Design of Trehalose-Based Amide/Sulfonamide C-type Lectin Receptor Signaling Compounds

Abstract

Mincle agonists have been shown to induce inflammatory cytokine production, such as tumor necrosis factor-alpha (TNF) and promote the development of a Th1/Th17 immune response that might be crucial to development of effective vaccination against pathogens such as Mycobacterium tuberculosis. As an expansion of our previous work, a library of 6,6'-amide and sulfonamide α,α-d-trehalose compounds with various substituents on the aromatic ring was synthesized efficiently in good to excellent yields. These compounds were evaluated for their ability to activate the human C-type lectin receptor Mincle by the induction of cytokines from human peripheral blood mononuclear cells. A preliminary structure-activity relationship (SAR) of these novel trehalose diamides and sulfonamides revealed that aryl amide-linked trehalose compounds demonstrated improved activity and relatively high potency cytokine production compared to the Mincle ligand trehalose dibehenate adjuvant (TDB) and the natural ligand trehalose dimycolate (TDM) inducing dose-dependent and human-Mincle-specific stimulation in a HEK reporter cell line.

Keywords: Mincle; TH-17; adjuvants; trehalose tuberculosis; vaccines.

Figure 1.

Representative Mincle ligands: Trehalose Dimycolates (TDM), α-Trehalose Diesters (αTDE’s, B16), Trehalose Dicorynomycolates (TDCM), Trehalose dibehenate (TDB), Brartemicin analogues, and UM1024.

Figure 2:

Synthesis of a focused library of 6,6’diaryl and alkyl amide trehalose Mincle signaling compounds

Figure 3:. Cytokine production of UM1024, TDB, TDM, UM1088, 5a-k.

Cytokine production from primary human mononuclear cells in response to stimulation with synthetic trehalose diamide compounds. (A) and (B) The indicated compound was dissolved in ethanol, serially diluted, and then dried to the bottom of a tissue culture plate. Fresh PBMCs were purified, applied to the compound-coated plates, and incubated at 37°C; the supernatant was harvested 24 h later. (C) and (D) Selected IL-6 positive compounds were subsequently analyzed for TNF, IL-1β via an ELISA. Graphs are mean values from 3 donors +/− SEM.

Figure 4:

Synthesis of a focused library of 6,6’diaryl sulfonamide trehalose compounds.

Figure 5:. Cytokine production of UM1088, TDB, TDM, and 7a-i.

Cytokine production from primary human mononuclear cells in response to stimulation with synthetic trehalose diamide compounds. (A) The indicated compound was dissolved in ethanol, serially diluted, and then dried to the bottom of a tissue culture plate. Fresh PBMCs were purified, applied to the compound-coated plates, and incubated at 37°C; the supernatant was harvested 24 h later. (B) and (C) Selected IL-6 positive compounds were subsequently analyzed for TNF, IL-1β via a ELISA. Graphs are mean values from 3 donors +/− SEM.

Figure 6:. Activation of human Mincle in response to TDM, TDB, and synthesized trehalose 6,6’diaryl amide trehalose derivatives.

A) and B) The indicated compounds were plate coated and hMincle HEK NF-κB-SEAP reporter cells were incubated with the compounds for 24 h followed by assessment of the supernatants for SEAP levels. C) results in HEK null cells Data are represented as fold change in OD650 over vehicle-treated cells. Graphs are mean values from three independent experiments ± SEM.