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

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, USA.
Biochemical Journal (Impact Factor: 4.4). 11/2008; 418(1):103-12. DOI: 10.1042/BJ20081427
Source: PubMed


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.

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    • "For example, several studies have focused on HMGA inactivation either using small molecules that competitively bind to HMGA1 DNA binding sites or cross-link HMGA1 to DNA [36] [37]. Others have developed drugs such as FR900482, Hoechst 33258 and netropsin that compete for binding with HMGA1 to the minor groove of AT-rich DNA sequences, which results in competitive inhibition of HMGA1 DNA binding and diminished HMGA1 activity [20] [38] [39], however, these molecules have been shown to have high toxicity. An alternative approach to counteract carcinogenic effects of HMGA protein in cancer cells has been to knock down HMGA1 or HMGA2 expression levels using either antisense RNA or RNA interference. "
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