NOD-IN-1 (Compound 4) is a potent mixed inhibitor of nucleotide-binding oligomerization domain (NOD)-like receptors.

NOD2:6.45 μM, NOD1:5.74 μM

NOD-IN-1, also known as compound 4, is a potent mixed inhibitor of NOD1 and NOD2, demonstrating balanced inhibitory effects on both targets within the low micromolar range. With IC50 values of 5.74 μM for NOD1 and 6.45 μM for NOD2, NOD-IN-1 is highlighted as the most effective compound in its series, showcasing significative inhibition capabilities in the lower micromolar spectrum. Comparative analysis reveals that NOD-IN-1 is seven times less effective than Noditinib-1 at inhibiting NOD1 and lacks selective inhibitory activity towards either NOD1 or NOD2, contrasting with Noditinib-1. Nonetheless, NOD-IN-1 maintains balanced dual inhibitory activities on both targets at concentrations below 10 μM[1].

NOD-IN-1 is dissolved in DMSO and stored, and then diluted with appropriate medium before use[1]. An MTS assay in which the proliferation rates of HEK-Blue NOD1 cells are measured in the presence of Noditinib-1 and of the synthesized potential NOD1 inhibitor NOD-IN-1 is employed to screen these compounds for potential cytotoxicity. Cells are treated for 24 h with the compound of interest at concentrations of up to 25 μM. Comparison of the resulting metabolic activities with that of the untreated control showed that all compounds are well tolerated by HEK-Blue NOD1 cells, since their residual metabolic activities do not fall below 80% at the maximum concentration tested[1].