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

Abstract

SYNOPSIS 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 high mobility group A1 (HMGA1) architectural transcription factor facilitates NOS2 induction by binding a specific AT-rich Oct sequence in the core NOS2 promoter via AT-hook motifs. The small-molecule, minor groove binder (MGB) netropsin selectively targets AT-rich DNA sequences and can interfere with transcription factor binding. Therefore we hypothesized that netropsin would improve survival from murine endotoxemia by attenuating NOS2 induction through interference with HMGA1-DNA binding to the core NOS2 promoter. Netropsin improved survival from endotoxemia 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 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, nuclear Overhauser effect spectroscopy (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 modeling 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 important for the development of critical illness.

Coordinate activation of inflammatory gene networks, alveolar destruction and neonatal death in AKNA deficient mice

“…Although homeobox proteins have been established as master gene transcription factors [46], DNA architecturemodulating AT-hook proteins constitute an evolutionarily conserved family with analogous global gene-regulation functions [1,2,4,6,9]. In accordance with this notion, AT-hook proteins bind a broad spectrum of AT-rich sequence-containing promoters [5] and regulate pathwayspecific gene expression networks [3,8,47,48]. The present in vivo studies aimed to assess the function of AKNA, a transcription factor with a theoretical N-terminus AT-hook and C-terminus ALM.…”

Overexpression of HMGA1 deregulates tumor growth via cdc25A and alters migration/invasion through a cdc25A-independent pathway in medulloblastoma

“…Thus, netropsin could function as an effective HMGA1 competitor to disrupt the HMGA1-DNA interaction [48] and thereby alter the expression of HMGA1-regulated genes. It was demonstrated that netropsin proved beneficial for survival during murine endotoxemia in vivo by interfering with the interaction between HMGA1 and the NOS2 promoter [20]. We showed that netropsin inhibited in vitro MB cell growth in a dose-dependent manner and importantly it inhibited the growth of intracranial MB xenografts in nude mice without any major toxicity, as reflected by no significant change of whole body weights.…”

“…Furthermore, interfering with HMGA1 expression by siRNA significantly inhibited MB cell growth and reduced migration and invasion potential, further supporting the rationale for the development of MB therapy targeting HMGA1 for the patients. HMGA1 is an architectural transcriptional factor that binds onto the minor groove of AT-rich DNA through its AT-hook to regulate target gene expression (reviewed in [39]), whereas netropsin is a smallmolecule DNA minor groove binder that structurally mimics the conformation of the highly conserved Pro-ArgGly-Arg-Pro core of the HGMA1 DNA binding domain AT-hooks [20]. Thus, netropsin could function as an effective HMGA1 competitor to disrupt the HMGA1-DNA interaction [48] and thereby alter the expression of HMGA1-regulated genes.…”

The high mobility group A1 molecular switch: turning on cancer – can we turn it off?

“…For example, distamycin [99] and netropsin [100] are minor groove-binding drugs that inhibit HMGA1 binding by blocking AT-rich regions located within minor grooves of DNA. Netropsin binds to an AT-rich region in the nitric oxide synthase-2 promoter [100].…”

“…For example, distamycin [99] and netropsin [100] are minor groove-binding drugs that inhibit HMGA1 binding by blocking AT-rich regions located within minor grooves of DNA. Netropsin binds to an AT-rich region in the nitric oxide synthase-2 promoter [100]. Distamycin was also tested in a murine model of endotoxemia where it blocks expression of the HMGA1 transcriptional target, P-selectin [99].…”