Dimensionally Enhanced Antibacterial Library Screening.

The emergence and spread of antimicrobial resistance is a major public health threat, and there is an urgent need to develop new strategies to address the issue. In this study, the possibility of enhancing a whole cell based antibacterial library screen by increasing the dimensionality of the screening effort is explored using methicillin-resistant Staphylococcus aureus (MRSA) as the target organism. One dimension involved generating and screening a human liver microsome metabolized FDA approved drug library. Comparative screening of the un-metabolized (UM) and pre-metabolized (PM) libraries allows identification of intrinsically active agents from the UM library screen and of agents with active metabolites from the PM library screen. To further enhance this screening effort, it was combined with a -/+ resistant to antibiotic screen (-/+ cefoxitin; Cef). This allows the identification of agents that can act synergistically with the resistant to antibiotic. This approach revealed five compounds with substantially improved activity after metabolism and four compounds with substantial synergistic activity with cefoxitin. Capecitabine in particular only had significant antibacterial activity after metabolism. Its metabolites were isolated, identified, and characterized for spectrum of activity along with several other anticancer drugs with anti-MRSA activity. Floxuridine, gemcitabine, novobiocin, and rifaximin were identified as having substantial synergy with cefoxitin from the -/+Cef screens. Checkerboard assays verified synergy for these agents. Floxuridine demonstrated a particularly high degree of synergy with cefoxitin (FIC = 0.14). This study demonstrates how a dimensionally enhanced comparative screening effort can identify new antibacterial agents and strategies for countering antibacterial agent resistance.

Design and evaluation of biological activities of 1,3-oxazolidinone derivatives bearing amide, sulfonamide, and thiourea moieties.

1,3-Oxazolidine-2-one is an important heterocyclic ring participating in the chemical structure of many drugs. In this research, 22 new amide/sulfonamide/thiourea derivatives (1-22) were obtained by the reaction of (S)-4-(4-aminobenzyl)-2(1H)-1,3-oxazolidinone with 4-substituted benzoyl chlorides, 4-substituted benzene sulfonyl chlorides, and 4-substituted phenyl isothiocyanates. The structures of all synthesized compounds were clarified by FT-IR, NMR, and mass spectroscopic and elemental analysis techniques. The synthesized compounds were screened for their antimicrobial activity. Antimicrobial susceptibility and cellular physiology were evaluated using the microbroth dilution assay and the flow cytometry method. As a result, it was determined that compound 16 displayed better antimicrobial activity than chloramphenicol against Gram-positive bacteria, especially Staphylococcus aureus. In order to understand the mechanism of effect of the compounds on the cell membrane, fluorescence microscopy was used. Cell membrane damage of the Gram positive bacteria treated with compound 16 was observed as a result of intense staining with propidium iodide. In addition, genotoxicity, cytotoxicity, and absorption, distribution, metabolism, and excretion (ADME) parameters of compound 16 were examined and it was found as non-mutagenic and non-cytotoxic at the concentration at which it showed antimicrobial activity. According to the calculated ADME parameters and drug likeness scores, the compounds can be good drug candidates, especially compound 16.

Modulation of hepatic cytochrome P450s by Citrobacter rodentium infection in interleukin-6- and interferon-{gamma}-null mice.

After infection with Citrobacter rodentium, murine hepatic cytochrome P450 (P450) mRNAs are selectively regulated. Several serum proinflammatory cytokines are elevated, the most abundant being interleukin-6 (IL6). To elucidate the role of cytokines in the regulation of P450s during infection, we orally infected wild-type, IL6(-/-), or interferon-γ(-/-) [IFNγ(-/-)] female C57BL/6J mice with C. rodentium and analyzed hepatic P450 expression 7 days later. The majority of P450 mRNAs were equally affected by infection in each genotype, indicating that IL6 and IFNγ are not the primary mediators of P450 down-regulation in this disease model. The down-regulation of CYP3A11 and CYP3A13 and induction of CYP2D9 mRNAs were attenuated in the IL6(-/-) mice, suggesting a role of IL6 in the regulation of only these P450s. Similar evidence implicated IFNγ in the regulation of CYP2D9, CYP2D22, CYP3A11, CYP3A25, and CYP4F18 mRNAs in C. rodentium infection and CYP2B9, CYP2D22, and CYP2E1 in the bacterial lipopolysaccharide model of inflammation. This is the first indication of an in vivo role for IFNγ in hepatic P450 regulation in disease states. The deficiency of IL6 or IFNγ affected serum levels of the other cytokines. Moreover, experiments in cultured hepatocytes demonstrated that tumor necrosis factor α (TNFα) is the most potent and efficacious of the cytokines tested in the regulation of murine P450 expression. It is therefore possible that part of the IFNγ(-/-) and IL6(-/-) phenotypes could be attributed to the reduced levels of TNFα and part of the IFNγ(-/-) phenotype could be caused by reduced levels of IL6.

Characterization of Escherichia coli nitroreductase NfsB in the metabolism of nitrobenzodiazepines.

Nitrobenzodiazepine (NBDZ) is a sedative-hypnotic drug used in the treatment of anxiety and sleep problems. Overdose of NBDZ may cause severe neurological effects, especially for people in drug abuse or addiction. In the present study, we investigated NBDZ nitroreduction in rat enteric contents and characterized the role of enterobacterial nitroreductase in the reductive pathway. Nitroreduction of flunitrazepam (FZ) was studied in the microsomal membrane fractions of rat liver, jejunum and jejunal microflora using HPLC analysis. In the jejunal microflora, FZ was demonstrated to be significantly reduced to its amino derivative under anaerobic condition. Escherichia coli type I nitroreductase NfsB (EC 1.5.1.34) was found in rat jejunal microflora via PCR technique and Western blotting. The participation of NfsB in FZ nitroreduction was demonstrated from inhibition studies. Kinetic study of the purified recombinant NfsB indicated that nitroreduction of FZ, nitrazepam (NZ) and clonazepam (CZ) are mediated by NfsB, where CZ shows lower k(cat)/K(M) ratio than that of the other two. Finally, two other nitroreductases E. cloacae NR (EC 1.6.99.7) and S. typhimurium Cnr were also found to be responsible for FZ nitroreduction. These results provide that the reduction of NBDZ in normal flora is catalyzed by type I nitroreductase NfsB.

OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice.

BACKGROUND: Tuberculosis (TB) is still a leading cause of death worldwide. Almost a third of the world's population is infected with TB bacilli, and each year approximately 8 million people develop active TB and 2 million die as a result. Today's TB treatment, which dates back to the 1970s, is long and burdensome, requiring at least 6 mo of multidrug chemotherapy. The situation is further compounded by the emergence of multidrug-resistant TB (MDR-TB) and by the infection's lethal synergy with HIV/AIDS. Global health and philanthropic organizations are now pleading for new drug interventions that can address these unmet needs in TB treatment. METHODS AND FINDINGS: Here we report OPC-67683, a nitro-dihydro-imidazooxazole derivative that was screened to help combat the unmet needs in TB treatment. The compound is a mycolic acid biosynthesis inhibitor found to be free of mutagenicity and to possess highly potent activity against TB, including MDR-TB, as shown by its exceptionally low minimum inhibitory concentration (MIC) range of 0.006-0.024 microg/ml in vitro and highly effective therapeutic activity at low doses in vivo. Additionally, the results of the post-antibiotic effect of OPC-67683 on intracellular Mycobacterium tuberculosis showed the agent to be highly and dose-dependently active also against intracellular M. tuberculosis H37Rv after a 4-h pulsed exposure, and this activity at a concentration of 0.1 microg/ml was similar to that of the first-line drug rifampicin (RFP) at a concentration of 3 microg/ml. The combination of OPC-67683 with RFP and pyrazinamide (PZA) exhibited a remarkably quicker eradication (by at least 2 mo) of viable TB bacilli in the lung in comparison with the standard regimen consisting of RFP, isoniazid (INH), ethambutol (EB), and PZA. Furthermore, OPC-67683 was not affected by nor did it affect the activity of liver microsome enzymes, suggesting the possibility for OPC-67683 to be used in combination with drugs, including anti-retrovirals, that induce or are metabolized by cytochrome P450 enzymes. CONCLUSIONS: We concluded that based on these properties OPC-67683 has the potential to be used as a TB drug to help combat the unmet needs in TB treatment.

Mutagenic potentials of dental cements as detected by the Salmonella/microsome test.

The potential mutagenicity of a zinc phosphate (Poscal), a polycarboxylate (Aqualox) and glass ionomer cements with (Argion) and without (Meron) silver reinforcement were characterized by employing the Ames Salmonella/microsome test. The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24h at 37 degrees C. Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver. Poscal and Aqualox elicited mutagenic effects on S. typhimurium TA 98 and TA 1535, whereas Meron exhibited mutagenic effects on S. typhimurium TA 98. No mutagenic effects were detected for Argion. The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1). We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities.

[The mutagenic effects of sodium nitrite and monosodium glutamate used as food additives demonstrated by the Salmonella microsome test system]. / Gida katki maddelerinden sodyum nitrit ve monosodyum glutamatin Salmonella/mikrozom test sistemiyle mutajenik etkilerinin arastirilmasi.

In this study, the mutagenicity of sodium nitrite (an antimicrobial agent) and monosodium glutamate (a flavoring agent), which are widely used as food additives, have been evaluated. They were tested for mutagenicity in the Salmonella/microsome test system by employing plate-incorporation test in the absence and presence of rat-liver fraction, S 9. No mutagenic activity was seen for monosodium glutamate in the Salmonella/microsome test system by using the tester strains Salmonella typhimurium TA98, TA100, TA1535 and TA1538. Sodium nitrite was found mutagenic on S. typhimurium TA1535, whereas it was found weakly mutagenic on S. typhimurium TA100, in the presence and absence of rat-liver fraction, S 9.

Involvement of cytochrome b5 in the cytotoxic response to Escherichia coli lipopolysaccharide.

Cytotoxic lesions, induced by Gram-negative lipopolysaccharides (LPS), occur mainly in liver where the microsomal compartment of hepatocytes is involved in the detoxification mechanisms as well as in the biosynthesis of different active metabolites. The alterations induced by LPS from E. coli 0111:B4 on cytochrome b5 and its correlation with cytochrome P450, have been studied using an in vivo reversible endotoxic shock model and 24 h non-replicative hepatocyte monolayers. Results show that cytochrome b5 is directly affected by LPS that induces also a membrane damage with an active release of lactate dehydrogenase (LDH). The increase of cytochrome b5 levels may enhance the efficiency of the electron transport, thus facilitating the cytochrome P450-associate oxidations and reactions involved in the repair mechanisms of membranes.

Inhibition of reverse transcription in rat liver intracisternal A-particles by thymidine derivatives.

The thymidine derivatives araAzT, dTTP(3'N3), TTP(3'NH2), and araTTP(3'N3), were studied as inhibitors of the reverse transcription taking place within endogenous retroviral A-type particles, where retroviral RNAs served as templates and primers, dTTP(3'N3) was shown to be the most efficient inhibitor of retroviral particle reverse transcription. Termination of DNA chain elongation is the basic mechanism of the inhibitory action of dTTP(3'N3). The compound has a very low inhibitory effect on mammalian DNA-dependent DNA polymerases alpha, beta and gamma.