Mark T. Livesay

Synthetic Toll-Like Receptor 4 Agonists Stimulate Innate Resistance to Infectious Challenge

ABSTRACT A compound family of synthetic lipid A mimetics (termed the aminoalkyl glucosaminide phosphates [AGPs]) was evaluated in murine infectious disease models of protection against challenge with Listeria monocytogenes and influenza virus. For the Listeria model, intravenous administration of AGPs was followed by intravenous bacterial challenge 24 h later. Spleens were harvested 2 days postchallenge for the enumeration of CFU. For the influenza virus model, mice were challenged with virus via the intranasal/intrapulmonary route 48 h after intranasal/intrapulmonary administration of AGPs. The severity of disease was assessed daily for 3 weeks following challenge. Several types of AGPs provided strong protection against influenza virus or Listeria challenge in wild-type mice, but they were inactive in the C3H/HeJ mouse, demonstrating the dependence of the AGPs on toll-like receptor 4 (TLR4) signaling for the protective effect. Structure-activity relationship studies showed that the activation of innate immune effectors by AGPs depends primarily on the lengths of the secondary acyl chains within the three acyl-oxy-acyl residues and also on the nature of the functional group attached to the aglycon component. We conclude that the administration of synthetic TLR4 agonists provides rapid pharmacologic induction of innate resistance to infectious challenge by two different pathogen classes, that this effect is mediated via TLR4, and that structural differences between AGPs can have dramatic effects on agonist activity in vivo.

Synthesis and biological evaluation of a new class of vaccine adjuvants: aminoalkyl glucosaminide 4-phosphates (AGPs)

A novel series of acylated omega-aminoalkyl 2-amino-2-deoxy-4-phosphono-beta-D-glucopyranosides (aminoalkyl glucosaminide 4-phosphates) was synthesized and screened for immunostimulant activity. Several of these compounds enhance the production of tetanus toxoid-specific antibodies in mice and augment vaccine-induced cytotoxic T cells against EG.7-ova target cells.

The ‘Ethereal’ nature of TLR4 agonism and antagonism in the AGP class of lipid A mimetics

To overcome the chemical and metabolic instability of the secondary fatty acyl residues in the AGP class of lipid A mimetics, the secondary ether lipid analogs of the potent TLR4 agonist CRX-527 (2) and TLR4 antagonist CRX-526 (3) were synthesized and evaluated along with their ester counterparts for agonist/antagonist activity in both in vitro and in vivo models. Like CRX-527, the secondary ether lipid 4 showed potent agonist activity in both murine and human models. Ether lipid 5, on the other hand, showed potent TLR4 antagonist activity similar to CRX-526 in human cell assays, but did not display any antagonist activity in murine models and, in fact, was weakly agonistic. Glycolipids 2, 4, and 5 were synthesized via a new highly convergent method utilizing a common advanced intermediate strategy. A new method for preparing (R)-3-alkyloxytetradecanoic acids, a key component of ether lipids 4 and 5, is also described.

Chemical Synthesis of the Major Constituents of Salmonella Minnesota Monophosphoryl Lipid A

Abstract Structural investigations have shown that lipid A constitutes the active principle of lipopoly saccharide (LPS, endotoxin), a complex amphipathic molecule located on the cell surface of Gram-negative bacteria.1 Lipid A elicits not only the typical endotoxic reactions such as fever and lethal shock but also adjuvant, antitumor and other beneficial effects.1 As a result, there has been a great deal of interest in the synthesis of lipid A derivatives possessing low toxicity.1,2

Structural requirements for TLR7-selective signaling by 9-(4-piperidinylalkyl)-8-oxoadenine derivatives.

We report the synthesis and biological evaluation of a new series of 8-oxoadenines substituted at the 9-position with a 4-piperidinylalkyl moiety. In vitro evaluation of the piperidinyl-substituted oxoadenines 3a-g in human TLR7- or TLR8-transfected HEK293 cells and in human PBMCs indicated that TLR7/8 selectivity/potency and cytokine induction can be modulated by varying the length of the alkyl linker. Oxoadenine 3f containing a 5-carbon linker was found to be the most potent TLR7 agonist and IFNα inducer in the series whereas 3b possessing a 1-carbon linker was the most potent TLR8 agonist.

An Efficient Synthesis of 6,6′-DI-O-Acylated α,α-Trehaloses †

The potent immunostimulant and antitumor activity of trehalose 6,6′-dimycolates from mycobacteria has led to a great deal of interest in the synthesis of these glycolipids and their analogs.1J To circumvent the formation of 3,6-anhydro derivatives and other by-products resulting from partial protection strategies, many of the synthetic routes to symmetrical 6,6′-diesters (including dimycolates) of trehalose have utilized trehalose intermediates fully protected at the secondary hydroxyl position.2,3 Recently, we described the synthesis of one such intermediate, 2,3,4,2′,3 ′,4′-hexakis-O-trimethylsilyl-aptrehalose (l), directly from trehalose dihydrate via a novel persilylatioddesilylation protocol.4 Diol 1 and related derivatives have been employed in the preparation of trehalose 6,6′-diesters both by direct acylation methods3,5-8 and by nucleophilic displacement of 6,6′-dideoxy derivatives possessing various leaving groups on the 6 and 6′-positions.7-12 †Dedicated to Mayer B. Goren on the occasion of his ...

New procedure for the preparation of 2-(trimethylsilyl)ethyl 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-β--glucopyranoside and other alkyl β-glycosides

Abstract The title compound and other alkyl β-glycosides 1a-g can be prepared in one step from commercially available 2- acetamido-2-deoxy-1,3,4,6-tetra -O- acetyl-β- d -glucopyranose ( 3 ) and simple alcohols using camphorsulfonic acid as a promoter together with azeotropic removal of acetic acid.

Characterization of TRIF Selectivity in the AGP Class of Lipid A Mimetics: Role of Secondary Lipid Chains

TLR4 agonists that favor TRIF-dependent signaling and the induction of type 1 interferons may have potential as vaccine adjuvants with reduced toxicity. CRX-547 (4), a member of the aminoalkyl glucosaminide 4-phosphate (AGP) class of lipid A mimetics possessing three (R)-3-decanoyloxytetradecanoyl groups and d-relative configuration in the aglycon, selectively reduces MyD88-dependent signaling resulting in TRIF-selective signaling, whereas the corresponding secondary ether lipid 6a containing (R)-3-decyloxytetradecanoyl groups does not. In order to determine which secondary acyl groups are important for the reduction in MyD88-dependent signaling activity of 4, the six possible ester/ether hybrid derivatives of 4 and 6a were synthesized and evaluated for their ability to induce NF-κB in a HEK293 cell reporter assay. An (R)-3-decanoyloxytetradecanoyl group on the 3-position of the d-glucosamine unit was found to be indispensable for maintaining low NF-κB activity irrespective of the substitutions (decyl or decanoyl) on the other two secondary positions. These results suggest that the carbonyl group of the 3-secondary lipid chain may impede homodimerization and/or conformational changes in the TLR4-MD2 complex necessary for MyD88 binding and pro-inflammatory cytokine induction.