Anti-endotoxin agents. 1. Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents.
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 ED(50)values.
Wood
Stewart J
SJ
Department of Medicinal Chemistry, University of Kansas, Life Sciences Research Laboratories, 1501 Wakarusa Drive, Lawrence, KS 66049, USA.
Miller
Kelly A
KA
David
Sunil A
SA
D000209:Acute-Phase Proteins; D000900:Anti-Bacterial Agents / Q000032:analysis*; D001896:Boron Compounds; D002103:Cadaverine / Q000031:analogs & derivatives; D002352:Carrier Proteins / Q000032:analysis*; D005456:Fluorescent Dyes; D006801:Humans; D008050:Lipid A / Q000032:analysis / Q000276:immunology; D008070:Lipopolysaccharides / Q000032:analysis* / Q000276:immunology; D008562:Membrane Glycoproteins; D023421:Models, Animal; D008968:Molecular Conformation; D011112:Polymyxin B; D013050:Spectrometry, Fluorescence; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000900:Anti-Bacterial Agents; D001896:Boron Compounds; D002103:Cadaverine; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008050:Lipid A; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011112:Polymyxin B; D000099231:Lipopolysaccharide-Binding Protein; C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; C488469:BODIPY TR cadaverine