4) at a concentration of 40 mg/ml (0.5 mM). The protein concentration was determined spectrophotometrically using the extinction coefficient of 36,500 M−1 cm−1 at 280 nm [28].

FTIR spectra of drug–BSA complexes

The complexation of tamoxifen and its metabolites with BSA was characterized by infrared spectroscopy and its derivative methods. Since there was no major spectral shifting for the protein amide I band at 1656 cm−1 (mainly Cdouble bondO stretch) and amide II band at 1545 cm−1 (C–N stretching coupled with N–H bending modes) [43], [44] upon drug interaction, the difference spectra [(protein solution + drug solution)  (protein solution)] were obtained to monitor the intensity variations of these vibrations and

Discussion

In serum, more than 98% of the tamoxifen is bound to albumin, however, the nature of tamoxifen–serum interaction is not yet known. The therapeutic efficacy of tamoxifen is determined by the distribution of the drug into tissues and the availability of the parent drug and its active metabolites in target tissues. However, there has been no report on the interaction and stability of tamoxifen metabolites, 4-hydroxytamoxifen and endoxifen, with bovine serum albumin.

Our comparative study showed for

Acknowledgments

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC), and by the Foundation of the University of Medicine and Dentistry of New Jersey (Grants 64-09 and PC28-11).

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