TLR4 is a member of the recently identified Toll-like receptor family of proteins and has been putatively identified as Lps, the gene necessary for potent responses to lipopolysaccharide in mammals. In order to determine whether TLR4 is involved in lipopolysaccharide-induced activation of the nuclear factor-B (NF-B) pathway, HEK 293 cells were transiently transfected with human TLR4 cDNA and an NF-B-dependent luciferase reporter plasmid followed by stimulation with lipopolysaccharide/CD14 complexes. The results demonstrate that lipopolysaccharide stimulates NF-Bmediated gene expression in cells transfected with the TLR4 gene in a dose-and time-dependent fashion. Furthermore, E5531, a lipopolysaccharide antagonist, blocked TLR4-mediated transgene activation in a dosedependent manner (IC 50 ϳ30 nM). These data demonstrate that TLR4 is involved in lipopolysaccharide signaling and serves as a cell-surface co-receptor for CD14, leading to lipopolysaccharide-mediated NF-B activation and subsequent cellular events. Lipopolysaccharide (LPS),1 a component of the outer membrane of Gram-negative bacteria, is a potent activator of a variety of mammalian cell types (1, 2). Activation by LPS constitutes the first step in a cascade of events believed to lead to the manifestation of Gram-negative sepsis, a condition that results in approximately 20,000 annual deaths in the United States (3). Activation of LPS-responsive cells, such as monocytes and macrophages, occurs rapidly after LPS interacts with circulating LPS-binding protein and CD14, a glycosylphosphatidylinositol-linked cell surface glycoprotein necessary for sensitive responses to LPS (1, 2). LPS has been shown to initiate multiple intracellular signaling events (4), including the activation of NF-B, which ultimately leads to the synthesis and release of a number of proinflammatory mediators, including interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-␣ (1). However, since CD14 is not a transmembrane protein, it lacks the ability to transduce cytoplasmic signals (2), and before the recent discovery of Toll-like receptors (TLRs), the identity of a transmembrane protein that could relay LPS-induced signals across the cell-surface membrane remained elusive.Toll is a transmembrane receptor in Drosophila that is involved in dorsal-ventral patterning in embryos and in the induction of an anti-fungal response (5, 6). Activation of the Toll receptor by its ligand Spä tzle results in the interaction and stimulation of several signaling molecules that are homologous to proteins involved in NF-B activation by the IL-1 receptor in mammalian cells (7,8). The cloning of a family of human receptors structurally related to Drosophila Toll revealed five proteins that have extracellular domains that contain multiple leucine-rich repeats and cytoplasmic domains with sequence homology to the intracellular portion of the IL-1 receptor (9). Furthermore, constitutively active mutants of TLR2, TLR4, and TLR5 can induce the activation of NF-B (10, 11)...

Summary There is pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury (ALI) caused by chemical or microbial insults is secondary to generation of host-derived, oxidized phospholipid that potently stimulates Toll-like Receptor 4 (TLR4)-dependent inflammation1. Subsequently, we reported that TLR4−/− mice are highly refractory to influenza-induced lethality2, and hypothesized that therapeutic antagonism of TLR4 signaling would protect against influenza-induced ALI. Herein, we report that therapeutic administration of Eritoran (E5564), a potent, well-tolerated, synthetic TLR4 antagonist3,4, blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titers. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signaling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other, infections.

␣-D-Glucopyranose,3-O-decyl-2-deoxy-6-O-[2-deoxy-3-O-[(3R)-3-methoxydecyl]-6-O-methyl-2-[[(11Z)-1-oxo-11-octadecenyl]amino]-4-O-phosphono-␤-D-glucopyranosyl]-2- [(1,3-dioxotetradecyl)amino]-1-(dihydrogen phosphate), tetrasodium salt (E5564) is a second-generation synthetic lipodisaccharide designed to antagonize the toxic effects of endotoxin, a major immunostimulatory component of the outer cell membrane of Gram negative bacteria. In vitro, E5564 dose dependently (nanomolar concentrations) inhibited lipopolysaccharide (LPS)-mediated activation of primary cultures of human myeloid cells and mouse tissue culture macrophage cell lines as well as human or animal whole blood as measured by production of tumor necrosis factor-␣ and other cytokines. E5564 also blocked the ability of Gram negative bacteria to stimulate human cytokine production in whole blood. In vivo, E5564 blocked induction of LPS-induced cytokines and LPS or bacterial-induced lethality in primed mice. E5564 was devoid of agonistic activity when tested both in vitro and in vivo and has no antagonistic activity against Gram positive-mediated cellular activation at concentrations up to 1 M. E5564 blocked LPSmediated activation of nuclear factor-B in toll-like receptor 4/MD-2-transfected cells. In a mouse macrophage cell line, activity of E5564 was independent of serum, suggesting that E5564 exerts its activity through the cell surface receptor(s) for LPS, without the need for serum LPS transfer proteins. Similar, another lipid A-like antagonist, E5564 associates with plasma lipoproteins, causing low concentrations of E5564 to be quantitatively inactivated in a dose-and time-dependent manner. However, compared with E5531, E5564 is a more potent inhibitor of cytokine generation, and higher doses retain activity for durations likely sufficient to permit clinical application. These results indicate that E5564 is a potent antagonist of LPS and lacks agonistic activity in human and animal model systems, making it a potentially effective therapeutic agent for treatment of disease states caused by endotoxin.

Antimalarial drugs have thus far been chiefly derived from two sources—natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

We previously reported that TLR4-/- mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist, Eritoran, blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post-infection. Herein, we extend these findings: anti-TLR4- or TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h prior to PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4-/- mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks HMGB1-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI, (iii) IL-1β contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the anti-viral drug, oseltamivir, were administered starting 4 days post-infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.