In vitro activity: NOD-IN-1 is a potent mixed inhibitor of nucleotide-binding oligomerization domain (NOD)-like receptors, NOD1 and NOD2 with IC50 of 5.74 μM and 6.45 μM, respectively. NOD1 and NOD2 are important members of the pattern recognition receptor family and play a crucial role within the context of innate immunity. However, overactivation of NODs, especially of NOD1, has also been implicated in a number of diseases. Surprisingly, NOD1 remains a virtually unexploited target in this respect.

Kinase Assay: NOD-IN-1 is a potent mixed inhibitor of nucleotide-binding oligomerization domain (NOD)-like receptors, NOD1 and NOD2 with IC50 of 5.74 μM and 6.45 μM, respectively.

Cell Assay: HEK-Blue NOD1 and NOD2 cells (Invivogen) were cultured in DMEM medium (Sigma) with 10 % heat-deactivated FBS (Gibco), 2 mM l-glutamine (Sigma), 50 U/mL penicillin (Sigma), 50 ng/mL streptomycin (Sigma), 4.5 g glucose (Sigma) and 100 μg/mL Normocin (Invivogen) for 2 passages. All subsequent passages were cultured in the medium supplemented with 30 μg/mL Blasticidin (Invivogen) and 100 μg/mL Zeocin (Invivogen). The experiments were carried out on passages 7–12. NOD-IN-1 (compound 4) is potent mixed inhibitor of NOD1 and NOD2, displaying a balanced inhibitory activity on both targets in the low micromolar range. NOD-IN-1 (IC50 (NOD1)=5.74 μM; IC50 (NOD2)=6.45 μM) is identified as the best of the series, possessing NOD1- and NOD2-inhibitory activities in the lower micromolar range. These results show that NOD-IN-1 is 7-fold less potent than Noditinib-1 in terms of NOD1 inhibition and completely devoid of selective activity for NOD1 or NOD2 as opposed to Noditinib-1. NOD-IN-1 exhibits balanced dual activities of less than 10 μM on the two targets.