Modulating Receptor Activity, Immune Response, and Kinetic Solubility: The Impact of Linker Chemistry in Conjugated NOD2/TLR4 Agonists

Abstract

Novel immunopotentiators are essential for advancing our understanding of immune receptor crosstalk and for addressing infectious diseases. Previous studies have suggested that coactivation of nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 4 (TLR4) can synergistically enhance the immune response. To investigate this synergy, we synthesized and evaluated a series of conjugated NOD2/TLR4 dual agonists comprising our in-house NOD2 agonist and two structurally distinct TLR4 agonists connected via flexible or rigid linkers. Our findings indicate that dual agonist activity toward both NOD2 and TLR4 is diminished upon conjugation. We also show that the linker chemistry significantly influences the kinetic solubility of these conjugates. Furthermore, the conjugates elicit distinct immunomodulatory effects in human primary peripheral blood mononuclear cells, characterized by a Th2-polarized cytokine response. These results provide insights into the structure-activity relationship of conjugated NOD2/TLR4 agonists and offer preliminary guidelines for tuning their solubility profiles.

1

Chemical structures of the first reported conjugated NOD2/TLR4 agonists 1 and 2.

2

Design of novel conjugated NOD2/TLR4 agonists.

1. Synthesis of Conjugated NOD2/TLR4 Agonists 17–21 Based on the TLR4 Agonist 4

2. Synthesis of Conjugated NOD2/TLR4 Agonists 27–31 Based on TLR4 Agonist 6

3

Metabolic activities/proliferation rates of HEK-Blue hNOD2 (A) and HEK-Blue hTLR4 (B) cells were measured after 18 h treatment with compounds (10 μM). Data are shown relative to the untreated control (0.1% DMSO). Data are means ± SEM of two independent experiments; ns, not significant versus control (one-way ANOVA post hoc Dunnett’s tests).

4

Concentration-dependent NF-κB transcriptional activities of conjugated NOD2/TLR4 agonists. SEAP activity was measured in (A) HEK-Blue hNOD2 cells after incubation for 18 h with MDP (positive control; 4 μM), and compounds (0.5–10 μM); and (B) HEK-Blue hTLR4 (B) cells after incubation for 18 h with LPS (positive control; 10 ng/mL) and compounds (0.5–10 μM or 1–100 μM concentration range as indicated). Data are shown as relative activities to the vehicle-treated control (0.1% DMSO) and are means ± SEM of three independent experiments.

5

Effects of conjugate treatments on the release of cytokines from human PBMCs. Cytokine concentrations were measured after 18 h stimulation with unlinked mixtures of corresponding agonists (1 μM), conjugated agonists (1 μM), or the control vehicle (0.1% DMSO). Data are expressed as mean ± SEM of two independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 versus control.