Synthetic glycan-based TLR4 agonists targeting caspase-4/11 for the development of adjuvants and immunotherapeutics.

Florian Adanitsch,Rudi Beyaert,Holger Heine,Alla Zamyatina,Feng Shao,Jianjin Shi

doi:10.1039/c7sc05323a

Abstract

Gram-negative bacterial lipopolysaccharide (LPS)-induced Toll-like receptor 4 (TLR4) mediated pro-inflammatory signaling plays a key role in immunoprotection against infectious challenges and boosts adaptive immunity, whereas the activation of the cytosolic LPS receptor caspase-4/11 leads to cell death by pyroptosis and is deeply implicated in the development of sepsis. Despite tremendous advances in the understanding of the LPS-TLR4 interaction, predictably regulated TLR4 activation has not yet been achieved. The structural basis for the induction of caspase-4/11 protease activity by LPS is currently unknown. The modulation of innate and adaptive immune responses through the controlled induction of TLR4 signaling without triggering caspase-4/11 activity would open novel perspectives in the development of safe vaccine adjuvants and immunotherapeutics. We report the discovery of highly potent glycan-based immunostimulants with picomolar affinity for TLR4 which interact with caspase-4/11 and promote caspase-4/11 oligomerization while abolishing caspase-11 protease activity. The rigidity and twisted molecular shape of the α,α-(1↔1')-linked disaccharide core of synthetic LPS mimicking anionic glycolipids accounted for both species-independent and adjustable TLR4-mediated NF-κB signaling and the modulation of caspase-4/11 activation. By the use of crystal structure based design and advanced synthetic chemistry we created a set of versatile probes for studying the structural basis of caspase-4/11 activation and established a chemical strategy for controllable TLR4 mediated cytokine release which is dissociable from the induction of caspase-11 protease activity.

Highlights

Innate immunity provides an instant protection against bacterial infection by detecting and responding to lipopolysaccharide (LPS), a major component of the outer membrane of Gramnegative bacteria, which proceeds through a germlineencoded transmembrane pattern recognition receptor Toll-like receptor 4 (TLR4) (ESI-Fig. 1‡).[1,2] TLR4 activation by LPS results in the induction of transcription factor NF-kB signaling leading to the upregulation of cytokines, chemokines and costimulatory molecules which generally facilitates recoveryThe immunostimulating portion of LPS – a native TLR4 agonist glycophospholipid lipid A – is built on the basis of a highly conserved bisphosphorylated b(1/6)-linked diglucosamine (GlcN) backbone [bGlcN(1/6)GlcN] which carries a variable number of long-chain (R)-3-hydroxyacyl- and (R)-3acyloxyacyl residues in asymmetric distribution (Fig. 1A)
By the use of crystal structure based design and advanced synthetic chemistry we created a set of versatile probes for studying the structural basis of caspase-4/11 activation and established a chemical strategy for controllable TLR4 mediated cytokine release which is dissociable from the induction of caspase-11 protease activity
The twisted molecular shape of the synthetic trehaloselike aGlcN(1410)aMan scaffold allowed for dissecting two hydrophobic clusters in our hybrid TLR4 agonist molecules: one large hydrophobic batch composed of two (R)-3-acyloxyacyl residues attached at positions 20 and 30 of the a-GlcN ring and a smaller detached hydrophobic cluster composed of an (R)-3acyloxyacyl residue linked at positions 4- or 6- of the a-Man moiety (Fig. 2B, 3B)

Summary

Introduction

Innate immunity provides an instant protection against bacterial infection by detecting and responding to lipopolysaccharide (LPS), a major component of the outer membrane of Gramnegative bacteria, which proceeds through a germlineencoded transmembrane pattern recognition receptor Toll-like receptor 4 (TLR4) (ESI-Fig. 1‡).[1,2] TLR4 activation by LPS results in the induction of transcription factor NF-kB signaling leading to the upregulation of cytokines, chemokines and costimulatory molecules which generally facilitates recoveryThe immunostimulating portion of LPS – a native TLR4 agonist glycophospholipid lipid A – is built on the basis of a highly conserved bisphosphorylated b(1/6)-linked diglucosamine (GlcN) backbone [bGlcN(1/6)GlcN] which carries a variable number of long-chain (R)-3-hydroxyacyl- and (R)-3acyloxyacyl residues in asymmetric distribution (Fig. 1A). The modulation of innate and adaptive immune responses through the controlled induction of TLR4 signaling without triggering caspase-4/11 activity would open novel perspectives in the development of safe vaccine adjuvants and immunotherapeutics.

Results

Conclusion