Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides

Jung Min Park, Tsunehiko Higuchi, Kazuya Kikuchi, Yasuteru Urano, Hiroyuki Hori, Takeshi Nishino, Junken Aoki, Keizo Inoue, Tetsuo Nagano

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14 Citations (Scopus)

Abstract

1. The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. 2. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. 3. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the α carbon atom. 4. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. 5. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay.

Original languageEnglish
Pages (from-to)1876-1882
Number of pages7
JournalBritish Journal of Pharmacology
Volume132
Issue number8
DOIs
Publication statusPublished - 2001

Keywords

  • Depeptide
  • Human DLD-1 cells
  • Human nitric oxide synthase
  • Hydrophobic L-amino acids
  • Isozyme-selective inhibition
  • Nitric oxide
  • Structure-activity relationship study

ASJC Scopus subject areas

  • Pharmacology

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  • Cite this

    Park, J. M., Higuchi, T., Kikuchi, K., Urano, Y., Hori, H., Nishino, T., Aoki, J., Inoue, K., & Nagano, T. (2001). Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides. British Journal of Pharmacology, 132(8), 1876-1882. https://doi.org/10.1038/sj.bjp.0704023