Matteo Piazza

Therapeutic targeting of innate immunity with Toll-like receptor 4 (TLR4) antagonists

Early recognition of invading bacteria by the innate immune system has a crucial function in antibacterial defense by triggering inflammatory responses that prevent the spread of infection and suppress bacterial growth. Toll-like receptor 4 (TLR4), the innate immunity receptor of bacterial endotoxins, plays a pivotal role in the induction of inflammatory responses. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, LBP, CD14 and MD-2 receptors, that bind lipopolysaccharide (LPS) and present it to TLR4 by forming the activated (TLR4-MD-2-LPS)(2) complex. Small molecules active in modulating the TLR4 activation process have great pharmacological interest as vaccine adjuvants, immunotherapeutics or antisepsis and anti-inflammatory agents. In this review we present natural and synthetic molecules active in inhibiting TLR4-mediated LPS signalling in humans and their therapeutic potential. New pharmacological applications of TLR4 antagonists will be also presented related to the recently discovered role of TLR4 in the insurgence and progression of neuropathic pain and sterile inflammations.

Exploring the LPS/TLR4 signal pathway with small molecules

The identification of the bacterial endotoxin receptors for innate immunity, most notably TLR4 (Toll-like receptor 4), has sparked great interest in therapeutic manipulation of the innate immune system. In the present mini-review, several natural and synthetic molecules that modulate the TLR4-mediated LPS (lipopolysaccharide) signalling in animals and humans are considered, and their mechanisms of action are discussed. The process of LPS sensing and signal amplification in humans is based on the sequential action of specific receptors situated in the extracellular side of the innate immunity cells, which bind and transfer LPS to TLR4: LBP (LPS-binding protein), CD14, MD-2 (myeloid differentiation protein 2). We classified the compounds active on TLR4 pathway depending on the specific molecular targets (LPS, LBP, CD14, MD-2 or TLR4). Small molecules developed by our group are described that inhibit LPS-stimulated TLR4 activation by selectively targeting the LPS-CD14 interaction. These compounds have an interesting antiseptic shock, anti-inflammatory and anti-neuropathic pain activity in vivo.

Glycolipids and Benzylammonium Lipids as Novel Antisepsis Agents: Synthesis and Biological Characterization

New glycolipids and a benzylammonium lipid were rationally designed by varying the chemical structure of a D-glucose-derived hit compound active as lipid A antagonist. We report the synthesis of these compounds, their in vitro activity as lipid A antagonists on HEK cells, and the capacity to inhibit LPS-induced septic shock in vivo. The lack of toxicity and the good in vivo activity suggest the use of some compounds of the panel as hits for antisepsis drug development.

Evidence of a specific interaction between new synthetic antisepsis agents and CD14

Synthetic molecules derived from natural sugars with a positively charged amino group or ammonium salt and two lipophilic chains have been shown to inhibit TLR4 activation in vitro and in vivo. To characterize the mechanism of action of this class of molecules, we investigated possible interactions with the extracellular components that bind and shuttle endotoxin [lipopolysaccharide (LPS)] to TLR4, namely, LBP, CD14, and MD-2. Molecules that inhibited TLR4 activation inhibited LBP.CD14-dependent transfer of endotoxin monomers derived from aggregates of tritiated lipooligosaccharide ([(3)H]LOS) from Neisseria meninigitidis to MD-2.TLR4, resulting in a reduced level of formation of a ([(3)H]LOS.MD-2.TLR4(ECD))(2) (M(r) approximately 190000) complex. This effect was due to inhibition of the transfer of [(3)H]LOS from aggregates in solution to sCD14 with little or no effect on [(3)H]LOS shuttling from [(3)H]LOS.sCD14 to MD-2. These compounds also inhibited transfer of the [(3)H]LOS monomer from full-length CD14 to a truncated, polyhistidine-tagged CD14. Dose-dependent inhibition of the transfer of [(3)H]LOS between the two forms of CD14 was observed with each of three different synthetic compounds that inhibited TLR4 activation but not by another structurally related analogue that lacked TLR4 antagonistic activity. Saturation transfer difference (STD) NMR data showed direct binding to CD14 by the synthetic TLR4 antagonist mediated principally through the lipid chains of the synthetic compound. Taken together, our findings strongly suggest that these compounds inhibit TLR4 activation by endotoxin by competitively occupying CD14 and thereby reducing the level of delivery of activating endotoxin to MD-2.TLR4.

