Received for publication August 31, 2007.
Revised January 17, 2008.
Accepted for publication January 17, 2008.

Benzoxathiole Derivative Blocks Lipopolysaccharide-Induced Nuclear Factor-B Activation and Nuclear Factor-B-Regulated Gene Transcription through Inactivating Inhibitory B Kinase

Abstract

Benzoxathiole derivatives have been used in the treatment of acne, and have shown cytostatic, antipsoriatic and antibacterial properties. However, little is known about the molecular basis for these pharmacological properties, although nuclear factor (NF)-B activation is closely linked to inflammation and cell proliferation. Here, we demonstrate that the novel small-molecule benzoxathiole BOT-64 inhibits NF-B activation with an IC₅₀ value of 1 uM by blocking inhibitory B (IB) kinase (IKK), and also suppresses NF-B-regulated expression of inflammatory genes in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. BOT-64 inhibits IKK-mediated IB phosphorylation in LPS-activated macrophages, resulting in sequential prevention of downstream events including proteolytic degradation of IB, DNA binding ability and transcriptional activity of NF-B. BOT-64 inhibits LPS-inducible IKK activity in the cells as well as catalytic activity of highly purified IKK. Moreover, the effect of BOT-64 on cell-free IKK was abolished by substitution of Ser-177 and Ser-181 residues in the activation loop of IKK to Glu residues, indicating a direct interaction site of benzoxathiole. BOT-64 attenuates NF-B-regulated expression of inflammatory genes such as inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-, interleukin (IL)-1 and IL-6 in LPS-activated or expression vector IKK-transfected macrophages. Furthermore, BOT-64 dose-dependently increases the survival rates of endotoxin LPS-shocked mice.

Key words: NFkappaB, Regulation of gene expression, Regulation - transcriptional, Cyclooxygenases, Transcription targets