Inhibitor E5564, which was used as a reference structure for its ability to bind to TLR4 in the initial in silico screen, has a 2-acylamidopyranoside substructure. Furthermore, the N-acetyl-glucosamine BAY 73-4506 VEGFR/PDGFR inhibitor scaffold has known antiinflammatory effects in a variety of cells, including retinal pigment epithelial cells, endothelial cells, mast cells and peritoneal mesothelial cells, providing further evidence for the role for this class of molecules as TLR4 inhibitors. Interestingly, Lee and colleagues also recently showed that a novel aminosaccharide has anti-TLR effects in macrophages in vitro. Based on these findings, we submit that per-acylated 2-aminopyranoses with alkyl side chains at the anomeric carbon, in particular those with similarity to our lead compound, C34, have the potential to elicit broad and beneficial clinical effects in diseases characterized by TLR4 hyperactivation. Given that the development of necrotizing enterocolitis occurs almost exclusively in premature infants after they have received oral feeds, and given our findings that the development of NEC reflects increased signaling via TLR4 in the intestinal epithelium of the premature gut, it is exciting to speculate that a feeding regimen which contains a TLR4 inhibitor such as C34 may be of benefit in the prevention of NEC in this population. An exciting and rather serendipitous discovery in the current work links our identification of this aminoglycoside scaffold to the treatment of experimental NEC, a disease that we and others have shown to be mediated via TLR4 activation in mice and humans. Although a cure for NEC remains elusive, there is consensus that the administration of breast milk is the most effective preventive strategy. While the precise mechanisms to explain the protective effects of breast milk remain unknown, an emerging body of literature has determined that breast milk-derived oligosaccharides exert anti-inflammatory properties, and that these molecules may be responsible in part for the protective effects of breast milk in NEC. For instance, human milk oligosaccharides reduce leukocyte adhesion to endothelial cells, reduce platelet-leukocyte adhesion, alter the composition of the microbiome, and alter the Y-27632 dihydrochloride side effects degree of adherence of certain microbes to the surface of the intestinal epithelium. The specific mechanisms by which human milk oligosaccharides exert these protective effects remain incompletely understood. Given our finding that simple aminomonosaccharides serve as TLR4 inhibitors, it is possible that human milk oligosaccharides convey their anti-inflammatory function in part through a previously unrecognized inhibitory effect on TLR4 signaling. In summary, we have identified a new class of small molecule inhibitors of TLR4, which show substantial benefits in models of experimental NEC. These findings suggest a viable approach for the development of novel therapies for a variety of infectious and inflammatory diseases in which TLR4 signaling plays a critical role.