Abstract: Worldwide spreading of drug-resistant pathogens makes mechanistic understanding of antibiotic action an urgent task. The macrocyclic antibiotic lipiarmycin (Lpm), which is under development for clinical use, inhibits bacterial RNA polymerase (RNAP) by an unknown mechanism. Using genetic and biochemical approaches, we show that Lpm targets the sigma(70) subunit region 3.2 and the RNAP beta' subunit switch-2 element, which controls the clamping of promoter DNA in the RNAP active-site cleft. Lpm abolishes isomerization of the 'closed'-promoter complex to the transcriptionally competent 'open' complex and blocks sigma(70)-stimulated RNA synthesis on promoter-less DNA templates. Lpm activity decreases when the template DNA strand is stabilized at the active site through the interaction of RNAP with the nascent RNA chain. Template DNA-strand fitting into the RNAP active-site cleft directed by the beta' subunit switch-2 element and the sigma(70) subunit region 3.2 is essential for promoter melting and for de novo initiation of RNA synthesis, and our results suggest that Lpm impedes this process.
Abstract: The first antibiotic of the ansamycin family, rifampicin (RIF), was isolated in 1959 and was introduced into therapy in 1962; it is still a first-line agent in the treatment of diseases such as tuberculosis, leprosy and various biofilm-related infections. The antimicrobial activity of RIF is due to its inhibition of bacterial RNA polymerase (RNAP). Most frequently, bacteria become resistant to RIF through mutation of the target; however, this mechanism is not unique. Other mechanisms of resistance have been reported, such as duplication of the target, action of RNAP-binding proteins, modification of RIF and modification of cell permeability. We suggest that several of these alternative resistance strategies could reflect the ecological function of RIF, such as autoregulation and/or signalling to surrounding microorganisms. Very often, resistance mechanisms found in the clinic have an environmental origin. One may ask whether the introduction of the RIF analogues rifaximin, rifalazil, rifapentine and rifabutin in the therapeutic arsenal, together with the diversification of the pathologies treated by these molecules, will diversify the resistance mechanisms of human pathogens against ansamycins.
Abstract: Entomopathogenic bacteria of the genus Xenorhabdus are known to be symbiotically associated with soil dwelling nematodes of the Steinernematidae family. These bacteria are transported by their nematode hosts into the hemocoel of the insect larvae, where they proliferate and produce insecticidal proteins, inhibitors of the insect immune system and antimicrobial molecules. In this study, we describe the discovery of a new family (PAX) of five antimicrobial compounds produced by fermentation of the Xenorhabdus nematophila F1 strain and purified by cation exchange chromatography and reversed phase chromatography. The chemical structure of PAX 3, a lysine-rich cyclolipopetide, was obtained from the analysis of homo and heteronuclear 2D NMR and confirmed by MS-MS experiments. The five members of the PAX family showed significant activity against plants and human fungal pathogens and moderate activity against few bacteria and yeast. No cytotoxicity was observed on CHO or insect cells.The Journal of Antibiotics advance online publication, 17 April 2009; doi:10.1038/ja.2009.31.
Abstract: Ensuring the availability of new antibiotics to eradicate resistant pathogens is a critical issue, but very few new antibacterials have been recently commercialized. In an effort to rationalize their discovery process, the industry has utilized chemical library and high-throughput approaches already applied in other therapeutical areas to generate new antibiotics. This strategy has turned out to be poorly adapted to the reality of antibacterial discovery. Commercial chemical libraries contain molecules with specific molecular properties, and unfortunately systemic antibacterials are more hydrophilic and have more complex structures. These factors are critical, since hydrophobic antibiotics are generally inactive in the presence of serum. Here, we review how the skewed distribution of systemic antibiotics in chemical space influences the discovery process.
Abstract: Evaluation of: Belogurov GA, Vassylyeva MN, Sevostyanova A et al.: Transcription inactivation through local refolding of the RNA polymerase structure. Nature 457, 332-335 (2008) and, Mukhopadhyay J, Das K, Ismail S et al.: The RNA polymerase 'switch region' is a target for inhibitors. Cell 135, 295-307 (2008). Bacterial RNA polymerase is an essential enzyme, which is responsible for synthesizing RNA from a DNA template and is targeted by a number of antibiotics. The mechanism of action of two closely related transcription inhibitors, myxopyronin B and a synthetic analog desmethyl-myxopyronin was elucidated, together with the structures of the antibiotic-RNA polymerase complexes. The studies reveal a new binding site and a new mechanism of action affecting the jaw domain of the enzyme. As the need for new antibiotics increase, these studies open new ways to the synthesis of more potent myxopyronin analogs.
Abstract: The pharmacologic effect of an antibiotic is directly related to its unbound concentration at the site of infection. Most commercial antibiotics have been selected in part for their low propensity to interact with serum proteins. These nonspecific interactions are classically evaluated by measuring the MIC in the presence of serum. As higher-throughput technologies tend to lose information, surface plasmon resonance (SPR) is emerging as an informative medium-throughput technology for hit validation. Here we show that SPR is a useful automatic tool for quantification of the interaction of model antibiotics with serum proteins and that it delivers precise real-time kinetic data on this critical parameter.
Abstract: The bacterial RNA polymerase (RNAP) is an essential enzyme that is responsible for making RNA from a DNA template and is targeted by several antibiotics. Rifampicin was the first of such antibiotics to be described and is one of the most efficient anti-tuberculosis drugs in use. In the past five years, structural studies of bacterial RNAP and the resolution of several complexes of drugs bound to RNAP subunits have revealed molecular details of the drug-binding sites and the mechanism of drug action. This knowledge opens avenues for the development of antibiotics. Here these drugs are reviewed, together with their mechanisms and their potential interest for therapeutic applications.
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 </=10 microM and have promising activities against various Gram-positive pathogens including Staphylococcus epidermidis biofilms, a major cause of nosocomial infection.
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.
Abstract: OBJECTIVES: Staphylococcus epidermidis biofilms form at the surface of implants and prostheses and are responsible for the failure of many antibiotic therapies. Only a few antibiotics are relatively active against biofilms, and rifampicin, a transcription inhibitor, is among the most effective molecules for treating biofilm-related infections. Having recently selected a new potential transcription inhibitor, we attempted to evaluate its efficacy against S. epidermidis biofilms. METHODS: Biofilm-forming S. epidermidis strains were grown planktonically or as biofilms and their susceptibility to this transcription inhibitor was compared with reference antibiotics with different mechanisms of action. CONCLUSIONS: 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.
Abstract: OBJECTIVES: Despite extensive functional screening of the bacterial RNA polymerase (RNAP) over the past years, very few novel inhibitors have been reported. We have, therefore, decided to screen with a radically different, non-enzymic, protein-protein interaction assay. Our target is the highly conserved RNAP-sigma interaction that is essential for transcription. METHODS: Small molecule inhibitors of the RNAP-sigma interaction were tested for their activity on transcription and on bacteria. RESULTS: These compounds have antibacterial activity against Gram-positive bacteria including multiresistant clinical isolates. CONCLUSIONS: This is, to our knowledge, the first example of a small molecule inhibitor of this interaction.
