title: 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.affiliation: Inflammatory and Viral Diseases Unit, Roche Bioscience, Palo Alto, California 94304, USA. david.swinney@roche.com
authors.forename: David C
authors.initials: DC
authors.lastname: Swinney
authors.forename: Yi-Zheng
authors.initials: YZ
authors.lastname: Xu
authors.forename: Liliana E
authors.initials: LE
authors.lastname: Scarafia
authors.forename: Ina
authors.initials: I
authors.lastname: Lee
authors.forename: Amy Y
authors.initials: AY
authors.lastname: Mak
authors.forename: Qing-Fen
authors.initials: QF
authors.lastname: Gan
authors.forename: Chakkodabylu S
authors.initials: CS
authors.lastname: Ramesha
authors.forename: Mary A
authors.initials: MA
authors.lastname: Mulkins
authors.forename: Jim
authors.initials: J
authors.lastname: Dunn
authors.forename: On-Yee
authors.initials: OY
authors.lastname: So
authors.forename: Teresa
authors.initials: T
authors.lastname: Biegel
authors.forename: Marie
authors.initials: M
authors.lastname: Dinh
authors.forename: Pamela
authors.initials: P
authors.lastname: Volkel
authors.forename: Jim
authors.initials: J
authors.lastname: Barnett
authors.forename: Stacie A
authors.initials: SA
authors.lastname: Dalrymple
authors.forename: Simon
authors.initials: S
authors.lastname: Lee
authors.forename: Martin
authors.initials: M
authors.lastname: Huber
mesh_terms: D000818:Animals; D016207:Cytokines / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins; D008297:Male; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors*; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity; D013454:Sulfoxides / Q000494:pharmacology*; D013777:Tetrazoles / Q000494:pharmacology*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D016207:Cytokines; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606206:Nfkbia protein, rat; C461648:Ro 106-9920; D013454:Sulfoxides; D013777:Tetrazoles; D025801:Ubiquitin; D000072000:NF-KappaB Inhibitor alpha