Identification of Two Tamoxifen Target Proteins by Photolabeling with 4-(2-Morpholinoethoxy)benzophenone

Abstract

Our quest to identify target proteins involved in the activity of tamoxifen led to the design of photoaffinity ligand analogues of tamoxifen able to cross-link such proteins. A new tritiated photoprobe, 4-(2-morpholinoethoxy)benzophenone (MBoPE), was synthesized and used to identify proteins involved in tamoxifen binding in rat liver. MBoPE, which has structural features in common with the potential antagonist of the intracellular histamine receptor (N,N-diethyl-2-[(4-phenylmethyl)phenoxy]ethanamine HCl: DPPE) is unable to bind the estrogen receptor although it does compete with tamoxifen for an antiestrogen binding site (AEBS). This tritiated benzophenone derivative was obtained by metal-catalyzed halogen-tritium replacement reaction. Because of its high specific activity, four target proteins could be photolabeled, three of which were identified with M(r) of 60,000, 49,500, and 14,000, while the fourth at 27,500 was in too low an amount and could not be sequenced. The 49.5 kDa protein corresponded by mass spectrometry to the microsomal epoxide hydrolase already identified with an aryl azide photoprobe [Mesange, F., et al. (1998) Biochem. J. 334, 107-112]. The 60 and 14 kDa proteins were identified as the carboxylesterase (ES10) and the liver fatty acid binding protein (L-FABP), respectively. The inhibitory effect of tamoxifen on carboxylesterase activity and the competitive efficacy of oleic acid on [(3)H]tamoxifen binding suggest that both proteins are AEBS subunits. Moreover, treatment of hepatocytes with antisense mRNA directed against ES10 or L-FABP abolished both tamoxifen and MBoPE binding. On the basis of previous pharmacological arguments, the 27.5 kDa protein might correspond to the sigma I receptor. Altogether, these results confirm that the microsomal epoxide hydrolase is a target for tamoxifen and provide evidence of two new target proteins implicated in cell lipid metabolism.

Structural Insights into Drug Processing by Human Carboxylesterase 1: Tamoxifen, Mevastatin, and Inhibition by Benzil

“…19 hCE1 was not thought to be involved in the trafficking of tamoxifen through the body until it was found that only four proteins in rat liver bound a labeled form of tamoxifen, and one of these proteins was the rat homologue of hCE1. 20 Thus, it is possible that the binding of tamoxifen to hCE1 may limit the bioavailability of the drug, or may lead to drug-drug interactions and/or side-effects associated with tamoxifen use, including impact on cholesterol levels. 21 In addition to its role in xenobiotic metabolism, hCE1 has been shown to catalyze reactions involved in cholesterol homeostasis and fatty acid metabolism.…”

Implications of the binding of tamoxifen to the coactivator recognition site of the estrogen receptor

“…In these tissues, a biomacromolecule was observed to bind tamoxifen and other estrogenic derivatives, including ICI 47699 (Murphy & Sutherland 1981a), and displays tissue-specific differences in cellular concentration . This substance was subsequently named the microsomal antiestrogen-binding site (AEBS); a hetero-oligomeric multiprotein complex found in most mammals in different tissue-dependent concentrations (Mésange et al 2002, Kedjouar et al 2004. For example, AEBS is w20-30 times more abundant in the liver when compared with tumor cell lines (Chailleux et al 1994).…”

Molecular Characterization of the Microsomal Tamoxifen Binding Site

“…For this reason, the liver have been chosen for the purification of the AEBS but the pharmacological profiles of the AEBS found in the liver and in tumor cell lines such as MCF-7 cells (a mam-mary adenocarcinoma cell line) have been reported to be different, suggesting a possible difference in the composition of the AEBS in the two systems (19). We have recently reported that the AEBS from rat liver was a hetero-oligomeric multiprotein complex that contained subunits that were not directly involved in the binding of tamoxifen such as the microsomal epoxide hydrolase (mEH) (20), the carboxyl-esterase (ES-10), and the liver fatty acid-binding protein (FABP) (21). Each of these proteins have been implicated in lipid metabolism: mEH is a bile acid transporter (22), and carboxylesterase has cholesterol esterification properties (23) but were related to the AEBS found in normal liver because ES-10 and FABP were not found in tumor cell lines.…”