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

J Biol Chem. 2002 Jun 28;277(26):23573-81. doi: 10.1074/jbc.M200842200. Epub 2002 Apr 11.

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.

MeSH terms

  • 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*

Substances

  • 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