Validation for high-throughput screening of a VanRS-based reporter gene assay for bacterial cell wall inhibitors.

AIMS: The present study was undertaken to validate, for antibiotic discovery, a reporter gene assay based on a Bacillus subtilis strain expressing the Enterococcusfaecium vanRS genes and a vanH-lacZ fusion, which produced beta-galactosidase activity in the presence of cell wall inhibitors (CWI) and lysozyme. METHODS AND RESULTS: The reporter assay was miniaturized, automated and validated with antibiotics and tested against portions of chemical and microbial extract libraries. The assay is simple, fast and reproducible and can detect all CWI, sometimes at concentrations lower than those necessary to inhibit bacterial growth. However, some membrane-interfering compounds also generate comparable signals. While most CWI elicit a signal that is transcription-dependent and abolished in an osmoprotective medium, transcription is not required for beta-galactosidase activity brought about by the membrane-interfering compounds. CONCLUSIONS: At least two distinct mechanisms appear to lead to enzymatic activity in the reporter strain. Effective counterscreens can be designed to discard the undesired classes of compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: Extensive validation is required before introducing a reporter assay in high-throughput screening. However, the ease of operation and manipulation makes the reporter assays powerful tools for antibiotic discovery.

Synthesis and biological evaluation of new fragments from kirromycin antibiotic.

New N-acyl derivatives of 1-N-desmethyl goldinamine were obtained from degradation of kirromycin. Periodate-oxidation of these derivatives provided new aldehydic fragments that were further elaborated. Both N-phenyl ureido and N-phthalimido derivatives of 1-N-desmethyl goldinamine are able to inhibit bacterial protein synthesis in cell-free assay and are active against whole microorganisms, although with lower potency than kirromycin. The derivatives from the aldehydic fragments are totally inactive.

Antibiotic GE37468 A: a new inhibitor of bacterial protein synthesis. I. Isolation and characterization.

GE37468 A is a new thiazolyl peptide antibiotic obtained by fermentation of Streptomyces sp. strain ATCC 55365. It inhibits bacterial protein synthesis by acting on elongation factor Tu and is structurally and functionally related to the GE2270 class of EF-Tu inhibitors. It is active in vitro against Gram-positive bacteria and Bacteroides fragilis, and protects mice against Staphylococcus aureus infection.

Antimicrobial activity of MDL 63,246, a new semisynthetic glycopeptide antibiotic.

MDL 63,246 is a semisynthetic derivative of the naturally occurring glycopeptide antibiotic MDL 62,476 (A40926). It was more active in vitro against Staphylococcus aureus and coagulase-negative staphylococci than MDL 62,476, teicoplanin, and vancomycin and was more active than mideplanin (MDL 62,873) against some isolates. MDL 63,246 had excellent activity against streptococci and teicoplanin-susceptible enterococci, and it also had in vitro activity against some VanA enterococcal isolates. It was more active than teicoplanin and vancomycin against acute staphylococcal, streptococcal, and enterococcal septicemia in immunocompetent and neutropenic mice. It was highly efficacious in reducing the bacterial load in the hearts of rats in staphylococcal endocarditis experiments and the bacterial load of Staphylococcus epidermis in a high infection model in neutropenic mice. The excellent in vivo activity of MDL 63,246 appears to correlate both with its in vitro antibacterial activity and with its long half-life in rodents.

Pharmacological modulation of neuropeptides in peripheral mononuclear cells.

The concentrations of beta-endorphin and cholecystokinin were measured in fresh resting peripheral mononuclear cells obtained from rats and human subjects in basal conditions and after different pharmacological treatments. Both in the human and the rat, beta-endorphin concentrations in mononuclear cells, increased after treatment with serotoninergic agonists, decreased after dopaminergic or GABAergic drugs, while the respective antagonists exerted the opposite effect. In vitro, serotoninergic and GABAergic compounds confirmed their roles in the modulation of beta-endorphin in mononuclear cells. Cholecystokinin was never affected by the pharmacological treatments.

Effect of psychoactive drugs on lymphocyte neuropeptides.

The concentrations of beta-endorphin and cholecystokinin were measured in lymphocytes obtained from young or old rats and from humans at different ages. Both in rats and humans, beta-endorphin and cholecystokinin increase with age; also in vitro, after 48-h culturing, the concentrations of beta-endorphin and cholecystokinin in lymphocytes obtained from humans of different ages changed with the same pattern observed in ex vivo experiments. In the human, beta-endorphin in lymphocytes shows a circadian rhythm that shifts approximately 6 h when compared to plasma ACTH and cortisol rhythm. The HPLC analysis of the molecular forms of beta-endorphin in lymphocytes revealed the presence of N-acetyl-beta-endorphin, with a ratio of beta-endorphin to N-acetyl-beta-endorphin ranging from 1 to 2. The concentrations of beta-endorphin and cholecystokinin were also measured in lymphocytes obtained from rats and human subjects undergoing different pharmacological treatments. In rat, the serotonin receptor antagonist metergoline decreased basal concentrations of the opioid peptide and blocked the increase of beta-endorphin concentrations induced by the serotonin precursor 5-hydroxytryptophan and the tricyclic antidepressant chlorimipramine. Also in the human, the antidepressant drug chlorimipramine increased lymphocyte beta-endorphin concentrations. In contrast to what was observed for beta-endorphin, cholecystokinin concentrations were not affected by the modulation of the serotoninergic system. Chronic treatment of rats with the dopamine receptor antagonist haloperidol induced an increase of beta-endorphin concentrations in lymphocytes that was reversed by the concomitant treatment with the dopamine receptor agonist bromocriptine, which when given alone decreased the basal concentrations of the peptide. In the human, haloperidol increased concentrations of beta-endorphin after both 24 h and chronic treatment, while cholecystokinin was never affected. Finally, beta-endorphin, but not cholecystokinin, increases both in rat and human lymphocytes after treatment with the GABA agonist sodium valproate.