Inhibitors of IkappaB kinase (IKK) have long been sought as specific regulators of NF-kappaB. A screening effort of the endogenous IKK complex allowed us to identify 5-bromo-6-methoxy-beta-carboline as a nonspecific IKK inhibitor. Optimization of this beta-carboline natural product derivative resulted in a novel class of selective IKK inhibitors with IC(50)s in the nanomolar range. In addition, we show that one of these beta-carboline analogues inhibits the phosphorylation of IkappaBalpha and subsequent activation of NF-kappaB in whole cells, as well as blocking TNF-alpha release in LPS-challenged mice.

TrCP . Here we show that Nedd8 modification of the Cul-1 component of SCFb TrCP is important for function of SCFb TrCP in ubiquitination of IkBa. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCFb TrCP , phosphorylated IkBa and b-catenin, indicating that Nedd8-Cul-1 conjugates are part of SCFb TrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed bTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkBa required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCFb TrCP containing a K720R mutant of Cul-1 only weakly supported IkBa ubiquitination compared to SCFb TrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCFb TrCP . These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkBa.

ABSTRACT Regulation of NF-κB occurs through phosphorylation-dependent ubiquitination of IκBα, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IκBα is carried out by a ubiquitin-ligase enzyme complex called SCFβTrCP . Here we show that Nedd8 modification of the Cul-1 component of SCFβTrCP is important for function of SCFβTrCP in ubiquitination of IκBα. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCFβTrCP , phosphorylated IκBα and β-catenin, indicating that Nedd8–Cul-1 conjugates are part of SCFβTrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed βTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IκBα required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCFβTrCP containing a K720R mutant of Cul-1 only weakly supported IκBα ubiquitination compared to SCFβTrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCFβTrCP . These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IκBα.

Regulation of NF-kB occurs through phosphorylation-dependent ubiquitination of IkBa, which is degraded by the 26S proteasome. Recent studies have shown that ubiquitination of IkBa is carried out by a ubiquitinligase enzyme complex called SCF TrCP . Here we show that Nedd8 modification of the Cul-1 component of SCF TrCP is important for function of SCF TrCP in ubiquitination of IkBa. In cells, Nedd8-conjugated Cul-1 was complexed with two substrates of SCF TrCP , phosphorylated IkBa and b-catenin, indicating that Nedd8–Cul-1 conjugates are part of SCF TrCP in vivo. Although only a minute fraction of total cellular Cul-1 is modified by Nedd8, the Cul-1 associated with ectopically expressed bTrCP was highly enriched for the Nedd8-conjugated form. Moreover, optimal ubiquitination of IkBa required Nedd8 and the Nedd8-conjugating enzyme, Ubc12. The site of Nedd8 ligation to Cul-1 is essential, as SCF TrCP containing a K720R mutant of Cul-1 only weakly supported IkBa ubiquitination compared to SCF TrCP containing WT Cul-1, suggesting that the Nedd8 ligation of Cul-1 affects the ubiquitination activity of SCF TrCP . These observations provide a functional link between the highly related ubiquitin and Nedd8 pathways of protein modification and show how they operate together to selectively target the signal-dependent degradation of IkBa.

It is becoming increasingly apparent that NF-kappa B plays a critical role in regulating the inflammatory response. Data obtained from studies in our laboratories demonstrate that the proteasome plays an important role in the inflammatory cascade by regulating the activation of NF-kappa B. Indeed, the availability of selective and orally active proteasome inhibitors should prove useful in delineating the roles of the proteasome and NF-kappa B in other pathophysiological conditions such as cancer and heart disease.

The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.