脂多糖
脂质A
化学
败血症
鲎试剂
细菌外膜
生物化学
生物物理学
生物
免疫学
基因
大肠杆菌
作者
Stewart J. Wood,Kelly A. Miller,Sunil A. David
出处
期刊:Combinatorial Chemistry & High Throughput Screening
[Bentham Science]
日期:2004-05-01
卷期号:7 (3): 239-249
被引量:63
标识
DOI:10.2174/1386207043328832
摘要
Lipopolysaccharides (LPS), otherwise termed ‘endotoxins’, are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of ‘Septic Shock’, a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS, and have shown using animal models of sepsis that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. Assays reported previously in the literature do not lend themselves well to the rapid screening of large numbers of structurally diverse compounds. In this report, we describe a highly sensitive and robust fluorescent displacement assay using BODIPY TR cadaverine (BC), which binds specifically to the toxic center of LPS, lipid A, and is competitively displaced by compounds displaying an affinity for lipid A. The assay clearly discriminates subtle differences in the binding of polymyxin B, and its nonapeptide derivative, with LPS. The spectral properties of the BODIPY fluorophore are ideally suited for screening diverse structural classes of compounds, including those with conjugated aromatic groups, or with chromophores in the 260-500 nm range. The fluorescent probe: LPS complex is stable under physiologically relevant salt concentrations, resulting in the rapid rejection of spurious binders interacting via non-specific electrostatic interactions, and, therefore, in greatly improved dispersion of ED50 values. Keywords: endotoxin, lipopolysaccharide, sepsis, shock, fluorescence, high-throughput screening
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