IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

Angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a recently identified zinc metalloprotease with carboxypeptidase activity that was identified using our genomics platform. We implemented a rational design approach to identify potent and selective ACE2 inhibitors. To this end, picomolar inhibitors of ACE2 were designed and synthesized.

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IB kinase (IKK) ␤ is essential for inflammatory cytokine-induced activation of nuclear factor B (NF-B). NF-B plays a pivotal role in the function of major cell types that contribute to the pathophysiological process of rheumatoid arthritis (RA). Here, we report the mechanism and the effect of the IKK␤ inhibitor N- (6-chloro-7-methoxy-9H-␤-carbolin-8-yl)-2-methylnicotinamide (ML120B), a ␤-carboline derivative, on NF-B signaling and gene activation in RA-relevant cell systems. ML120B is a potent, selective, reversible, and ATP-competitive inhibitor of IKK␤ with an IC 50 of 60 nM when evaluated in an IB␣ kinase complex assay. ML120B does not inhibit other IKK isoforms or a panel of other kinases. ML120B concentrationdependently inhibits tumor necrosis factor ␣ (TNF␣)-stimulated NF-B signaling via inhibition of IB␣ phosphorylation, degradation, and NF-B translocation into the nucleus. For the first time, we have demonstrated that in human fibroblast-like synoviocytes, TNF␣-or interleukin (IL)-1␤-induced monocyte chemoattractant protein-1 regulated on activation, normal T cell expressed and secreted and production is IKK␤-dependent. In addition, for the first time, we have demonstrated that lipopolysaccharide-or peptidoglycan-induced cytokine production in human cord blood-derived mast cells is IKK␤-dependent. In addition, in human chondrocytes, ML120B inhibited IL-1␤-induced matrix metalloproteinase production with an IC 50 of approximately 1 M. ML120B also blocked IL-1␤-induced prostaglandin E 2 production. In summary, ML120B blocked numerous NF-B-regulated cell responses that are involved in inflammation and destructive processes in the RA joint. Our findings support the evaluation of IKK␤ inhibitors as anti-inflammatory agents for the treatment of RA.

A genetic approach utilizing the yeast Saccharomyces cerevisiae was used to identify the target of antifungal compounds. This analysis led to the identification of small molecule inhibitors of RNA polymerase (Pol) III from Saccharomyces cerevisiae. Three lines of evidence show that UK-118005 inhibits cell growth by targeting RNA Pol III in yeast. First, a dominant mutation in the g domain of Rpo31p, the largest subunit of RNA Pol III, confers resistance to the compound. Second, UK-118005 rapidly inhibits tRNA synthesis in wild-type cells but not in UK-118005 resistant mutants. Third, in biochemical assays, UK-118005 inhibits tRNA gene transcription in vitro by the wild-type but not the mutant Pol III enzyme. By testing analogs of UK-118005 in a template-specific RNA Pol III transcription assay, an inhibitor with significantly higher potency, ML-60218, was identified. Further examination showed that both compounds are broad-spectrum inhibitors, displaying activity against RNA Pol III transcription systems derived from Candida albicans and human cells. The identification of these inhibitors demonstrates that RNA Pol III can be targeted by small synthetic molecules.Defining the mechanism of action of small molecules in higher eukaryotes is hindered by their complexity, limited genetic methods, and protracted life cycles. In contrast, model eukaryotes are amenable to extensive genetic analyses in relatively short time frames. For example, unicellular eukaryotes, such as Saccharomyces cerevisiae and Aspergillus nidulans, have been used to rapidly elucidate the mechanism of action of many compounds, including drugs that are relevant to human therapeutics. The targets of the immunosuppressive compounds cyclosporine, FK506, and rapamycin were discovered in studies with yeast (9, 24). Resistance to cerulenin in S. cerevisiae was mapped to FAS2, and inhibition of the encoded enzyme, fatty acid synthase, was demonstrated biochemically (21). Similarly, isolation of an A. nidulans mutant resistant to a novel agricultural antifungal compound led to identification of dihydroorotate dehydrogenase as the target (15).In the present report, we extend the utility of S. cerevisiae for discovering and characterizing antifungal compounds. Our initial studies focused on UK-118005, a compound that has broad-spectrum antifungal activity. Using classical genetics, molecular biology, and biochemistry, we show that UK-118005 inhibits RNA polymerase (Pol) III. Although natural products have been identified that inhibit different RNA Pols, such as ␣-amanitin (32, 33) and tagetitoxin (34,35), this is the first example of a synthetic small molecule inhibiting RNA Pol. This finding demonstrates that cell-based screening can be a powerful method for identifying novel druggable targets.Additional antifungal structural analogs of UK-118005 were identified and further characterized. These results showed that whereas some analogs inhibit RNA Pol III as expected, others caused growth inhibition by an entirely different mechanism. Thus, yeast can be us...

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T-cell receptors (TCRs) are created by a stochastic gene rearrangement process during thymocyte development, generating thymocytes bearing useful, as well as unwanted, specificities. Within the latter group, autoreactive thymocytes arise which are subsequently eliminated via a thymocyte-specific apoptotic mechanism, termed negative selection. The molecular basis of this deletion is unknown. Here, we show that TCR triggering by peptide/MHC ligands activates a caspase in double-positive (DP) CD4+ CD8+ thymocytes, resulting in their death. Inhibition of this enzymatic activity prevents antigen-induced death of DP thymocytes in fetal thymic organ culture (FTOC) from TCR transgenic mice as well as apoptosis induced by anti-CD3epsilon monoclonal antibody and corticosteroids in FTOC of normal C57BL/6 mice. Hence, a common caspase mediates immature thymocyte susceptibility to cell death.

Rip2 (Rick, Cardiak, CCK2, and CARD3) is a serine/ threonine kinase containing a caspase recruitment domain (CARD) at the C terminus. Previous reports have shown that Rip2 is involved in multiple receptor signaling pathways that are important for innate and adaptive immune responses. However, it is not known whether Rip2 kinase activity is required for its function. Here we confirm that Rip2 participates in lipopolysaccharide (LPS)/Toll-like receptor (TLR4) signaling and demonstrate that its kinase activity is not required. Upon LPS stimulation, Rip2 was transiently recruited to the TLR4 receptor complex and associated with key TLR signaling mediators IRAK1 and TRAF6. Furthermore, Rip2 kinase activity was induced by LPS treatment. These data indicate that Rip2 is directly involved in the LPS/ TLR4 signaling. Whereas macrophages from Rip2-deficient mice showed impaired NF-B and p38 mitogenactivated protein kinase activation and reduced cytokine production in response to LPS stimulation, LPS signaling was intact in macrophages from mice that express Rip2 kinase-dead mutant. These results demonstrate that Rip2-mediated LPS signaling is independent of its kinase activity. Our findings strongly suggest that Rip2 functions as an adaptor molecule in transducing signals from immune receptors.