Co-administration of Antimicrobial Peptides Enhances Toll-like Receptor†4 Antagonist Activity of a Synthetic Glycolipid.

This study examines the effect of co-administration of antimicrobial peptides and the synthetic glycolipid FP7, which is active in inhibiting inflammatory cytokine production caused by TLR4 activation and signaling. The co-administration of two lipopolysaccharide (LPS)-neutralizing peptides (a cecropin A-melittin hybrid peptide and a human cathelicidin) enhances by an order of magnitude the potency of FP7 in blocking the TLR4 signal. Interestingly, this is not an additional effect of LPS neutralization by peptides, because it also occurs if cells are stimulated by the plant lectin phytohemagglutinin, a non-LPS TLR4 agonist. Our data suggest a dual mechanism of action for the peptides, not exclusively based on LPS binding and neutralization, but also on a direct effect on the LPS-binding proteins of the TLR4 receptor complex. NMR experiments in solution show that peptide addition changes the aggregation state of FP7, promoting the formation of larger micelles. These results suggest a relationship between the aggregation state of lipid A-like ligands and the type and intensity of the TLR4 response.

Amphiphilic Guanidinocalixarenes Inhibit Lipopolysaccharide (LPS)- and Lectin-Stimulated Toll-like Receptor 4 (TLR4) Signaling.

We recently reported on the activity of cationic amphiphiles in inhibiting TLR4 activation and subsequent production of inflammatory cytokines in cells and in animal models. Starting from the assumption that opportunely designed cationic amphiphiles can behave as CD14/MD-2 ligands and therefore modulate the TLR4 signaling, we present here a panel of amphiphilic guanidinocalixarenes whose structure was computationally optimized to dock into MD-2 and CD14 binding sites. Some of these calixarenes were active in inhibiting, in a dose-dependent way, the LPS-stimulated TLR4 activation and TLR4-dependent cytokine production in human and mouse cells. Moreover, guanidinocalixarenes also inhibited TLR4 signaling when TLR4 was activated by a non-LPS stimulus, the plant lectin PHA. While the activity of guanidinocalixarenes in inhibiting LPS toxic action has previously been related to their capacity to bind LPS, we suggest a direct antagonist effect of calixarenes on TLR4/MD-2 dimerization, pointing at the calixarene moiety as a potential scaffold for the development of new TLR4-directed therapeutics.

TLR4 antagonist FP7 inhibits LPS-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection.

Dysregulated Toll-like receptor (TLR)-4 activation is involved in acute systemic sepsis, chronic inflammatory diseases, such as atherosclerosis and diabetes, and in viral infections, such as influenza infection. Thus, therapeutic control of the TLR4 signalling pathway is of major interest. Here we tested the activity of the small-molecule synthetic TLR4 antagonist, FP7, in vitro on human monocytes and monocyte-derived dendritic cells (DCs) and in vivo during influenza virus infection of mice. Our results indicate that FP7 antagonized the secretion of proinflammatory cytokines (IL-6, IL-8, and MIP-1ß) by monocytes and DCs (IC50 < 1 µM) and prevented DC maturation upon TLR4 activation by ultrapure lipopolysaccharide (LPS). FP7 selectively blocked TLR4 stimulation, but not TLR1/2, TLR2/6, or TLR3 activation. TLR4 stimulation of human DCs resulted in increased glycolytic activity that was also antagonized by FP7. FP7 protected mice from influenza virus-induced lethality and reduced both proinflammatory cytokine gene expression in the lungs and acute lung injury (ALI). Therefore, FP7 can antagonize TLR4 activation in vitro and protect mice from severe influenza infection, most likely by reducing TLR4-dependent cytokine storm mediated by damage-associated molecular patterns (DAMPs) like HMGB1.

Glycolipid-based TLR4 Modulators and Fluorescent Probes: Rational Design, Synthesis, and Biological Properties.

The cationic glycolipid IAXO-102, a potent TLR4 antagonist targeting both MD-2 and CD14 co-receptors, has been used as scaffold to design new potential TLR4 modulators and fluorescent labels for the TLR4 receptor complex (membrane TLR4.MD-2 dimer and CD14). The primary amino group of IAXO-102, not involved in direct interaction with MD-2 and CD14 receptors, has been exploited to covalently attach a fluorescein (molecules 1 and 2) or to link two molecules of IAXO-102 through diamine and diammonium spacers, obtaining 'dimeric' molecules 3 and 4. The structure-based rational design of compounds 1-4 was guided by the optimization of MD-2 and CD14 binding. Compounds 1 and 2 inhibited TLR4 activation, in a concentration-dependent manner, and signaling in HEK-Blue TLR4 cells. The fluorescent labeling of murine macrophages by molecule 1 was inhibited by LPS and was also abrogated when cell surface proteins were digested by trypsin, thus suggesting an interaction of fluorescent probe 1 with membrane proteins of the TLR4 receptor system.

Synthetic and natural small molecule TLR4 antagonists inhibit motoneuron death in cultures from ALS mouse model.

