INH14 is a novel inhibitor of the IKKα/β-dependent TLR inflammatory response (IC50s: 8.97/3.59 μM for IKKα/IKKβ).

IKKβ:3.59 μM (cell free), IKKα:8.97 μM (cell free)

在用INH14处理的细胞中，TLR2通路部分蛋白质的过表达显示目标位于复合体TAK1/TAB1下游。INH14减少了由脂肽（TLR2配体）激活的细胞中IkBα的降解。通过激酶实验确认，INH14的靶点是激酶IKKα和/或IKKβ（IC50 IKKα=8.97μM; IC50 IKKβ=3.59μM）。

体内实验显示，INH14减少了脂肽诱导炎症后形成的TNFα，治疗卵巢癌细胞时，INH14降低了NF-kB的固有活性，并减少了这些细胞的伤口愈合能力。

IKKα and IKKβ kinase assays (ADP-GloTM 109 kinase assay) were purchased from Promega and used following the manufacturer′s instructions. The quantification of the ADP produced in the reactions (chemiluminescence) was measured with a Victor plate reader. IKKα (15 ng per reaction) or IKKβ (20 ng per reaction) were incubated with ATP (50 μM or 25 μM, respectively) and substrate-peptide (0.2 ng/ml) in the presence of vehicle or increasing concentrations of INH14 at room temperature for one hour.

Human primary monocytes (8 x 10^4 cells/well) were seeded and incubated overnight with the compound, media control, or SDS (0.02%). Then, the tetrazolium salt WST-8 was added, and the cells were incubated for one additional hour at 37°C. During this period, the dehydrogenase activity of viable cells led to the production of the coloured product (formazan). The cell viability was measured with a Victor plate reader as an increase in the absorbance at 450 nm.

8-week old, male, pathogen-free C57BL/6J mice were maintained at the animal facility (12 h light/dark cycle; standard rodent chow and water available ad libitum). For lipopeptide-induced inflammation, 5 μg/g of INH14 or vehicle were administered intraperitoneally. After one hour, P2 (2.5 μg/g) was injected and 25 μl of tail vein blood were collected at that time point (0 h) and 2 h after. The blood was centrifuged at 5 x 10^3 x g, and the supernatant frozen at -20 °C until further cytokine measurement by ELISA.