Gram-negative sepsis, a common and serious sequel of systemic bacterial infections is the leading cause of mortality, accounting for some 200,000 fatalities annually in the US alone. The pathogenesis of Gram-negative septic shock is due to the host response to endotoxins, or lipopolysaccharides (LPS), present on the surface of gram-negative bacteria. There are, to date, no FDA-approved therapeutic options targeting the endotoxin itself to prevent or treat this disease. We have shown that relatively simple, and synthetically easily accessible molecules of the lipopolyamine class specifically bind to LPS and neutralize its toxicity both in vitro and in animal models of septic shock. The affinity of the lipopolyamines toward LPS, however, is relatively weak (2-10 (M). In this proposal, our goal is to identify high-affinity LPS binders with nonlipopolyamine scaffolds as novel leads for the therapy of Gram-negative sepsis. A focused library of ~6000 compounds, each possessing the primary pharmacophore for LPS binding will be screened using a well-established fluorescence displacement method implemented in HTS formats. Binding, however, does not necessarily manifest in neutralization of LPS toxicity. For neutralization, an additional, appropriately positioned long-chain aliphatic group is essential. High-affinity binders (""""""""hits"""""""") identified in HTS will be alkylated appropriately to generate LPS-neutralizing compounds (sequestrants). In in vitro assays, the potency of lead compounds in inhibiting the release of LPS-mediated proinflammatory cytokines such as tumor necrosis factor will be characterized. In a select subset of promising leads identified in the screens described above, we will verify that the mechanism of action of inhibition of LPS toxicity is via its sequestration by showing that relevant upstream cell-signaling events are blocked. The protective effects of particularly promising molecules will then be examined in two well-established murine models of gram-negative sepsis. We will systematically evaluate the toxicity of the test-compounds in a carefully chosen panel of in vitro assays. Molecular modeling techniques will be applied in an effort to correlate experimentally observed binding affinities of the test compounds with features of molecular interaction such as binding geometry, H-bonds, electrostatic, hydrophobic, and van der Waals contributions to the free energy of binding. Based on the data from the primary screen, in silico modeling, and biological assays, we will synthesize a series of analogues around promising leads using a combination of focused virtual library screening and classical medicinal chemistry approaches.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01AI056476-03
Application #
6848719
Study Section
Special Emphasis Panel (ZAI1-ALR-M (M3))
Program Officer
Korpela, Jukka K
Project Start
2003-08-01
Project End
2008-01-31
Budget Start
2005-02-01
Budget End
2006-01-31
Support Year
3
Fiscal Year
2005
Total Cost
$360,000
Indirect Cost
Name
University of Kansas Lawrence
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
076248616
City
Lawrence
State
KS
Country
United States
Zip Code
66045
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Schuerholz, Tobias; Dömming, Sabine; Hornef, Mathias et al. (2012) Bacterial cell wall compounds as promising targets of antimicrobial agents II. Immunological and clinical aspects. Curr Drug Targets 13:1131-7
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Wood, Stewart J; Miller, Kelly A; David, Sunil A (2004) Anti-endotoxin agents. 1. Development of a fluorescent probe displacement method optimized for the rapid identification of lipopolysaccharide-binding agents. Comb Chem High Throughput Screen 7:239-49