Abstract
Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.
Highlights
The innate immune receptor toll-like receptor 4 (TLR4) has been recognized as the receptor on hematopoietic and nonhematopoietic cells for bacterial endotoxin [1], as well as for a variety of endogenous molecules that are released during inflammatory or infectious disorders [2]
We report the results of a computational similarity searchassisted drug discovery approach for the identification of a new class of small organic molecules with shared structural identities based on E5564
Through a series of confirmatory studies, we established that our lead compound C34 inhibits TLR4 in vitro in macrophages and enterocytes, and in vivo in experimental models of endotoxemia and necrotizing enterocolitis (NEC)
Summary
The innate immune receptor toll-like receptor 4 (TLR4) has been recognized as the receptor on hematopoietic and nonhematopoietic cells for bacterial endotoxin (lipopolysaccharide, ‘‘LPS’’) [1], as well as for a variety of endogenous molecules that are released during inflammatory or infectious disorders [2]. A number of diseases have been attributed to exaggerated TLR4 signaling, including both infectious and non-infectious processes. The elucidation of the structure of LPS led to the identification of the synthetic lipid A analogue eritoran (E5564), as well as the lipid A mimetic CRX-526 in which the reducing sugar on lipid A was replaced with an N-acylated aminoalkyl saccharide [10]. Both E5564 and CRX-526 inhibited LPS signaling, but, lacked efficacy in clinical trials [11,12]
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