Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter.
Netropsin improves survival from endotoxaemia by disrupting HMGA1 binding to the NOS2 promoter.
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DOI：
10.1042/bj20081427
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发表时间：
2009-02-15
期刊：
影响因子：
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通讯作者：
Rigby AC
中科院分区：
文献类型：
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作者：
Grant MA;Baron RM;Macias AA;Layne MD;Perrella MA;Rigby AC
诱导型一氧化氮合酶(NOS2)在感染产生内毒素的革兰氏阴性细菌引起的脓毒症中起着重要作用。高迁移率族A1(HMGA1)结构转录因子通过AT-HOOK基序与NOS2核心启动子中特定的富含AT的OCT序列结合，从而促进NOS2的诱导。小分子、小沟槽结合蛋白(MGB)的netropsin选择性地靶向富含AT的DNA序列，并能干扰转录因子的结合。因此，我们假设Netropsin可以通过干扰核心NOS2启动子上的HMGA1-DNA结合来减弱NOS2的诱导，从而提高小鼠内毒素血症的存活率。Netropsin改善了野生型小鼠内毒素血症的存活率，但在NOS2缺陷小鼠中却没有，这支持了NOS2在MGB给药的有益效果中的重要作用。在巨噬细胞瞬时转染研究中，Netropsin显著减弱了NOS2启动子的活性，而富含AT的HMGA1-DNA结合位点对这一作用至关重要。EMSA证明netropsin干扰了HMGA1-NOS2启动子的结合，并用核磁共振波谱表征了这种破坏。化学位移扰动分析表明，netropsin有效地与富含AT的NOS2启动子序列中的两个HMGA1 DNA结合AT-挂钩竞争。此外，核Overhauser效应光谱(NOESY)数据发现netropsin与NOS2启动子HMGA1结合位点上的A/T碱基对之间存在直接的分子相互作用。最后，我们通过分子模拟和动力学计算确定了netropsin/NOS2启动子OCT位点复合体的结构。这些发现代表着朝着改进基于结构的配体设计新化合物的方向迈出了重要的一步，这些化合物具有治疗效益，可以选择性地针对对危重疾病的发展至关重要的基因内的关键调控区域。
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.