Department of Medicinal Chemistry

Research Interests

The design and development of anti-endotoxin agents for the prevention and therapy of Gram-negative septic shock.

 

The primary focus of research in Dr. David’s laboratory is the development of novel drugs for the therapy and prophylaxis of Septic Shock. Sepsis is the leading cause of mortality in the intensive care unit. A common and serious sequel of systemic bacterial infections, sepsis accounts for some 200,000 fatalities annually in the US alone, a figure higher than that attributable to AIDS and breast cancer combined. The pathogenesis of Gram-negative septic shock, a leading cause of mortality in critically ill patients, is a consequence of the host response to endotoxins, or lipopolysaccharides (LPS), present on the surface of gram-negative bacteria. We have shown that relatively simple, and synthetically easily accessible molecules of the lipopolyamine class specifically bind to the toxic "Lipid A" portion of LPS and neutralize its toxicity both in vitro and, importantly, in well-established animal models of septic shock (Antimicrob. Agents Chemother. (1999) 43: 912-919). Using experimentally determined structural leads as our point of departure, and utilizing a battery of established and biophysical and biological assays, we are testing hypotheses pertaining to specific structural requirements that ascribe endotoxin-binding and -neutralizing properties in synthetic small molecules, with the aim of developing promising leads as candidate endotoxin sequestrants of potential clinical value. For a recent review, please see: "Towards a rational development of anti-endotoxin agents: novel approaches to sequestration of bacterial endotoxins with small molecules". J. Molec. Recognition. (2001) 14: 370-387.

Figure 2

In the first project (R01AI050107), in collaboration with Dr. Apurba Dutta, we are evaluating eleven classes of lipopolyamines, the design of which not only incorporates structural features that dictate the specific molecular recognition of LPS, but also avoids those features that correspond to toxicity. The interactions of the test-compounds with LPS are being analyzed quantitatively using a rapid-throughput fluorescence displacement assay. In a panel of in vitro assays, the potency of the test compounds in inhibiting the release of LPS-mediated proinflammatory cytokines are being 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. We will systematically evaluate the toxicity of the test-compounds in a carefully chosen panel of in vitro assays. The protective effects of particularly promising molecules will then be examined in two well-established murine models of gram-negative sepsis. In 5U01AI056476, a focused library of several thousand compounds, each possessing the primary pharmacophore for LPS binding, and bearing non-polyamine scaffolds are being screened using a fluorescence displacement method implemented in high throughput screening (HTS) format. Binding, however, is necessary, but not sufficient for the 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 acylated or alkylated appropriately to generate LPS-neutralizing compounds. These compounds will be evaluated in a hierarchical biological assay panel. In 1U01AI054785, in collaboration with MediQuest Therapeutics, Inc., we are synthesizing large libraries of novel compounds rationally designed to maximize binding affinity and neutralization potency, and to exhibit desirable pharmacokinetic and toxicological profiles, based on optimal structural templates that we have already established with the lipopolyamines.

Although the outcome of both Gram-negative and Gram-positive infections are indistinguishable in that they both lead to the clinical syndrome of shock, the causative factor in the Gram-positive organism was unknown until recently. Compelling evidence now points to a major role for lipoteichoic acid, an integral component of the cell wall in Gram-positive organisms. In the course of our continuing efforts in identifying small-molecules that would specifically bind and neutralize Gram-negative lipopolysaccharide, we have found that certain classes of compounds also inhibit lethality in murine models of Gram-positive shock. In 1R03AI055725, we have initiated preliminary screening of select compounds based on the leads we have already obtained. Focused libraries of these compounds will be subjected to two levels of screening. In the primary screen, the inhibition of key cytokines induced by lipoteichoic acid in human peripheral blood mononuclear cells will be quantified. Active compounds will be subjected to a secondary screen in which upstream cellular events (cytokine mRNA transcription) will be examined.

Exploratory work has also begun on developing novel angiogenesis inhibitors via a high-throughput screen designed to identify small molecules that bind heparan/heparin with high affinity. Heparans are important cofactors necessary for the binding of pro-angiogenic factors such as VEGF and FGF to their cell-surface receptors.