No abstract

The dramatic rise of antibiotic-resistant bacteria over the past two decades has stressed the need for completely novel classes of antibacterial agents. Accordingly, recent advances in the study of prokaryotic transcription open new opportunities for such molecules. This paper reports the structure-activity relationships of a series of phenyl-furanyl-rhodanines (PFRs) as antibacterial inhibitors of RNA polymerase (RNAP). The molecules have been evaluated for their ability to inhibit transcription and affect growth of bacteria living in suspension or in a biofilm and for their propensity to interact with serum albumin, a critical parameter for antibacterial drug discovery. The most active of these molecules inhibit Escherichia coli RNAP transcription at concentrations of

This is, to our knowledge, the first example of a small molecule inhibitor of this interaction.

No abstract

Staphylococcus epidermidis is a major cause of nosocomial infections because of its ability to form biofilms on the surface of medical devices. Only a few antibacterial agents are relatively active against biofilms, and rifampin, a transcription inhibitor, ranks among the most effective molecules against biofilm-related infections. Whether this efficacy is due to advantageous structural properties of rifampin or to the fact that the RNA polymerase is a favorable target remains unclear. In an attempt to answer this question, we investigated the action of different transcription inhibitors against S. epidermidis biofilm, including the newest synthetic transcription inhibitors. This comparison suggests that most of the antibiotics that target the RNA polymerase are active on S. epidermidis biofilms at concentrations close to their MICs. One of these compounds, CBR703, despite its high MIC ranks among the best antibiotics to eradicate biofilm-embedded bacteria.

We have developed a multiwell assay for the detection of modulators of prokaryotic transcription based on the quantification of protein-protein interaction. This assay consists of three steps: (a) the immobilization of the Escherichia coli protein sigma70 in the well, (b) the incubation of the immobilized protein with core RNA polymerase and a potential inhibitor, and (c) washing and quantification of the binding of core to sigma70 with a monoclonal antibody conjugated to horseradish peroxidase. We show that this assay is sensitive, reproducible, and robust, and is able to discriminate between control competitors with different affinities. We demonstrate the usefulness of the assay to screen for microbial RNA polymerase inhibitors as potential new drugs for the treatment of emerging antibiotic-resistant bacteria.

Our results demonstrate that this new molecule is active; its effects are fast and kinetically related to those of rifampicin, but unlike rifampicin it does not select for resistant bacteria.

A 37-kDa binding polypeptide accumulates in peripheral blood mononuclear cell (PBMC) extracts from chronic fatigue syndrome (CFS) patients and is being considered as a potential diagnostic marker (De Meirleir, K., Bisbal, C., Campine, I., De Becker, P., Salehzada, T., Demettre, E., and Lebleu, B. (2000) Am. J. Med. 108, 99 -105). We establish here that this low molecular weight 2-5A-binding polypeptide is a truncated form of the native 2-5A-dependent ribonuclease L (RNase L), generated by an increased proteolytic activity in CFS PBMC extracts. RNase L proteolysis in CFS PBMC extracts can be mimicked in a model system in which recombinant RNase L is treated with human leukocyte elastase. RNase L proteolysis leads to the accumulation of two major fragments with molecular masses of 37 and 30 kDa. The 37-kDa fragment includes the 2-5A binding site and the N-terminal end of native RNase L. The 30-kDa fragment includes the catalytic site in the C-terminal part of RNase L. Interestingly, RNase L remains active and 2-5A-dependent when degraded into its 30-and 37-kDa fragments by proteases of CFS PBMC extract or by purified human leukocyte elastase. The 2-5A-dependent nuclease activity of the truncated RNase L could result from the association of these digestion products, as suggested in pull down experiments.Chronic fatigue syndrome is characterized by long-lasting and debilitating fatigue, myalgia, impairment of neurocognitive functions, and flu-like symptoms, which are often severely worsened after physical exercise. A case definition has been proposed by the Center for Disease Control in Atlanta under the name of Holmes (2) and Fukuda (3) criteria. Most of these symptoms are unfortunately common to other diseases, thus complicating a diagnosis that still relies on extensive clinical testing to exclude other pathologies (4). A large proportion of the patients report an infectious episode at the onset of their chronic fatigue. No single agent has been conclusively associated with the disease, although several candidates including human T-cell lymphotrophic virus-1, human herpesvirus-6, Enterovirus, or mycoplasma have been proposed (5-8). Dysregulation of immune functions has also been suggested and natural killer cell cytotoxicity was significantly diminished in patients (when compared with normal controls) (9).These observations have prompted studies of possible dysfunctioning of interferon-induced responses. An up-regulation of the 2-5A/RNase L antiviral pathway in peripheral blood mononuclear cells (PBMC) 1 of CFS patients has been described (10). RNase L is the terminal enzyme in the 2-5A synthetase/ RNase L antiviral pathway and plays an essential role in the elimination of viral mRNAs (for review, see Ref. 11). Activation of RNase L requires the binding of a small 2Ј,5Ј-linked oligoadenylate (2-5A). Intriguingly, a low molecular weight 2-5A-binding polypeptide has been observed in a subset of patients diagnosed for CFS (12). Similar observations have been made in a larger study including CFS patients and control ...