Linker Chemistry and Connectivity Fine-Tune the Immune Response and Kinetic Solubility of Conjugated NOD2/TLR7 Agonists

Abstract

There is a growing interest in developing novel immune potentiators capable of eliciting a cellular immune response. We tackle this challenge by harnessing the synergistic cross-activation between two innate immune receptors─the nucleotide-binding oligomerization domain-containing protein 2 (NOD2) and Toll-like receptor 7 (TLR7). Herein, we investigate the structure-activity relationship of a series of novel conjugated NOD2/TLR7 agonists incorporating a variety of flexible aliphatic, poly(ethylene glycol)-based and triazole-featuring linkers. Our findings reveal potent immune-enhancing properties of conjugates in human primary peripheral blood mononuclear cells, characterized by a Th1/Th17 polarized cytokine response. Importantly, we demonstrate that both the chemistry of the linker and the site of linkage affect the immune fingerprint and the kinetic solubility of these conjugated agonists. These results shed further light on the immunostimulatory potential of NOD2/TLR7 cross-activation and provide insights for designing innovative immune potentiators.

Figure 1

Chemical structures of conjugated NOD2/TLR7 agonists 1, 2, and 3.

Figure 2

Design of novel conjugated NOD2/TLR7 and NOD2/TLR7/8 agonists.

Scheme 1. Synthesis of Conjugated NOD2/TLR7 Agonists 22-26

Reagents and conditions: (a) 8, COMU, DIPEA, DMF, rt.; (b) 9, COMU, DIPEA; DMF, rt.; (c) 10, COMU, DIPEA, DMF, rt.; (d) 13, COMU, DIPEA, DMF, rt.; (e) TFA/DCM (1:5), rt.; (f);K₂CO₃, MeOH (g) 5a, COMU, DIPEA, DMF, rt; (h) 5b, COMU, DIPEA, DMF, rt.

Scheme 2. Assembly of Conjugated NOD2/TLR7 Agonist 29

Reagents and conditions: (a) propargyl amine, COMU, DIPEA, DMSO, rt., 2h, (b) azido-PEG₃-amine, HATU, DIPEA, DMF, rt., (c) CuSO₄·5H₂O, Na-ascorbate, DMF, rt.

Scheme 3. Synthesis of Conjugated NOD2/TLR7/8 Agonists 32 and 33

Reagents and conditions: (a) 11, HATU, DIPEA, DMF, rt.; (b) 12, HATU, DIPEA, DMF, rt.; (c) TFA/DCM (1:5), rt.; (d) H₂, Pd/C, EtOH; (e) 5a, HATU, DIPEA, DMF, rt.

Figure 3

Heatmap of the cytokines logarithmic concentrations measured after 18 h of stimulation of human PBMCs with individual NOD2 and TLR7 agonists, their mixture, and conjugated NOD2/TLR7 agonists (1 μM). The data are mean values of three independent experiments. *, p < 0.05, **, p < 0.001, ***, p < 0.001 versus combination of 4 + 5 (one-way ANOVA post hoc Dunnett’s test).