Distinctive Immune Signatures Driven by Structural Alterations in Desmuramylpeptide NOD2 Agonists

Herein we report on the design, synthesis and biological evaluation of a series of nucleotide-binding oligomerization-domain-containing protein 2 (NOD2) desmuramylpeptide agonists. The structural prerequisites that shape both physicochemical and immunomodulatory profiles of desmuramylpeptide NOD2 agonists have been delineated. Within this context, we identified <b>3</b>, a butyrylated desmuramylpeptide, as a potent <i>in vitro</i> NOD2 agonist (EC₅₀ = 4.6 nM), exhibiting an almost 17-fold enhancement in potency compared to its unsubstituted counterpart <b>1</b> (EC₅₀ = 77.0 nM). The novel set of desmuramylpeptides demonstrate unique <i>in vitro</i> immunomodulatory activities. They elicited cytokine production in peripheral blood mononuclear cells (PBMCs), both alone and in conjunction with lipopolysaccharide (LPS). The spermine-decorated <b>32</b> also stimulated the LPS-induced cytotoxic activity (2.95-fold) of PBMCs against K562 cancer cells. Notably, the cholesterol-conjugate <b>26</b> displayed anti-inflammatory actions, highlighted by its capacity to convert the inflammatory monocyte subset into an anti-inflammatory phenotype. Finally, the eicosapentaenoylated derivative <b>23</b> augmented antigen presentation by mouse bone marrow-derived dendritic cells (BMDCs), thus highlighting its potential as a vaccine adjuvant.