Lipid A Mimetics Based on Unnatural Disaccharide Scaffold as Potent TLR4 Agonists for Prospective Immunotherapeutics and Adjuvants

Abstract

TLR4 is a key pattern recognition receptor that can sense pathogen‐ and danger‐ associated molecular patterns to activate the downstream signaling pathways which results in the upregulation of transcription factors and expression of interferons and cytokines to mediate protective pro‐inflammatory responses involved in immune defense. Bacterial lipid A is the primary TLR4 ligand with very complex, species‐specific, and barely predictable structure‐activity relationships. Given that therapeutic targeting of TLR4 is an emerging tool for management of a variety of human diseases, the development of novel TLR4 activating biomolecules other than lipid A is of vast importance. We report on design, chemical synthesis and immunobiology of novel glycan‐based lipid A‐mimicking molecules that can activate human and murine TLR4‐mediated signaling with picomolar affinity. Exploiting crystal structure ‐ based design we have created novel disaccharide lipid A mimetics (DLAMs) where the inherently flexible β(1→6)‐linked diglucosamine backbone of lipid A is exchanged with a conformationally restrained non‐reducing βGlcN(1↔1′)βGlcN scaffold. Excellent stereoselectivity in a challenging β,β‐1,1′ glycosylation was achieved by tuning the reactivities of donor and acceptor molecules using protective group manipulation strategy. Divergent streamlined synthesis of β,β‐1,1′‐linked diglucosamine‐derived glycolipids entailing multiple long‐chain (R)‐3‐ acyloxyacyl residues and up two three phosphate groups was developed. Specific 3D‐molecular shape and conformational rigidity of unnatural β,β‐1,1′‐linked diglucosamine combined with carefully optimized phosphorylation and acylation pattern ensured efficient induction of the TLR4‐mediated signaling in a species‐independent manner.

Therapeutic Targeting of TLR4 for Inflammation, Infection, and Cancer: A Perspective for Disaccharide Lipid A Mimetics

“…As changes in the phosphorylation status of the carbohydrate backbone of native lipid A can markedly enhance or reduce the predisposition of LPS to interact with the TLR4 complex, we set out to investigate the influence of increased electronegativity of the sugar head group of DLAMs on the TLR4 activation and synthesized hyperphosphorylated ββ-DLAMs by attaching a third phosphate group at position 3 of the distal GlcN ring (along with 4, 4´ phosphate groups) ( Figure 9 ). As expected, the characteristic geometry of the βGlcN(1↔1)βGlcN disaccharide conferred specific immunobiological behavior to the glycolipid immunomodulator ββ-DLAMs based thereon, and the TLR4-mediated cell activation induced by ββ-DLAMs could be fine-tuned by adding/removing a P-3 phosphate and/or shortening the length of a secondary acyl chain [ 164 ].…”

“…In our approach, the flexible GlcNβ(1→6)GlcN backbone of natural lipid A was exchanged for a rigid nonreducing disaccharide scaffold imitating the 3D-molecular shape of the diglucosamine skeleton of a protein-bound lipid A, which is strikingly different for TLR4 agonist and antagonist ligands ( Figure 6 A,B). Along these lines, the β,α-1,1´-linked diglucosamine disaccharide with co-planar-arranged sugar rings mimicking the molecular shape of the diglucosamine backbone of hMD-2-bound antagonists lipid IVa and Eritoran (PDB codes: 2E59, 2Z65, respectively) was considered an excellent carbohydrate scaffold for synthetic TLR4 antagonists [ 155 , 159 ] ( Figure 6 A,C), whereas the α,α-1,1´-linked or β,β-1,1´-linked nonreducing disaccharide scaffolds reflecting the 3D-tertiary structure of the diglucosamine backbone of endotoxic lipid A found in the active [TLR4/MD-2/( E.coli lipid A)] 2 complexes (PDB codes: 3FXI, 3VQ1, 3VQ2) ( Figure 6 B) showed a skewed relative orientation of two GlcN rings and thus were believed to provide a perfect scaffold for carbohydrate-based TLR4 agonists [ 157 , 164 ] ( Figure 6 D,E).…”

“…Further advanced modulation of TLR4-mediated pro-inflammatory responses in a variety of cell types was achieved with the synthesis of ββ-DLAMs—the TLR4-activating glycolipids based on a ββ-1,1´-linked diglucosamine scaffold differing in its 3D-topology from the 1,1´-linked αα- counterpart ( Figure 9 ). Moreover, the primary chemical structure of the βGlcN(1↔1)βGlcN scaffold—with an equatorial 2- N -acetamide group instead of an axial 2- O -Me group in position 2 of the proximal sugar ring as well as a varying number of phosphate groups attached at positions 3/4 of the proximal GlcN residue—conferred specific immunobiological properties to the ββ-DLAMs based thereon [ 164 ]. As changes in the phosphorylation status of the carbohydrate backbone of native lipid A can markedly enhance or reduce the predisposition of LPS to interact with the TLR4 complex, we set out to investigate the influence of increased electronegativity of the sugar head group of DLAMs on the TLR4 activation and synthesized hyperphosphorylated ββ-DLAMs by attaching a third phosphate group at position 3 of the distal GlcN ring (along with 4, 4´ phosphate groups) ( Figure 9 ).…”

Exploring Species-Specificity in TLR4/MD-2 Inhibition with Amphiphilic Lipid A Mimicking Glycolipids

“…In general, fluoride-ion-mediated deprotection of the TBDMS group can be carried out under mild conditions at a slightly acidic pH (HF‧Py, pH 3–4), near neutral conditions (TREAT-HF, 3HF‧Et 3 N, pH 5–6) or under more hash conditions (TBAF), while the outcome of the reaction and the stability of other protective groups in the molecule additionally depend on the concentration of fluoride reagent in the reaction solution [ 54 ]. Thus, the 3- O -TBDMS group in 9 could be readily cleaved using a concentrated solution of HF‧Py (as for 4 → 5 ) or TREAT to obtain 10 .…”

Novel TLR4-Activating Vaccine Adjuvant Enhances the Production of Enterococcus faecium-binding Antibodies

“…TLR4 agonists have shown the ability to initiate potent pro-inflammatory responses after the activation of intracellular pathways, namely, MyD88 and TRAM/TRIF pathways. Several lipid A mimetics and other compounds with a chemical structure unrelated to lipid A have been developed as small molecular TLR4 agonists and potential vaccine adjuvants. Interestingly, TLR4-activating molecules, such as MPLA, are also able to increase resistance to infection through immune cell modulation and increased bacterial clearance. − Thus, TLR4-directed adjuvants are attractive molecules to include in novel antimicrobial vaccine formulations.…”