The proteasome regulates cellular processes as diverse as cell cycle progression and NF-B activation. In this study, we show that the potent antitumor natural product epoxomicin specifically targets the proteasome. Utilizing biotinylated-epoxomicin as a molecular probe, we demonstrate that epoxomicin covalently binds to the LMP7, X, MECL1, and Z catalytic subunits of the proteasome. Enzymatic analyses with purified bovine erythrocyte proteasome reveal that epoxomicin potently inhibits primarily the chymotrypsin-like activity. The trypsin-like and peptidyl-glutamyl peptide hydrolyzing catalytic activities also are inhibited at 100-and 1,000-fold slower rates, respectively. In contrast to peptide aldehyde proteasome inhibitors, epoxomicin does not inhibit nonproteasomal proteases such trypsin, chymotrypsin, papain, calpain, and cathepsin B at concentrations of up to 50 M. In addition, epoxomicin is a more potent inhibitor of the chymotrypsin-like activity than lactacystin and the peptide vinyl sulfone NLVS. Epoxomicin also effectively inhibits NF-B activation in vitro and potently blocks in vivo inf lammation in the murine ear edema assay. These results thus define epoxomicin as a novel proteasome inhibitor that likely will prove useful in exploring the role of the proteasome in various in vivo and in vitro systems.

In recent years, the multi-subunit IKK complex has been shown to be responsible for cytokine-mediated stimulation of genes involved in inflammation and as such represents an attractive target for pharmaceutical intervention. Our finding that parthenolide targets this kinase complex provides a possible molecular basis for the anti-inflammatory properties of parthenolide. In addition, these results may be useful in the development of additional anti-inflammatory agents.

The type III secretion (TTS) system is used by several animal and plant pathogens to deliver effector proteins into the cytosol of the eukaryotic target cell as a strategy to evade the defense reactions elicited by the infected organism. The fact that these systems are highly homologous implies that novel antibacterial agents that chemically attenuate the pathogens via a specific interaction with the type III secretion mechanism can be identified. Type III secretion (TTS) constitutes a common virulence system present in many gram-negative species, including Yersinia spp., Salmonella spp., Shigella spp., Pseudomonas aeruginosa, entheropathogenic Escherichia coli, enterohemoragic E. coli, and Chlamydia spp. (11,24). The bacteria depend on their respective TTS system to invade the host, resist phagocytosis, grow in deep tissues, and cause disease. Furthermore, studies have revealed that several components of the TTS systems are conserved between different species (11, 42). These findings offer a possibility to develop novel antibacterial agents that target TTS-based virulence (32, 50). Moreover, small molecules that interfere with TTS can be utilized as tools in efforts aiming at increasing our understanding of complex bacterial virulence systems by using a chemical genetics approach (29,50). The strategy of identifying and using small molecules in functional studies of microbial virulence is attractive and complements current methods in the field, as illustrated by some recent publications (7,26,27,47).The well-studied, 70-kb-plasmid-encoded Ysc (for Yersinia secretion) TTS system of Yersinia (51) represents a suitable target for both drug development (32) and a small-molecule approach to address protein function (50). Of the 11 known species of Yersinia, Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis are pathogenic to mammals (51). The Ysc TTS apparatus is essential for the bacteria to evade the host immune defense, and compounds targeting this mechanism will result in attenuation without affecting bacterial growth. Interestingly 10 of the Ysc proteins have counterparts in almost all TTS systems, and it has been shown that some components of the secretion systems are interchangeable among different species (20), demonstrating evolutionary conservation. Since the TTS systems are conserved among the gram-negative bacteria utilizing this virulence mechanism it is likely that compounds targeting TTS machinery in Yersinia will also affect the TTS system in other species and that data generated with one species would also be valid for others. The importance of TTS studies is further stressed by the fact that the number of multiresistant strains in different species that utilize this virulence system is rising (38). Moreover, multiresistant strains of Y. pestis, a potential weapon in biological warfare and bioterrorism (25), have been isolated (18).During the progress of an infection the Yersinia bacterium adheres to eukaryotic cells, e.g., macrophages, and injects a set of effector proteins, called Yops (for Y...

The intracellular pathogen Chlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs).

A collection of nine salicylidene acylhydrazide compounds were tested for their ability to inhibit the activity of virulence-associated type III secretion systems (T3SSs) in Salmonella enterica serovar Typhimurium. The compounds strongly affected Salmonella pathogenicity island 1 (SPI1) T3SS-mediated invasion of epithelial cells and in vitro secretion of SPI1 invasion-associated effector proteins. The use of a SPI1 effector ␤-lactamase fusion protein implicated intracellular entrapment of the protein construct upon application of a salicylidene acylhydrazide, whereas the use of chromosomal transcriptional gene fusions revealed a compound-mediated transcriptional silencing of SPI1. Salicylidene acylhydrazides also affected intracellular bacterial replication in murine macrophage-like cells and blocked the transport of an epitope-tagged SPI2 effector protein. Two of the compounds significantly inhibited bacterial motility and expression of extracellular flagellin. We conclude that salicylidene acylhydrazides affect bacterial T3SS activity in S. enterica and hence could be used as lead substances when designing specific inhibitors of bacterial T3SSs in order to pharmaceutically intervene with bacterial virulence.