A Small Molecule Ubiquitination Inhibitor Blocks NF-κB-dependent Cytokine Expression in Cells and Rats

Abstract

A small molecule inhibitor of NF-B-dependent cytokine expression was discovered that blocked tumor necrosis factor (TNF) ␣-induced IB␣ degradation in MM6 cells but not the degradation of ␤-catenin in Jurkat cells. Ro106-9920 blocked lipopolysaccharide (LPS)-dependent expression of TNF␣, interleukin-1␤, and interleukin-6 in fresh human peripheral blood mononuclear cells with IC 50 values below 1 M. Ro106-9920 also blocked TNF␣ 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 ␤TRCP but associates with IB␣ and will ubiquitinate IB␣ S32E,S36E (IB␣ee) specifically at lysine 21 or 22. Ro106-9920 was identified in a cell-free system as a time-dependent inhibitor of IB␣ee ubiquitination with an IC 50 value of 2.3 ؎ 0.09 M. 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 ␤TRCP , the putative E3 for IB␣ ubiquitination. Ro106-9920 was observed to be selective for IB␣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 LPSand TNF␣-induced IB␣ degradation and NF-B activation.

Regulation of Neutrophil Extracellular Trap Formation by Anti-Inflammatory Drugs

“…Because the effects of high ASA concentrations are not only related to cyclo-oxygenase acetylation and include inhibition of NF-kB activation (Yin et al, 1998), we next investigated the role of NF-kB blockade on NET formation. Neutrophils were incubated with BAY 11-7082 or Ro 106-9920, two structurally unrelated specific inhibitors of IkBa (inhibitor of k B) phosphorylation (Pierce et al, 1997) and IkBa ubiquitination (Swinney et al, 2002), respectively. Figure 2, A and B, shows that the release of DNA traps was markedly impaired by these drugs.…”

Inhibitors of NF-κB signaling: 785 and counting

“…In addition, the small molecule R0196-9920 has been reported to be an inhibitor of IkBa ubiquitination, and can act as an oral inflammation inhibitor in two mouse models of induced inflammation (Swinney et al, 2002).…”

Bacterial DNA Activates Endothelial Cells and Promotes Neutrophil Adherence through TLR9 Signaling

“…In some experiments, HUVECs were pretreated for 30 min with bafilomycin A (30 nM), chloroquine (5 g/ml), PD98059 (50 M), wortmannin (100 nM), SB203580 (2 M), the TLR9 inhibitory ODN 5Ј-tttagggttagggttaggg-3Ј (iODN, 6.6 or 20 M; InvivoGen) (25), a negative control ODN 5Ј-tgctgctgcttgcaagcagcttgat-3Ј (ctrlODN; InvivoGen), or the selective NF-B inhibitors BAY 11-7082 (10 M) (26) or Ro 106-9920 (5 M) (27), then cultured with CpG DNA. In additional experiments, responsiveness of nonpassaged primary culture of HUVECs to CpG ODN was also assessed.…”