To contribute to this effort, we have developed a high-throughput assay for PBPs that measures the fluorescence polarization of Bocillin-FL and used it to screen for potential inhibitors of N. gonorrhoeae PBP 2 from a library of 50,080 compounds. After eliminating those that acted promiscuously, did not demonstrate a concentration-dependent inhibition, or were b-lactams, 18 compounds were examined in more detail, and 7 of these exhibited antimicrobial activity against N. gonorrhoeae, including PenR and CephI strains. To the best of our knowledge, this is the first report of high-throughput screening against a PBP. Interestingly, this compound contains a thiazolidine ring, a feature of penicillin antibiotics and arylalkylidene iminothiazolidin-4-ones, which inhibit some PBPs in vitro and shows antimicrobial activity. Compound 7 was also successful in the docking simulations and there are predicted interactions with Ser310. The resemblance of compound 7 to a b-lactam-like compound led us to determine whether there is a covalent interaction with PBP 2, but no increase in mass of the protein was observed by mass spectrometry. Finally, compound 3 is probably the least promising candidate at this stage since it has a relatively high IC50 against PBP 2 and its MIC values against the N. gonorrhoeae strains are correspondingly weak. Overall, there was relatively weak correlation between IC50 and MIC values for the seven compounds, but this is expected for initial hits from HTS because there are a variety of factors that determine the MIC. Since N. gonorrhoeae expresses two essential PBPs, PBP 1 could be the lethal target for a given compound in addition to, or independent of, PBP 2. The outer membrane of Gram-negative bacteria is a SP600125 molecular weight barrier to hydrophobic compounds and the lipooligosaccharide structure of N. gonorrhoeae also impacts permeability. In addition, entry of hydrophilic compounds into the periplasmis influenced by porins and the action of efflux systems can remove compounds from the periplasm. Both of the antibiotic resistant strains contain a mutation in the promoter of mtrCDE that increases expression of the MtrC-MtrD-MtrE effluxpump. The development of this assay paves the way to screen using larger compound libraries and against variants of PBP 2 that contribute to third-generation cephalosporin resistance. They catalyze the hydrolysis of b- glycosidic bonds between the N-acetylmuramic acid and Nacetylglucosamine repeating units composing the backbone of peptidoglycan, the major constituent of bacterial cell walls. Lysozyme is a component of both phagocytic and secretory granules of neutrophils and is also produced by monocytes, macrophages and epithelial cells. It is found in significant concentrations in saliva, airway mucus, milk and other secretions, and is considered to be an important first line barrier against bacterial infection. While many gram-positive bacteria are rapidly killed by lysozyme in vitro, gram-negative bacteria are not because they have an outer membrane that prevents direct access of lysozyme to the peptidoglycan sacculus. However, in vivo, gramnegative bacteria are sensitized to lysozyme by accessory antimicrobial peptides of the innate immunity system such as defensins and complement which disrupt the outer membrane barrier. Several structurally different lysozymes have been described and the major types within the animal kingdom are the c-type, the R428 g-type and the i-type lysozymes. Vertebrates have genes for both c- and g-type lysozyme, but their spatio-temporal expression is species-specific. The chicken genome for instance comprises a single c-type and two g-type lysozyme genes.