Uniform Lipopolysaccharide (LPS)‐Loaded Magnetic Nanoparticles for the Investigation of LPS–TLR4 Signaling

The interaction of highly conserved microbial constituents with the innate immune system contributes greatly to the recognition and reaction against intruding pathogens by mammals. Examples of such microbial constituents include the lipopolysaccharides (LPS) and lipooligosaccharides (LOS), also known as endotoxin (E), of Gram-negative bacteria. These potent pro-inflammatory molecules include a hydrophilic oligosaccharide chain of variable length and a hydrophobic, membrane anchoring moiety, termed lipid A.[1–3] The interaction of E with cells of the innate immune system leads to the formation and release of endogenous mediators initiating inflammatory and immune responses essential for optimal antibacterial defense.[4,5] Such a cascade of events is triggered by activation of the Toll-like receptor 4 (TLR4) by E. This is the last step of a sequential process of E recognition and interaction with extracellular and cell surface host proteins, including LPS-binding protein (LBP), soluble and membrane-associated CD14, secreted and TLR4-associated MD-2, and TLR4 itself.[6–8] The amphiphilic character of both LPS and LOS results in the formation of micelles in aqueous environment above their critical micellar concentration (CMC) (Scheme 1, upper).[9] CMC values between 10−8 M and 10−7 M for deep rough mutant LPS Re, [10,11] and between 1.3 and 1.6 μM for E. coli LPS, [12] were reported. In balanced salts solutions containing physiologic extracellular concentrations of Mg2+ and Ca2+, CMC values of 1 nM or lower are likely.[13,14] From these data and from the fact that LPS aggregates are usually highly stable, aggregated forms of LPS should predominate in the concentration range relevant for biological responses. In physiological fluids, LPS aggregates were also found as membrane “blebs”, which are constitutively released from growing Gram-negative bacteria.[15] Transmission electron microscopy revealed that blebs exist predominantly as vesicles with an average size of 40–80 nm.[15] The current view of mammalian LPS sensing and signaling is that it is initiated by the LBP-catalyzed extraction and transfer of an LPS monomer from aggregates[16] to CD14[17] and subsequent transfer of the LPS monomer from CD14 to MD-2 and to the MD-2-TLR4 heterodimer.[7] While monomeric E-CD14 and E-MD-2 complexes are therefore the proximal vehicles for activation of MD-2-TLR4 and TLR4, respectively, by E, preceding interactions of host E-binding proteins that preferentially interact with E-rich interfaces and either promote (LBP) or preclude (BPI) transfer of E to CD14 play key roles in determining the potency of TLR4 activation by E.[18] Accordingly, variables in the aggregation state and the 3D form of E aggregates may directly influence the kinetics and potency of TLR4 activation and signaling.[9]

Hemin and a metabolic derivative coprohemin modulate the TLR4 pathway differently through different molecular targets

Heme is a prosthetic group in a large number of essential proteins that have a pivotal role in oxygen transport, storage and electron shuttling. High amounts of free heme are associated with pathological states. Recently, it has been suggested that activation of Toll-like receptor 4 (TLR4) is one of the ways in which the ‘danger signal’ of free heme is detected. Here, we examine the biochemical basis of the modulation of the TLR4 pathway by hemin (iron(III)-protoporphyrin IX) and its metabolic, oxidated derivative coprohemin (iron(III)-coproporphyrin I). High concentrations of hemin (50 μM) triggered TLR4-mediated IL-8 production in the human HEK293/TLR4 cell line in the absence of the co-receptors CD14 and MD-2; the latter an essential co-receptor for TLR4 activation by endotoxin. Hemin and endotoxin have additive effects when co-administrated to HEK/TLR4 cells, suggesting that hemin and endotoxin activate TLR4 by different mechanisms. Coprohemin, in contrast to hemin, is unable to trigger TLR4-dependent activation of HEK/TLR4 cells, but instead causes dose-dependent inhibition of endotoxin-stimulated IL-8 production. The inhibitory effect of coprohemin is paralleled by reduced delivery of endotoxin to MD-2 (-TLR4) that is necessary for activation of TLR4 by endotoxin. Thus, despite their similar chemical structure, hemin and coprohemin have very different effects on the TLR4 pathway, the former acting as a mild agonist of TLR4, the latter as an antagonist selectively targeting the endotoxin—MD-2 interaction.

The cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine inhibits LPS signaling by competing with endotoxin for CD14 binding.

