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

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.