Metabolic Activation and Major Protein Target of a 1-Benzyl-3-carboxyazetidine Sphingosine-1-phosphate-1 Receptor Agonist

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• 作者：Herve Aloysius ; Vincent W. Tong ; Jocelyn Yabut ; Scott A. Bradley ; Jackie Shang ; Yan Zou ; Richard A. Tschirret-Guth
• 刊名：Chemical Research in Toxicology
• 出版年：2012
• 出版时间：July 16, 2012
• 年：2012
• 卷：25
• 期：7
• 页码：1412-1422
• 全文大小：517K
• 年卷期：v.25,no.7(July 16, 2012)
• ISSN：1520-5010

文摘

1-{4-[(4-Phenyl-5-trifluoromethyl-2-thienyl)methoxy]benzyl}azetidine-3-carboxylic acid (MRL-A) is a potent sphingosine-1-phosphate-1 receptor agonist, with potential application as an immunosuppressant in organ transplantation or for the treatment of autoimmune diseases. When administered orally to rats, radiolabeled MRL-A was found to undergo metabolism to several reactive intermediates, and in this study, we have investigated its potential for protein modification in vivo and in vitro. MRL-A irreversibly modified liver and kidney proteins in vivo, in a dose- and time-dependent manner. The binding was found to occur selectively to microsomal and mitochondrial subcellular fractions. Following a nonspecific proteolytic digestion of liver and kidney proteins, a single major amino acid adduct was observed. This adduct was characterized with LC/MS/UV and NMR spectroscopy and was found to be the product of an unprecedented metabolic activation of the azetidine moiety leading to the formation of a ring-opened 伪,尾-unsaturated imine conjugated to the 蔚-amino group of a lysine residue. The formation of this adduct was not inhibited when rats were pretreated with 1-aminobenzotriazole, indicating that P450 enzymes were not involved in the metabolic activation of MRL-A. Rather, our findings suggested that MRL-A underwent bioactivation via a 尾-oxidation pathway. Several other minor adducts were identified from protein hydrolysates and included lysine, serine, and cysteine conjugates of MRL-A. These minor adducts were also detected in microsomal incubations fortified with the cofactors for acyl-CoA synthesis and in hepatocytes. Trypsin digestion of crude liver homogenates from rats treated with radiolabeled MRL-A led to the identification of a single radioactive peptide. Its sequence, determined by LC/MS analysis, revealed that the target of the major reactive species of MRL-A in vivo is Lys676 of long chain acyl-CoA synthetase-1 (ACSL1). This lysine residue has been found to be critical for ACSL1 activity, and its modification has the potential to lead to biological consequences such as cardiac hypertrophy or thermogenesis dysregulation.