Suppression of Murine Endotoxin Response by E5531, a Novel Synthetic Lipid A Antagonist

Abstract

As a consequence of blood-borne bacterial sepsis, endotoxin or lipopolysaccharide (LPS) from the cell walls of gram-negative bacteria can trigger an acute inflammatory response, leading to a series of pathological events and often resulting in death. To block this inflammatory response to endotoxin, a novel lipid A analogue, E5531, was designed and synthesized as an LPS antagonist, and its biological properties were examined in vitro and in vivo. In murine peritoneal macrophages, E5531 inhibited the release of tumor necrosis factor alpha (TNF-α) by Escherichia coli LPS with a 50% inhibitory concentration (IC50) of 2.2 nM, while E5531 elicited no significant increases in TNF-α on its own. In support of a mechanism consistent with antagonism of binding to a cell surface receptor for LPS, E5531 inhibited equilibrium binding of radioiodinated LPS ([125I]2-(r-azidosalicylamido)-1, 3′-dithiopropionate-LPS) to mouse macrophages with an IC50 of 0.50 μM. E5531 inhibited LPS-induced increases in TNF-α in vivo when it was coinjected with LPS into C57BL/6 mice primed with Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this model, the efficacy of E5531 was inversely correlated to the LPS challenge dose, consistent with a competitive antagonist-like mechanism of action. Blockade of the inflammatory response by E5531 could further be demonstrated in other in vivo models: E5531 protected BCG-primed mice from LPS-induced lethality in a dose-dependent manner and suppressed LPS-induced hepatic injury in Propionibacterium acnes-primed or galactosamine-sensitized mice. These results argue that the novel synthetic lipid A analogue E5531 can antagonize the action of LPS in in vitro and suppress the pathological effects of LPS in vivo in mice.

Effective Dosing of Lipid A Analogue E5564 in Rats Depends on the Timing of Treatment and the Route ofEscherichia coliInfection

“…Previous studies testing the inhibition of lipid A by use of this agent or the related one, E5531, via intraperitoneal E. coli challenge were performed in mice that had been pretreated with BCG [31,32]; this pretreatment increases the sensitivity of rats to the toxic inflammatory effects of LPS [32].…”

“…In contrast, E5531, an analogue derived from Rhodobacter bacteria and a naturally occurring lipid A antagonist, demonstrated substantial antagonistic activity against endotoxins produced by a range of pathogenic bacteria, with few agonistic effects [31]. E5531 inhibited the release of cytokines, oxygen free radicals, and endothelial selectin molecules and prevented mitogen-response activation in mice, resulting in improved survival after lethal endotoxin challenge [31][32][33]. In healthy humans, E5531 inhibited the release of tumor necrosis factor (TNF)-a into the bloodstream and abolished almost all of the septic-like physiological responses after safe, low-dose endotoxin chal- lenges [34].…”

“…However, antibodies to lipid A and recombinant bactericidal/permeability-increasing protein (rBPI21), which are also capable of inhibiting endotoxin, showed promise in early studies but failed in later clinical trials [31][32][33]. Although there may be several reasons for such failures, clinically relevant factors, such as the timing of treatment and the route of infection, could alter the dose of drug needed to be effective during gram-negative bacterial sepsis.…”

Induction of TNF-α and MnSOD by endotoxin: effect of E5531, a synthetic non-toxic lipid A analog

“…10,11 E5531 by itself does not induce TNF-α production by murine macrophages or human monocytes in vitro, and is not toxic to mice in vivo. [10][11][12][13] Thus, E5531 may be useful for the prevention of LPS-mediated toxicity. The effect of E5531 on the induction of MnSOD has not been previously investigated.…”

Examination of chlorpromazine and other amphipathic drugs on the activity of lipopolysaccharide antagonists, E5564 and E5531

“…2 Based on previous studies, it is believed that E5531 antagonizes LPS activity at its cell-surface receptor, leading to inhibition of transmembrane signal transduction. [3][4][5]18 Expression of a functional Toll-like receptor 4 (TLR4) pro-tein is critical for sensitive physiological responses to LPS, which is evident in the spontaneous TLR4 mutant mice (C3H/HeJ and C57/10ScCr) and TLR4 knockout mice. 6,7 We discovered that E5531 inhibits the stimulation of TLR4-transfected cells by LPS in a dose-dependent manner, with an IC 50 of ~30 nM, 5 suggesting that the LPS antagonists target TLR4.…”