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.

No abstract

In recent years mounting problems related to antibiotic-resistant bacteria have resulted in the prediction that we are entering the preantibiotic era. A way of preventing such a development would be to introduce novel antibacterial medicines with modes of action distinct from conventional antibiotics. Recent studies of bacterial virulence factors and toxins have resulted in increased understanding of the way in which pathogenic bacteria manipulate host cellular processes. This knowledge may now be used to develop novel antibacterial medicines that disarm pathogenic bacteria. The type III secretion system (T3SS) is known to be a potent virulence mechanism shared by a broad spectrum of pathogenic Gram-negative bacteria that interact with human, animal and plant hosts by injecting effector proteins into the cytosol of host cells. Diseases, such as bubonic plague, shigellosis, salmonellosis, typhoid fever, pulmonary infections, sexually transmitted chlamydia and diarrhoea largely depend on the bacterial proteins injected by the T3SS machinery. Recently a number of T3SS inhibitors have been identified using screening-based approaches. One class of inhibitors, the salicylidene acylhydrazides, has been subjected to chemical optimization and evaluation in several in vitro and ex vivo assays in multiple bacterial species including Yersinia spp., Chlamydia spp., Salmonella spp. and Pseudotuberculosis aeruginosa. Reports published up to date indicate that T3SS inhibitors have the potential to be developed into novel antibacterial therapeutics.

INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 M, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of >20 M; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle-to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.The type III secretion (T3S) system is known to be a potent virulence mechanism shared by several pathogenic bacteria, including the Chlamydiaceae (10). All T3S systems share common structural components, while their effector proteins and methods of gene regulation vary widely. Targeting and inactivating common T3S components has been proposed as a strategy to fight infections caused by pathogens that require a T3S system for virulence (13). In an attempt to identify such compounds, Kauppi et al. (13) used a chemical genetics approach to screen a large number of synthetic compounds for the ability to inhibit Yersinia T3S gene expression. They identified compounds with the general structure of an acylated hydrazone of salicylaldehydes that were able to inhibit the pathogenic Yersinia T3S system, neutralizing the virulence while not affecting the growth of the organism (13, 17).We have previously reported that INP 0400 was able to inhibit the growth of Chlamydophila pneumoniae (27a). We reported that this compound inhibited C. pneumoniae development in a dose-dependent manner, was not cytotoxic, was not directly toxic to elementary bodies (EBs), and was effective at inhibiting the growth of Chlamydia trachomatis and Chlamydia muridarum. The appearance of inclusions at lower concentrations (Ͻ20 M) of INP 0400 resembled the appearance of inclusions seen in persistent infections resulting...