Abstract
New monosaccharide-based lipid A analogues were rationally designed through MD-2 docking studies. A panel of compounds with two carboxylate groups as phosphates bioisosteres, was synthesized with the same glucosamine-bis-succinyl core linked to different unsaturated and saturated fatty acid chains. The binding of the synthetic compounds to purified, functional recombinant human MD-2 was studied by four independent methods. All compounds bound to MD-2 with similar affinities and inhibited in a concentration-dependent manner the LPS-stimulated TLR4 signaling in human and murine cells, while being inactive as TLR4 agonists when provided alone. A compound of the panel was tested in vivo and was not able to inhibit the production of proinflammatory cytokines in animals. This lack of activity is probably due to strong binding to serum albumin, as suggested by cell experiments in the presence of the serum. The interesting self-assembly property in solution of this type of compounds was investigated by computational methods and microscopy, and formation of large vesicles was observed by cryo-TEM microscopy.
Highlights
The Toll-like Receptor 4 (TLR4) is the mammalian receptor responsible for the Gram-negative bacterial endotoxin recognition
As the TLR4 antagonist Eritoran presents an unsaturated chain (C18, cis-11) that has the role to increase the binding affinity for the molecular dynamics (MD)-2 hydrophobic pocket[47], we aimed to investigate the effect of unsaturated chains linked to glucosamine C2 and C3
FP13-17 were computationally studied to predict their TLR4/MD-2 binding properties. To explore their possible binding modes, the compounds were computationally docked in a 3D model of the TLR4/MD-2 complex in antagonist conformation previously reported by us[48], by means of the Vina docking program[49]
Summary
The Toll-like Receptor 4 (TLR4) is the mammalian receptor responsible for the Gram-negative bacterial endotoxin recognition (lipopolysaccharide, LPS and lipooligosaccharide, LOS). TLR4 activation by endogenous factors (DAMPs) has been associated to several inflammatory disorders and auto-immune diseases affecting a variety of organs and body functions[16,17,18]. In this context, the development of hit or lead compounds that are able to modulate TLR4 signaling is attracting increasing interest for a wide range of possible therapeutic settings[19]. Compound E5564 (Eritoran, Fig. 1)[31] is one of the most potent TLR4 inhibitors so far The activity of this molecule is associated to its capacity to mimic the lipid A moiety, competing with LPS for MD-2 binding. In the case of aminoalkyl glucosaminide-4-phosphates (AGPs, or Corixa compounds, CRX)[44], the whole reducing sugar and phosphate have been replaced by an acylated diethanolamine bound to a phosphate or a carboxylic acid[42,43]
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