The identification of the bacterial endotoxin receptors for innate immunity, most notably the Toll-like receptor 4 (TLR4), has sparked great interest in therapeutic manipulation of innate immune system. We have recently developed synthetic molecules that have been shown to inhibit TLR4 activation in vitro and in vivo. Here we present the synthesis and the biological characterization of a new molecule, the cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine, with a structure strictly related to the previously developed TLR4 modulators. This compound is able to inhibit in a dose-dependent manner the LPS-stimulated TLR4 activation in HEK cells. In order to characterize the mechanism of action of this compound, we investigated possible interactions with the extracellular components that bind and shuttle LPS to TLR4, namely LBP, CD14, and MD-2. This compound inhibited LBP/CD14-dependent LPS transfer to MD-2.TLR4, resulting in reduced formation of a (LPS-MD-2-TLR4)(2) complex. This effect was due to inhibition of the transfer of LPS from aggregates in solution to sCD14 with little or no effect on LPS shuttling from LPS/CD14 to MD-2. This compound also inhibited transfer of LPS monomer from full-length CD14 to a truncated, polyhistidine tagged CD14. Taken together, our findings strongly suggest that this compound inhibits LPS-stimulated TLR4 activation by competitively occupying CD14 and thereby reducing the delivery of activating endotoxin to MD-2.TLR4.

A Synthetic Lipid A Mimetic Modulates Human TLR4 Activity

Innate immunity recognition relies on a diverse set of germ line encoded receptors, termed pattern recognition receptors (PRR), which recognize broad classes of molecular structures common to groups of microorganisms. One of the largest and best studied families of PRR are the Toll family of receptors (Toll-like receptors, TLRs) that detect microbial components with high sensitivity and selectivity[1]. Among TLRs, TLR4 selectively responds to bacterial endotoxin (E) (Gram-negative bacterial lipopolysaccharides (LPS) or lipooligosaccharides (LOS)),[2] resulting in the rapid triggering of pro-inflammatory processes necessary for optimal host immune responses to invading Gram-negative bacteria (GNB). TLR4 does not bind directly to endotoxin: LBP,[3] CD14,[4] MD-2[5] are required for efficient extraction and transfer of endotoxin monomers from the GNB outer membrane or aggregates of purified endotoxin to MD-2. The resulting monomeric E·MD-2 complex is the ligand that, depending on the structural properties of E and MD-2, specifies TLR4 activation or antagonism.[6] Although TLR4 plays a key physiologic role in host response to Gram-negative bacterial infection, an excessively potent and/or prolonged TLR4 response can promote life-threatening pathology such as septic shock.[7] TLR4 activation has also been associated with certain autoimmune diseases, non-infectious inflammatory disorders, and neuropathic pain, suggesting a wide range of possible clinical settings for application of TLR4 antagonists.[8] Conversely, agonists of TLR4 can be useful as adjuvants in vaccine development and in cancer immunotherapy [9]. Lipid A[10] (Scheme 1), the hydrophobic part of LPS, is responsible for TLR4-dependent proinflammatory activity.[11] Underacylated lipid A variants, such as tetraacylated lipid IVa[12] and E5564 (Eritoran)[13] are potent LPS antagonists (Scheme 1). The β(1→6) diglucosamine backbone of lipid A can be replaced by an aminoalkyl glucosamine moiety in aminoalkyl glucosaminide 4-phosphates (AGPs)[14] or by other non-carbohydrate structures[15] and the lipid A analogue retains TLR4 agonist or antagonist activity. One or two phosphates are typically present in synthetic lipid A mimics, but these groups could be, in principle, substituted by negatively charged isosteres. A carboxylic acid group replaces the C-1 phosphate in AGP derivatives,[14a] while a sulfate group is present in the monosaccharide lipid A mimic ONO-4007 (Scheme 1) developed by Ono Pharmaceutical Co (Osaka, Japan).[16] This compound showed TLR4 agonist activity inducing TNF-α production in tumour cells, but further clinical development was precluded by the compounds limited water solubility.

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 molecules and functionalized nanoparticles targeting the LPS-TLR4 signaling: A new generation of immunotherapeutics*

Toll-like receptor 4 (TLR4), the receptor of bacterial endotoxins in mammalians, plays a pivotal role in the induction of innate immunity and inflammation. TLR4 activation by bacterial lipopolysaccharide (LPS) is achieved by the coordinate and sequential action of three other proteins, the lipopolysaccharide binding protein (LBP), the cluster differentiation antigen CD14, and the myeloid differentiation protein (MD-2) receptors, that bind LPS and present it in a monomeric form to TLR4 by forming the activated [TLR4·MD-2·LPS]2 complex. Small molecules and nanoparticles active in modulating the TLR4 signal by targeting directly the MD-2·TLR4 complex or by interfering in other points of the TLR4 signaling are presented in this paper. These compounds have great pharmacological interest as vaccine adjuvants, immunotherapeutics, anti-sepsis, and anti-inflammatory agents.