A new class of small molecule RNA polymerase inhibitors with activity against rifampicin-resistant Staphylococcus aureus

The RNA polymerase holoenzyme is a proven target for antibacterial agents. A high-throughput screening program based on this enzyme from Staphylococcus aureus had previously identified a 2-ureidothiophene-3-carboxylate as a low micromolar inhibitor. An investigation of the relationships between the structures of this class of compounds and their inhibitory- and antibacterial activities is described here, leading to a set of potent RNA polymerase inhibitors with antibacterial activity. Characterization of this bioactivity, including studies of the mechanism of action, is provided, highlighting the power of the reverse chemical genetics approach in providing tools to inhibit the bacterial RNA polymerase.     

Syntheses of novel myxopyronin B analogs as potential inhibitors of bacterial RNA polymerase

Based upon observations from our initial findings, additional myxopyronin B analogs have been prepared and tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against Escherichia coli and Staphylococcus aureus.     

Genetic analysis of Staphylococcus aureus RNA polymerase mutants.

Spontaneous mutants of Staphylococcus aureus resistant to rifampin, rifamycin SV, streptovaricin, or streptolydigin were isolated and shown to be resistant due to chromosomal rather than plasmid mutations. Based on data concerning spontaneous mutation rates, genetic cotransduction rates, and in vitro sensitivity studies, four major antibiotic cross-resistance patterns were found. The genetic markers responsible for these cross-resistance patterns were shown to be separable by transduction. Nonpurified RNA polymerase activity in lysates of mutants showed the same sensitivity to these antibiotics as shown by the mutants on solid media. A model is proposed explaining possible structure-function relationships involved in the binding of these antibiotics to the RNA polymerase molecule and the mutations resulting in resistance to these antibiotics. This model includes generally overlapping but different-sized binding sites on the RNA polymerase protein coded for by similarly arranged mutable sites on the DNA.     

Antibacterial activity of flavanostilbenes against methicillin-resistant Staphylococcus aureus

Three phytochemical compounds (alopecurone A-C), flavanostilbenes which are produced by condensation between a hydroxyflavanone and a hydroxystilbene, were isolated as major components from the root of Sophora alopecuroides . They uniformly inhibited the growth of 21 strains of methicillin-resistant Staphylococcus aureus with minimum inhibitory concentrations of 3·13–6·25 μg ml^-1.     

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5?mg/l HA could completely inhibit the growth of 10^7?CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen?Ctungsten lamp for 120?min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5?mg/l HA under a halogen?Ctungsten lamp for 120?min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.     

Antibacterial activity of flavonoids against methicillin-resistant Staphylococcus aureus strains

An experimental and theoretical study was performed on the anti-staphylococcal activity of 18 natural and synthetic flavonoids against methicillin-resistant Staphylococcus aureus strains. The analysed flavonoids belong to three well-differentiated structural patterns: chalcones, flavanones and flavones. The quantitative analysis of the anti-staphylococcal activity of the compounds was carried out by determining their percent inhibition degree. The hierarchical cluster analysis method was used to analyse the anti-MRSA activity of the compounds. With this methodology, the flavonoids were classified into four groups according to their anti-staphylococcal activity (high, sufficient, intermediate and low). The carbonylic region is of importance because it is part of the bioactive region inducing anti-MRSA activity in the flavonoid molecules. The introduction of OH groups in positions 2' of chalcones and 5 of flavanones (or flavones) increases flavonoid activity, while the OCH(3)groups produce the reverse effect. Using the experimental anti-MRSA activity data of flavonoids and six quantum chemical parameters calculated by means of the AM1 semiempirical molecular orbital method, a very good quantitative structure-activity relationship was obtained (confidence range: 95%; significance level for tests: 0.05; correlation coefficient=0.9842). The selected parameters explain 96.86% of the percent inhibition degree. The obtained relation is consistent with the conclusions formulated in this paper and serves as a theoretical support for some of them. Finally, it is concluded that the flavonoids chalcone, 2'(OH)-chalcone, 2',4'(OH)(2)-chalcone and 2',4(OH)(2)-chalcone might constitute promising therapeutic agents against infections with methicillin-resistant S. aureus strains.     

Potentiation of methicillin activity against methicillin-resistant Staphylococcus aureus by diterpenes

Totarol is a diterpene compound extracted from the totara tree. Totarol and eight other diterpenes were found to potentiate methicillin, one reducing the minimum inhibitory concentration of methicillin against resistant Staphylococcus aureus 256-fold. Totarol did not inhibit the synthesis of DNA or peptidoglycan in S. aureus, but reduced the respiration rate by 70%. Under potentiation conditions, diterpenes had only a slight effect on the respiration rate, but had a significant effect on expression of PBP 2a. We conclude that the primary staphylococcal target for totarol is the respiratory chain, but that potentiation of methicillin by diterpenes is by interference with PBP 2a expression.     

Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.     

Sesquiterpene farnesol as a competitive inhibitor of lipase activity of Staphylococcus aureus

Staphylococcus aureus lipase (SAL) is known to possess broad substrate specificity for triacylglycerides. We found that a sub-minimum inhibitory concentration of farnesol (1000 mg L(-1)) inhibits this lipase activity on a Mueller-Hinton agar containing 1% Tween substrates. A quantitative lipase assay using p-nitrophenyl palmitate (pNPP) revealed that the inhibitory action of farnesol appears to be the result of the inhibition of lipase activity rather than of its secretion into the culture medium. The inhibition was observed in all the tested 8 methicillin-susceptible S. aureus and 31 methicillin-resistant S. aureus clinical isolates. Using homogeneous lipase purified by hydrophobic interaction chromatography, it was revealed that farnesol could competitively inhibit the lipase activity against the substrate pNPP.     

