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Metabolic activation and major protein target of a 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist.

Abstract：

: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 α,β-unsaturated imine conjugated to the ε-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 β-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.

journal_name

Chem Res Toxicol

journal_title

Chemical research in toxicology

authors

Aloysius H,Tong VW,Yabut J,Bradley SA,Shang J,Zou Y,Tschirret-Guth RA

doi

10.1021/tx300017s

subject

Has Abstract

pub_date

2012-07-16 00:00:00

pages

1412-22

issue

eissn

0893-228X

issn

1520-5010

journal_volume

pub_type

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Metabolic activation and major protein target of a 1-benzyl-3-carboxyazetidine sphingosine-1-phosphate-1 receptor agonist.