Nodinitib-1 (CID-1088438) is a potent and selective NOD1 inhibitor with an IC50 of 0.56 μM.

NOD1:0.56 μM

Nodinitib-1展现出对NOD1诱导的NF-κB激活在HEK293细胞中的选择性抑制作用，且未表现出细胞毒性，因此被选为探针候选分子。Nodinitib-1还通过选择性抑制NOD1依赖的IL-8分泌以及选择性抑制NOD1依赖的通向NF-κB激活的路径，在次级检测中得到确认。[1] 在另一项研究中，Nodinitib-1被证实可引起NOD1的构象变化，并在体外以及细胞内改变NOD1的亚细胞定位，为探讨调节NOD1活性的机制提供化学探针，并为探索NOD1在各种传染性和炎症性疾病中的作用提供工具。[2]

NOD1 Dose Response assay: Day 1 Procedure 1) Harvest HEK-293-T NFKB-Luc at 100% confluency at 100% confluency. 2) Add NOD assay media with Multidrop. 3) Spin down plates at 1000 rpm for 1 min in centrifuge. 4) Serial compound dilutions. 5) Add gamma-tri-DAP to cell suspension at 0.75 ug/mL. 6) Seed 13000 cells/well in 4 uL/well to full plate HEK-293-T NFKB-Luc to TC-treated plate. 7) Spin down plates 500 RPM for 5 min on centrifuge. 8) Lid Plates. Sandwich 4 plates between 2 lidded 384 plates filled with Water. 9) Wrap plates securely in single layer of Plastic Wrap. 10) Incubate overnight (14 hours) in 37 ℃ and 5% CO2 incubator. Day 2 Procedure 1) Add 3 ul/well of SteadyGlo solution with Multidrop. 2) Shake plates on a plate shaker for 20 min. 3) Spin plates 1000 RPM for 1 min using centrifuge. 4) Read luminescence.IC50 values are calculated using GraphPad Prism 5.0.The average Z' for the screen is 0.6, the signal to background is 11.1, signal to noise is 78.6 and signal to window is 6.0.

Hepatic toxicity of compounds is determined with Fa2N-4 immortalized human hepatocytes using the ATP-lite 1-step assay. Fa2N-4 cells are seeded at 50,000 cells/well, and incubated with a range of concentrations of the test compound (0.01 μM-50 μM) in MFE support medium for 24 h at 37 ℃, 5% CO2. At the end of the experiment D-luciferin and luciferase are added. The emitted luminescent signal produced as a result of the reaction with cellular ATP is captured on the Infinite M200 plate reader. The concentration of each compound that killed 50% of the cells (LC50) is calculated by non-linear regression analysis using a log (inhibitior) vs response equation with a variable slope, using the statistic software package Prism4.(Only for Reference)