Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial biofilm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed "second-generation" antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB, N-mercaptoacetyl-Phe-Tyr-amide (K i ‫؍‬ 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in biofilm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal biofilms, and to eradicate biofilm completely when used in combination with conventional antibiotics.

An endonuclease of Escherichia coli active on a DNA treated with methylmethane sulfonate has been separated from an endonuclease active on depurinated sites. The former enzyme is designated here as endonuclease II, while the latter enzyme is designated as apurinic acid endonuclease. Endonuclease II is also active on DNA treated with-methylnitrosourea, 7-bromomethyl-12-methylbenz[a anthracene, and 'y-irradiation. A third fraction which contains activities for both depurinated and alkylated sites needs further study. Endonuclease II, molecular weight 33,000, has been purified 12,500-fold and does not have exonuclease III activity. Apurinic acid endonuclease, molecular weight 31,500, has been purified 11,000-fold and does not have exonuclease III activity. Exonuclease III, molecular weight 26,000, has been purified 2300-fold and does not have endonucleolytic activity at depurinated reduced sites or at alkylated sites in DNA. Therefore, these are three separate proteins. Exonuclease III can produce, presumably by its exonucleolytic activity, double-strand breaks in heavily alkylated DNA under conditions where it does not make single-strand endonucleolytic breaks at either depurinatedreduced or alkylated sites. The first purpose of this paper is to define endonuclease II of Escherichia coli as an activity different from the apurinic acid endonuclease of E. coli. Strauss and Robbins first described an endonucleolytic activity in extracts of Bacillus subtilis that recognized alkylated DNA (1). In this laboratory, an enzyme in extracts of E. coli, active on heavily alkylated DNA, was partially purified, characterized, and designated endonuclease II of E. coli (2, 3). The substrate used for these experiments was DNA that was entrapped in a polyacrylamide gel and then alkylated with methylmethane sulfonate [MeSO2OMe (MMS)] at an MeSO2OMe-to-nucleotide ratio of 6000 to 1. A partially purified preparation of endonuclease II was also found to have an endonucleolytic activity on depurinated reduced DNA (4), and this activity was thought to be due to the same enzyme that was active on MeSO20Me-treated DNA. However, Verly et al. (5, 6), using the purification procedure originally described in this laboratory, obtained an enzyme that was active on depurinated DNA but not on alkylated DNA. Subsequently, we succeeded in separating the activity on depurinated sites in DNA from the activity on MeSO2OMe-treated DNA (7,8). The former we designate as the apurinic acid endonuclease of E. coli, while the latter we designate as endonuclease II of E. coli. Endonuclease II of E. coli is also active on DNA treated with methylnitrosourea, 7-bromomethyl-12-methvlbenz-[alanthracene, and y-irradiation (7-11).The second purpose of this paper is to demonstrate that endonuclease II, the apurinic acid endonuclease, and exonuclease III are separate proteins. Originally, Yajko and Weiss (12) demonstrated that a number of E. coli mutants deficient in exonuclease III were also deficient in "endonuclease II" and vice versa. The "endonucleas...

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In this study, we report on a novel, expedited solid-phase approach for the synthesis of biotinylated and fluorescently tagged irreversible affinity based probes for the chymotrypsin and elastase-like serine proteases. The novel solid-phase biotinylation or fluorescent labeling of the aminoalkane diphenyl phosphonate warhead using commercially available Biotin-PEG-NovaTag or EDANS NovaTag resin permits rapid, facile synthesis of these reagents. We demonstrate the kinetic evaluation and utilization of a number of these irreversible inactivators for chymotrypsin-like (chymotrypsin/human cathepsin G) and elastase-like serine proteases. Encouragingly, these compounds display comparable potency against their target proteases as their N-benzyloxycarbonyl (Cbz)-protected parent compounds, from which they were derived, and function as efficient active site-directed inactivators of their target proteases. We subsequently applied the biotinylated reagents for the sensitive detection of protease species via Western blot, showing that the inactivation of the protease was specifically mediated through the active site serine. Furthermore, we also demonstrate the successful detection of serine protease species with the fluorescently labeled derivatives "in-gel", thus avoiding the need for downstream Western blotting. Finally, we also show the utility of biotinylated and pegylated affinity probes for the isolation/enrichment of serine protease species, via capture with immobilized streptavidin, and their subsequent identification via de novo sequencing. Given their selectivity of action against the serine proteases, we believe that these reagents can be exploited for the direct, rapid, and selective identification of these enzymes from biological milieu containing multiple protease subclasses.