The human organism’s immune system is the main defense against pathogens, that is why rapid recognition and removal of foreign bodies is crucial for a successful response. Immune cells recognize pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs), including the nucleotide-binding oligomerization domain 2 (NOD2) receptor. NOD2 recognizes muramyl dipeptide (MDP; MurNAc-L-Ala-D-isoGln), which is an integral part of the bacterial peptidoglycan. Binding the agonist to NOD2 triggers the activation of both innate and specific immune responses, making NOD2 agonists clinically useful as potential adjuvants in vaccines. Previous research conducted at the Department of Pharmaceutical Chemistry at the Faculty of Pharmacy, resulted in a construction of an extensive library of synthetic NOD2 agonists, among which the trans-feruloyl derivatives of tripeptide Gly-L-Val-D-Glu SG8 and SG29 emerged as the most potent, therefore they were chosen as our lead compounds for further optimization.
In the Master's thesis, we first examined whether it is possible to improve the NOD2 activation capacity of lead compound SG8, via pharmacodynamic optimization, by introducing the following changes: i) the central amino acid L-Val was replaced by larger and more hydrophobic amino acids ii) trans-ferulic acid moiety was either replaced with cinnamic acid derivatives substituted at positions 3 and 4 or with rigid heterocycles. Both lead compounds were subjected to pharmacokinetic optimization by preparing prodrugs through the introduction of cyclopentyl ester and adamantane moieties into the parent molecules to facilitate the passive absorption of compounds across the membrane to its target. The effects of all introduced changes were biologically evaluated in HEK-Blue hNOD2 cells, which simultaneously detect the ability of compound to cross the membrane and, foremost, its ability to activate the intracellular target NOD2.
A significant decrease in activity was observed for derivates in which central amino acid was replaced by 3-cyclohexyl-L-alanine, adamantylglycine, 3-(pyridin-4-yl)-L-alanine and L-homophenylalanine. A similar trend was also observed when trans-ferulic acid was replaced with benzofuran, 5-methyl-2-phenyloxazole and 5-phenyl-1,2,4-oxadiazole. Among the derivatives incorporating cinnamic acid, 3-substituted analogues showed higher NOD2 activity than 4-substituted analogues, moreover the activity was also strongly depended on the size of incorporated functional groups. In particular, the introduction of larger aromatic groups such as phenyl and phenoxy to the 3-position of cinnamic acid resulted in potent NOD2 agonists. The pharmacokinetic optimization that was carried out by introducing cyclopentyl esters to the parent structures of 3-phenylcinnamic and 3-phenoxycinnamic derivates yielded no significant improvement of activity. On the other hand, decoration of lead compound SG8 with an adamantane moiety, led to compound 41 (EC50 = 4,5 nM), that is 10-20-fold more potent than our lead compounds, thus making it the most potent synthetic desmuramylpeptide NOD2 agonist to date.
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