Increasing evidence indicates that inflammatory responses could play a critical role in the pathogenesis of motor neuron injury in amyotrophic lateral sclerosis (ALS). Recent findings have underlined the role of Toll-like receptors (TLRs) and the involvement of both the innate and adaptive immune responses in ALS pathogenesis. In particular, abnormal TLR4 signaling in pro-inflammatory microglia cells has been related to motoneuron degeneration leading to ALS. In this study the effect of small molecule TLR4 antagonists on in vitro ALS models has been investigated. Two different types of synthetic glycolipids and the phenol fraction extracted from commercial extra-virgin olive oil (EVOO) were selected since they efficiently inhibit TLR4 stimulus in HEK cells by interacting with the TLR4·MD-2 complex and CD14 co-receptor. Here, TLR4 antagonists efficiently protected motoneurons from LPS-induced lethality in spinal cord cultures, and inhibited the interleukine-1ß production by LPS-stimulated microglia. In motoneurons/glia cocultures obtained from wild type or SOD1 G93A mice, motoneuron death induced by SOD1mut glia was counteracted by TLR4 antagonists. The release of nitric oxide by LPS treatment or SOD1mut glia was also inhibited by EVOO, suggesting that the action of this natural extract could be mainly related to the modulation of this inflammatory mediator.

Trehalose- and glucose-derived glycoamphiphiles: small-molecule and nanoparticle Toll-like receptor 4 (TLR4) modulators.

An increasing number of pathologies have been linked to Toll-like receptor 4 (TLR4) activation and signaling, therefore new hit and lead compounds targeting this receptor activation process are urgently needed. We report on the synthesis and biological properties of glycolipids based on glucose and trehalose scaffolds which potently inhibit TLR4 activation and signaling in vitro and in vivo. Structure-activity relationship studies on these compounds indicate that the presence of fatty ester chains in the molecule is a primary prerequisite for biological activity and point to facial amphiphilicity as a preferred architecture for TLR4 antagonism. The cationic glycolipids here presented can be considered as new lead compounds for the development of drugs targeting TLR4 activation and signaling in infectious, inflammatory, and autoimmune diseases. Interestingly, the biological activity of the best drug candidate was retained after adsorption at the surface of colloidal gold nanoparticles, broadening the options for clinical development.

Clicked and long spaced galactosyl- and lactosylcalix[4]arenes: new multivalent galectin-3 ligands.

Four novel calix[4]arene-based glycoclusters were synthesized by conjugating the saccharide units to the macrocyclic scaffold using the CuAAC reaction and using long and hydrophilic ethylene glycol spacers. Initially, two galactosylcalix[4]arenes were prepared starting from saccharide units and calixarene cores which differ in the relative dispositions of the alkyne and azido groups. Once the most convenient synthetic pathway was selected, two further lactosylcalix[4]arenes were obtained, one in the cone, the other one in the 1,3-alternate structure. Preliminary studies of the interactions of these novel glycocalixarenes with galectin-3 were carried out by using a lectin-functionalized chip and surface plasmon resonance. These studies indicate a higher affinity of lactosyl- over galactosylcalixarenes. Furthermore, we confirmed that in case of this specific lectin binding the presentation of lactose units on a cone calixarene is highly preferred with respect to its isomeric form in the 1,3-alternate structure.

Functional characterization of E. coli LptC: interaction with LPS and a synthetic ligand.

Lipopolysaccharide (LPS), the main cell-surface molecular constituent of Gram-negative bacteria, is synthesized in the inner membrane (IM) and transported to the outer membrane (OM) by the Lpt (lipopolysaccharide transport) machinery. Neosynthesized LPS is first flipped by MsbA across the IM, then transported to the OM by seven Lpt proteins located in the IM (LptBCFG), in the periplasm (LptA), and in the OM (LptDE). A functional OM is essential to bacterial viability and requires correct placement of LPS in the outer leaflet. Therefore, LPS biogenesis represents an ideal target for the development of novel antibiotics against Gram-negative bacteria. Although the structures of Lpt proteins have been elucidated, little is known about the mechanism of LPS transport, and few data are available on Lpt­LPS binding. We report here the first determination of the thermodynamic and kinetic parameters of the interaction between LptC and a fluorescent lipo-oligosaccharide (fLOS) in vitro. The apparent dissociation constant (Kd) of the fLOS­LptC interaction was evaluated by two independent methods. The first was based on fLOS capture by resin-immobilized LptC; the second used quenching of LptC intrinsic fluorescence by fLOS in solution. The Kd values by the two methods (71.4 and 28.8 µm, respectively) are very similar, and are of the same order of magnitude as that of the affinity of LOS for the upstream transporter, MsbA. Interestingly, both methods showed that fLOS binding to LptC is mostly irreversible, thus reflecting the fact that LPS can be released from LptC only when energy is supplied by ATP or in the presence of a higher-affinity LptA protein. A fluorescent glycolipid was synthesized: this also interacted irreversibly with LptC, but with lower affinity (apparent Kd=221 µM). This compound binds LptC at the LPS binding site and is a prototype for the development of new antibiotics targeting LPS transport in Gram-negative bacteria.