A small regulatory RNA alters Staphylococcus aureus virulence by titrating RNAIII activity

Staphylococcus aureus is an opportunistic human and animal pathogen with an arsenal of virulence factors that are tightly regulated during bacterial infection. The latter is achieved through a sophisticated network of regulatory proteins and regulatory RNAs. Here, we describe the involvement of a novel prophage-carried small regulatory S. aureus RNA, SprY, in the control of virulence genes. An MS2-affinity purification assay reveals that SprY forms a complex in vivo with RNAIII, a major regulator of S. aureus virulence genes. SprY binds to the 13th stem-loop of RNAIII, a key functional region involved in the repression of multiple mRNA targets. mRNAs encoding the repressor of toxins Rot and the extracellular complement binding protein Ecb are among the targets whose expression is increased by SprY binding to RNAIII. Moreover, SprY decreases S. aureus hemolytic activity and virulence. Our results indicate that SprY titrates RNAIII activity by targeting a specific stem loop. Thus, we demonstrate that a prophage-encoded sRNA reduces the pathogenicity of S. aureus through RNA sponge activity.     

铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌的抑菌活性研究

目的观察铜绿假单胞菌抗菌物质对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcusaureus,MRSA)的体外抑菌活性。方法用交叉划线接种方法进行铜绿假单胞菌对32株耐甲氧西林金葡菌的体外抗菌活性的测定。结果铜绿假单胞菌抗菌物质对MRSA的体外抑菌活性良好,产生色素的菌株的抗菌活性最好,15株铜绿假单胞菌中,7株产蓝绿色色素的铜绿假单胞菌,对MRSA的抑制率均达到了100%,平均抑菌带的宽度为37.7 mm。结论铜绿假单胞菌抗菌物质对32株MRSA具有较强的抗菌活性,无疑对MRSA感染的抗菌药物研制方面开辟了一条新的途径。这是国内的首次研究报道。     

Antibacterial activity of isoflavonoids isolated from Erythrina variegata against methicillin-resistant Staphylococcus aureus

AIMS: To screen 16 isoflavonoids isolated from Erythrina variegata (Leguminosae) for their antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). METHODS AND RESULTS: The roots of E. variegata were macerated with acetone. The chloroform-soluble fraction of the residue was subjected to repeated silica gel column chromatography followed by elution with various solvents. Structures of the isolated compounds were determined by extensive spectroscopic studies. Each compound was dissolved in dimethyl sulphoxide and added to agar plates (final concentration 1.56-100 microg ml(-1) and suspensions of MRSA spotted onto the agar plates to determine the minimum inhibitory concentration (MIC). Repeated silica gel chromatography yielded 16 compounds and spectroscopic studies revealed that all were isoflavonoids. Whilst 14 compounds showed antibacterial activity in this concentration range, the MIC values varied significantly among them. Of the active compounds, 3,9-dihydroxy-2,10-di(gamma,gamma-dimethylallyl)-6a,11a-dehydropterocarpan (erycristagallin) and 9-hydroxy-3-methoxy-2-gamma,gamma-dimethylallylpterocarpan (orientanol B) exhibited the highest activity with MIC values of 3.13-6.25 microg ml(-1). CONCLUSIONS: Erycristagallin and orientanol B showed the highest anti-MRSA activity (3.13-6.25 microg ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: Erycristagallin and orientanol B could be leading compounds for phytotherapeutic agents against MRSA infections.     

Antibacterial activity of chalcones, hydrazones and oxadiazoles against methicillin-resistant Staphylococcus aureus

The increase in antibiotic resistance due to multiple factors has encouraged the search for new compounds which are active against multidrug-resistant pathogens. In this context, chalcones, dihydrochalcones, hydrazones and oxadiazoles were tested against Staphylococcus aureus ATCC 25923 and methicillin-resistant S. aureus (MRSA) isolates, which were obtained from clinical laboratories and were characterized as MRSA using traditional and molecular methods. Among 65 tested compounds, two chalcones, one dihydrochalcone and two hydrazones were active against MRSA. Based on the minimal inhibitory concentration and cytotoxicity, hydrazones provided a better selectivity index than chalcones. Active hydrazones are promising antibiotic-like substances and they should be the subject of further microbiological studies.     

Enhancement of antibiotic activity against Staphylococcus aureus by exposure to hyperbaric oxygen

Growth of Staphylococcus aureus ATCC 6538P was studied in stationary broth cultures (11 mm deep) exposed to hyperbaric oxygen (100% O₂ at 3 atm absolute). The minimal inhibitory concentration (MIC) of the following antibiotics was determined after exposure to high-pressure oxygen (HPO) for 3, 6, and 12 hr: penicillin, streptomycin, tetracycline, oxytetracycline, kanamycin, and cephalothin. Logarithmic growth during exposure to HPO was retarded 60%. Air at 3 atm absolute did not retard growth. The longer the exposure of tube dilution tests to HPO, the lower the MIC. Regardless of the antibiotic used, MIC values relative to 100% for unexposed controls were similar for given exposures, and averaged 73% after 3 hr of exposure to HPO, 53% after 6 hr, and 34% after 12 hr. Similar enhancement with HPO and an iodophor suggests occurrence of a general phenomenon with antibacterial agents. Although HPO alone is primarily bacteriostatic, combined therapy with antibiotics and HPO may be useful against bacterial infections because the therapeutic effectiveness of a maximal dosage of antibiotic could be increased.