TY - JOUR
T1 - Anti-endotoxin agents. 1. Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents
AU - Wood, Stewart J.
AU - Miller, Kelly A.
AU - David, Sunil A.
PY - 2004/5
Y1 - 2004/5
N2 - 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.
AB - 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.
KW - Endotoxin
KW - Fluorescence
KW - High-throughput screening
KW - Lipopolysaccharide
KW - Sepsis
KW - Shock
UR - http://www.scopus.com/inward/record.url?scp=2942616397&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=2942616397&partnerID=8YFLogxK
U2 - 10.2174/1386207043328832
DO - 10.2174/1386207043328832
M3 - Article
C2 - 15134530
AN - SCOPUS:2942616397
SN - 1386-2073
VL - 7
SP - 239
EP - 249
JO - Combinatorial Chemistry and High Throughput Screening
JF - Combinatorial Chemistry and High Throughput Screening
IS - 3
ER -