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Research article

Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter

Marianne A. Grant, Rebecca M. Baron, Alvaro A. Macias, Matthew D. Layne, Mark A. Perrella, Alan C. Rigby
Biochemical Journal Feb 15, 2009, 418 (1) 103-112; DOI: 10.1042/BJ20081427
Marianne A. Grant
Division of Molecular and Vascular Medicine, Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215, U.S.A.Harvard Medical School, Boston, MA 02215, U.S.A.
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Rebecca M. Baron
Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, U.S.A.Harvard Medical School, Boston, MA 02215, U.S.A.
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Alvaro A. Macias
Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, U.S.A.Harvard Medical School, Boston, MA 02215, U.S.A.
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Matthew D. Layne
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, U.S.A.
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Mark A. Perrella
Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, MA 02115, U.S.A.Harvard Medical School, Boston, MA 02215, U.S.A.
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  • For correspondence: mperrella@rics.bwh.harvard.eduarigby@bidmc.harvard.edu
Alan C. Rigby
Division of Molecular and Vascular Medicine, Center for Vascular Biology Research, Department of Medicine, Beth Israel Deaconess Medical Center, 99 Brookline Avenue, Boston, MA 02215, U.S.A.Harvard Medical School, Boston, MA 02215, U.S.A.
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  • For correspondence: mperrella@rics.bwh.harvard.eduarigby@bidmc.harvard.edu
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Abstract

The inducible form of nitric oxide synthase (NOS2) plays an important role in sepsis incurred as a result of infection with Gram-negative bacteria that elaborate endotoxin. The HMGA1 (high-mobility group A1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct (octamer) sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule MGB (minor-groove binder) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. We therefore hypothesized that netropsin would improve survival from murine endotoxaemia by attenuating NOS2 induction through interference with HMGA1 DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxaemia in wild-type mice, yet not in NOS2-deficient mice, supporting an important role for NOS2 in the beneficial effects of MGB administration. Netropsin significantly attenuated NOS2 promoter activity in macrophage transient transfection studies and the AT-rich HMGA1 DNA-binding site was critical for this effect. EMSAs (electrophoretic mobility-shift assays) demonstrated that netropsin interferes with HMGA1 NOS2 promoter binding and NMR spectroscopy was undertaken to characterize this disruption. Chemical shift perturbation analysis identified that netropsin effectively competes both HMGA1 DNA-binding AT-hooks from the AT-rich NOS2 promoter sequence. Furthermore, NOESY data identified direct molecular interactions between netropsin and A/T base pairs within the NOS2 promoter HMGA1-binding site. Finally, we determined a structure of the netropsin/NOS2 promoter Oct site complex from molecular modelling and dynamics calculations. These findings represent important steps toward refined structure-based ligand design of novel compounds for therapeutic benefit that can selectively target key regulatory regions within genes that are important for the development of critical illness.

  • endotoxaemia
  • high-mobility group A protein
  • lipopolysaccharide
  • minor-groove binder
  • netropsin
  • nitric oxide synthase (NOS)

Abbreviations: AT, adenine thymine; DBD, DNA-binding domain; dsDNA, double-stranded DNA; EMSA, electrophoretic mobility-shift assay; HMGA, high-mobility group A; HSQC, heteronuclear single-quantum correlation; i.p., intraperitoneally; LPS, lipopolysaccharide; MGB, minor-groove binder; NOE, nuclear Overhauser effect; NOS2, inducible nitric oxide synthase; Oct, octamer; Sp1, specificity protein 1; SPR, surface plasmon resonance; TF, transcription factor; Tm, melting temperature; VSM, vascular smooth muscle; WT, wild-type

  • © The Authors Journal compilation © 2009 Biochemical Society
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February 2009

Volume: 418 Issue: 1

Biochemical Journal: 418 (1)
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Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter
Marianne A. Grant, Rebecca M. Baron, Alvaro A. Macias, Matthew D. Layne, Mark A. Perrella, Alan C. Rigby
Biochemical Journal Feb 2009, 418 (1) 103-112; DOI: 10.1042/BJ20081427
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Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter
Marianne A. Grant, Rebecca M. Baron, Alvaro A. Macias, Matthew D. Layne, Mark A. Perrella, Alan C. Rigby
Biochemical Journal Feb 2009, 418 (1) 103-112; DOI: 10.1042/BJ20081427

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Keywords

endotoxaemia
high-mobility group A protein
lipopolysaccharide
minor-groove binder
netropsin
nitric oxide synthase (NOS)

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