Anti-Endotoxin Agents. 1. Development of a Fluorescent Probe Displacement Method Optimized for the Rapid Identification of Lipopolysaccharide-Binding Agents

Abstract

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.

Interaction of the HIV-1 gp120 Viral Protein V3 Loop with Bacterial Lipopolysaccharide

“…Specific binding of V3 to lipid A was also demonstrated using a fluorescent displacement assay with BC. BC binds to lipid A and can be displaced by compounds displaying an affinity for lipid A (42). We showed similar concentration-dependent competition with BC for S-LPS binding by V3 or polymyxin B (Fig.…”

“…Lipid A is the central and essential moiety responsible for the biological activity of LPS; therefore we tested whether V3 binds to LPS through its lipid A moiety. For this purpose, we used polymyxin B, a cyclic polycationic lipopeptide antibiotic, which binds to lipid A with high affinity and is considered as the "gold standard" for LPS-sequestering agents (42). Polymyxin B displaced different chemotypes of LPS from the immobilized V3 (Fig.…”

New Amphiphilic Neamine Derivatives Active against Resistant Pseudomonas aeruginosa and Their Interactions with Lipopolysaccharides

“…Binding to the lipid A region of LPS was determined using the BODIPY-TR-cadaverine displacement assay (34,35), in which the probe bound to cell-free LPS is self-quenched but fluoresces when released in solution. Stock solutions of BODIPY-TRcadaverine (500 M) and LPS from P. aeruginosa (5 mg/ml) were prepared by dissolution in Tris buffer (50 mM, pH 7.4) (the Tris buffer was supplemented with 0.5% methanol for the former).…”

Interaction of Antimicrobial Peptide Temporin L with Lipopolysaccharide In Vitro and in Experimental Rat Models of Septic Shock Caused by Gram-Negative Bacteria

“…The difference between the initial surface pressure and the value observed after the penetration of temporin L into the film was taken as ⌬. Measurement of the temporin L ability to bind LPS and lipid A (diphosphoryl; from E. coli F583 [Sigma-Aldrich]) was performed by a fluorescent displacement assay using the probe BODIPY TR cadaverine (BC; Molecular Probes, Eugene, Oregon) as described elsewhere (54). All measurements were performed at room temperature.…”

“…To collect additional information on the temporin L-LPS modes of interaction, we used a fluorescent probe displacement method recently developed by Wood and colleagues (54). The fluorescent probe BC binds LPS, interacting specifically with its toxic center lipid A, probably via salt bridges with its glycosidic phosphate group, and the binding results in a progressive quenching of fluorescence.…”