A small molecule ubiquitination inhibitor blocks NF-kappa B-dependent cytokine expression in cells and rats.

Abstract

A small molecule inhibitor of NF-kappaB-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) alpha-induced IkappaB(alpha) degradation in MM6 cells but not the degradation of beta-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNFalpha, interleukin-1beta, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC(50) values below 1 microm. Ro106-9920 also blocked TNFalpha production in a dose-dependent manner following oral administration in two acute models of inflammation (air pouch and LPS challenge). Ro106-9920 was observed to inhibit an ubiquitination activity that does not require betaTRCP but associates with IkappaB(alpha) and will ubiquitinate IkappaB(alpha) S32E,S36E (IkappaB(alpha)(ee)) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IkappaB(alpha)(ee) ubiquitination with an IC(50) value of 2.3 +/- 0.09 microm. The ubiquitin E3 ligase activity is inhibited by cysteine-alkylating reagents, supported by E2UBCH7, and requires cIAP2 or a cIAP2-associated protein for activity. These activities are inconsistent with what has been reported for SCF(betaTRCP), the putative E3 for IkappaB(alpha) ubiquitination. Ro106-9920 was observed to be selective for IkappaB(alpha)(ee) ubiquitination over the ubiquitin-activating enzyme (E1), E2UBCH7, nonspecific ubiquitination of cellular proteins, and 97 other molecular targets. We propose that Ro106-9920 selectively inhibits an uncharacterized but essential ubiquitination activity associated with LPS- and TNFalpha-induced IkappaB(alpha) degradation and NF-kappaB activation.

• Authors: David C Swinney, Yi-Zheng Xu, Liliana E Scarafia, Ina Lee, Amy Y Mak, Qing-Fen Gan, Chakkodabylu S Ramesha, Mary A Mulkins, Jim Dunn, On-Yee So, Teresa Biegel, Marie Dinh, Pamela Volkel, Jim Barnett, Stacie A Dalrymple, Simon Lee, Martin Huber
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 277 Issue 26 Pg. 23573-81 (Jun 28 2002) ISSN: 0021-9258 [Print] United States
• PMID: 11950839 (Publication Type: Journal Article)

Chemical References

• Cytokines
• DNA-Binding Proteins
• Enzyme Inhibitors
• I-kappa B Proteins
• NF-kappa B
• NFKBIA protein, human
• Nfkbia protein, rat
• Ro 106-9920
• Sulfoxides
• Tetrazoles
• Ubiquitin
• NF-KappaB Inhibitor alpha

Topics

• Animals
• Cytokines (biosynthesis)
• DNA-Binding Proteins (metabolism)
• Enzyme Inhibitors (pharmacology)
• Humans
• I-kappa B Proteins
• Male
• NF-KappaB Inhibitor alpha
• NF-kappa B (antagonists & inhibitors)
• Rats
• Rats, Wistar
• Substrate Specificity
• Sulfoxides (pharmacology)
• Tetrazoles (pharmacology)
• Ubiquitin (metabolism)