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

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 alpha,alpha-(1 1')-linked disaccharide core of synthetic LPS mimicking anionic glycolipids accounted for both species-independent and adjustable TLR4-mediated NF-kappa B 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

Rational Vaccine Design in Times of Emerging Diseases: The Critical Choices of Immunological Correlates of Protection, Vaccine Antigen and Immunomodulation.

Vaccines are the most effective medical intervention due to their continual success in preventing infections and improving mortality worldwide. Early vaccines were developed empirically however, rational design of vaccines can allow us to optimise their efficacy, by tailoring the immune response. Establishing the immune correlates of protection greatly informs the rational design of vaccines. This facilitates the selection of the best vaccine antigens and the most appropriate vaccine adjuvant to generate optimal memory immune T cell and B cell responses. This review outlines the range of vaccine types that are currently authorised and those under development. We outline the optimal immunological correlates of protection that can be targeted. Finally we review approaches to rational antigen selection and rational vaccine adjuvant design. Harnessing current knowledge on protective immune responses in combination with critical vaccine components is imperative to the prevention of future life-threatening diseases

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

The Toll-like receptor 4 (TLR4) signaling pathway plays a central role in the prompt defense against infectious challenge and provides immediate response to Gram-negative bacterial infection. The TLR4/MD-2 complex can sense and respond to various pathogen-associated molecular patterns (PAMPs) with bacterial lipopolysaccharide (LPS) being the most potent and the most frequently occurring activator of the TLR4-mediated inflammation. TLR4 is believed to be both a friend and foe since improperly regulated TLR4 signaling can result in the overactivation of immune responses leading to sepsis, acute lung injury, or pathologic chronic inflammation involved in cancer and autoimmune disease. TLR4 is also considered a legitimate target for vaccine adjuvant development since its activation can boost the adaptive immune responses. The dual action of the TLR4 complex justifies the efforts in the development of both TLR4 antagonists as antisepsis drug candidates or remedies for chronic inflammatory diseases and TLR4 agonists as vaccine adjuvants or immunotherapeutics. In this review, we provide a brief overview of the biochemical evidences for possible pharmacologic applications of TLR4 ligands as therapeutics and report our systematic studies on the design, synthesis, and immunobiological evaluation of carbohydrate-based TLR4 antagonists with nanomolar affinity for MD-2 as well as disaccharide-based TLR4 agonists with picomolar affinity for the TLR4/MD-2 complex

Synthesis of Zwitterionic 1,1′-Glycosylphosphodiester: A Partial Structure of Galactosamine-Modified <i>Francisella</i> Lipid A

Synthesis of a “double glycosidic” phosphodiester comprising anomeric centers of two 2-amino-2-deoxy-sugars is reported. The carbohydrate epitope of <i>Francisella</i> lipid A modified with α-d-galactosamine at the anomerically linked phosphate has been stereoselectively prepared and coupled to maleimide-activated bovine serum albumin via an amide-linked thiol-terminated spacer group. H-Phosphonate and phosphoramidite approaches have been explored for the coupling of 4,6-DTBS-2-azido-protected GalN lactol and peracetylated spacer-equipped reducing βGlcN(1→6)­GlcN disaccharide via phosphodiester linkage. Deprotection conditions preserving the integrity of the labile glycosidic zwitterionic phosphodiester were elaborated

Stereoselective Synthesis of α- and β‑l‑Ara4N Glycosyl H‑Phosphonates and a Neoglycoconjugate Comprising Glycosyl Phosphodiester Linked β‑l‑Ara4N

Stereoselective synthesis of variably protected α- and β-l-Ara4N glycosyl H-phosphonates as key intermediates in the syntheses of β-l-Ara4N-modified LPS structures and α-l-Ara4N-containing biosynthetic precursors is reported. A facile one-pot approach toward β-l-Ara4N glycosyl H-phosphonates includes anomeric deallylation of protected 4-azido β-l-Ara4N via terminal olefin isomerization followed by ozonolysis and methanolysis of formyl groups to furnish exclusively β-configured lactols that are phosphitylated with retention of configuration. The carbohydrate epitope of β-l-Ara4N-modified Lipid A, βGlcN(1→6)­αGlcN­(1→P←1)­β-l-Ara4N, was stereoselectively synthesized and linked to maleimide-activated bovine serum albumin

DataSheet1_Versatile approach towards fully desymmetrized trehalose with a novel set of orthogonal protecting groups.pdf

Trehalose-containing glycans play an essential role in bacterial pathogenesis, host-pathogen interaction, and cell signaling. The investigation of trehalose uptake and metabolism in Mycobacteria using synthetic desymmetrized trehalose probes is an important approach for the development of diagnostic tools and potential therapeutics for tuberculosis. Trehalose-derived mycobacterial glycolipids activate the innate immune response through recognition by the C-type lectin Mincle, justifying efforts to develop novel trehalose-based Mincle-dependent adjuvants. The chemical synthesis of trehalose-based glycoconjugates, glycolipids, and small-molecule trehalose probes requires the challenging chemical desymmetrization of eight hydroxyl groups in a C2-symmetric disaccharide αGlc(1↔1)αGlc. Using a novel set of orthogonal protecting groups, we developed a flexible multiscale synthetic approach to a collection of differently and variably protected fully desymmetrized trehalose derivatives, ready for final chemical modification with relevant functional or reporter groups. Using a regioselective and site-specific protecting group strategy, we performed multiple symmetry-breaking operations, resulting in a library of trehalose-derived orthogonally protected building blocks as a versatile source for the synthesis of complex trehalose-containing glycans.</p

Disaccharide-based anionic amphiphiles as potent inhibitors of lipopolysaccharide-induced inflammation

Despite significant advances made in the last decade in the understanding of molecular mechanisms of sepsis and in the development of clinically relevant therapies, sepsis remains the leading cause of mortality in intensive care units with increasing incidence worldwide. Toll-like receptor 4 (TLR4)-a transmembrane pattern-recognition receptor responsible for propagating the immediate immune response to Gram-negative bacterial infection-plays a central role in the pathogenesis of sepsis and chronic inflammation-related disorders. TLR4 is complexed with the lipopolysaccharide (LPS)-sensing protein myeloid differentiation-2 (MD-2) which represents a preferred target for establishing new anti-inflammatory treatment strategies. Herein we report the development, facile synthesis, and biological evaluation of novel disaccharide-based TLR4-MD-2 antagonists with potent anti-endotoxic activity at micromolar concentrations. A series of synthetic anionic glycolipids entailing amide-linked beta-ketoacyl lipid residues was prepared in a straightforward manner by using a single orthogonally protected nonreducing diglucosamine scaffold. Suppression of the LPS-induced release of interleukin-6 and tumor necrosis factor was monitored and confirmed in human immune cells (MNC and THP1) and mouse macrophages. Structure-activity relationship studies and molecular dynamics simulations revealed the structural basis for the high-affinity interaction between anionic glycolipids and MD-2, and highlighted two compounds as leads for the development of potential anti-inflammatory therapeutics