To examine several poorly understood or contentious aspects of the antibacterial activity of silver (Ag(+)), including its cidality, mode of action, the prevalence of resistance amongst clinical ...staphylococcal isolates and the propensity for Staphylococcus aureus to develop Ag(+) resistance.
The effects of Ag(+) on the viability, macromolecular synthesis and membrane integrity of S. aureus SH1000 were assessed using established methodology. Silver nitrate MICs were determined for a collection of staphylococcal isolates (n = 1006) collected from hospitals across Europe and Canada between 1997 and 2010. S. aureus biofilms were grown using the Calgary Biofilm Device. To examine the in vitro development of staphylococcal resistance to Ag(+), bacteria were subjected to continuous subculture in the presence of sub-MIC concentrations of Ag(+).
Silver was bactericidal against S. aureus in buffered solution, but bacteriostatic in growth medium, and was unable to eradicate staphylococcal biofilms in vitro. Challenge of S. aureus with Ag(+) caused rapid loss of membrane integrity and inhibition of the major macromolecular synthetic pathways. All clinical staphylococcal isolates were susceptible to ≤ 16 mg/L silver nitrate and prolonged exposure (42 days) to Ag(+) in vitro failed to select resistant mutants.
The rapid and extensive loss of membrane integrity observed upon challenge with Ag(+) suggests that the antibacterial activity results directly from damage to the bacterial membrane. The universal susceptibility of staphylococci to Ag(+), and failure to select for resistance to Ag(+), suggest that silver compounds remain a viable option for the prevention and treatment of topical staphylococcal infections.
The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add
l-Ala,
d-Glu,
meso-A
2pm or
l-Lys, and
d-Ala-
d-Ala to the nucleotide precursor UDP-Mur
NAc, and they represent promising ...targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute ‘Diversity Set’ on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC
50
=
10
μM) and one novel MurF inhibitor (IC
50
=
63
μM).
Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first ...committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.
The terminal dipeptide, d-Ala-d-Ala, of the peptidoglycan precursor UDPMurNAc-pentapetide is a crucial building block involved in peptidoglycan cross-linking. It is synthesized in the bacterial ...cytoplasm by the enzyme d-alanine:d-alanine ligase (Ddl). Structure-based virtual screening of the NCI diversity set of almost 2000 compounds was performed with a DdlB isoform from Escherichia coli using the computational tool AutoDock 4.0. The 130 best-ranked compounds from this screen were tested in an in vitro assay for their inhibition of E. coli DdlB. Three compounds were identified that inhibit the enzyme with K i values in micromolar range. Two of these also have promising antibacterial activities against Gram-positive and Gram-negative bacteria.
ATP-dependent D-alanine:D-alanine ligase (Ddl) is a part of biochemical machinery involved in peptidoglycan biosynthesis, as it catalyzes the formation of the terminal D-ala-D-ala dipeptide of the ...peptidoglycan precursor UDPMurNAc-pentapeptide. Inhibition of Ddl prevents bacterial growth, which makes this enzyme an attractive and viable target in the urgent search of novel effective antimicrobial drugs. To address the problem of a relentless increase in resistance to known antimicrobial agents we focused our attention to discovery of novel ATP-competitive inhibitors of Ddl.
Encouraged by recent successful attempts to find selective ATP-competitive inhibitors of bacterial enzymes we designed, synthesized and evaluated a library of 6-arylpyrido2,3-dpyrimidine-based compounds as inhibitors of Escherichia coli DdlB. Inhibitor binding to the target enzyme was subsequently confirmed by surface plasmon resonance and studied with isothermal titration calorimetry. Since kinetic analysis indicated that 6-arylpyrido2,3-dpyrimidines compete with the enzyme substrate ATP, inhibitor binding to the ATP-binding site was additionally studied with docking. Some of these inhibitors were found to possess antibacterial activity against membrane-compromised and efflux pump-deficient strains of E. coli.
We discovered new ATP-competitive inhibitors of DdlB, which may serve as a starting point for development of more potent inhibitors of DdlB that could include both, an ATP-competitive and D-Ala competitive moiety.
A series of new inhibitors of the
d-alanine-
d-alanine ligase (Ddl) is presented. Thirteen diazenedicarboxamides were better inhibitors than
d-cycloserine, and some of them also possess antibacterial ...activity, which makes them a promising starting point for further development.
d-Alanine-
d-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor
d-alanyl-
d-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from
Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure–activity relationships. Thirteen diazenedicarboxamides were better inhibitors than
d-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.
Objectives: The post-antibiotic effect (PAE) is an important parameter of antibiotic action that is widely used as a predictor of pharmacodynamic activity. Traditionally, PAE has been determined by a ...labour-intensive method involving determination of viable cell numbers. New methods using spectrophotometric procedures could offer significant advantages for PAE determinations, particularly in terms of speed. A number of such methods have been described in the literature, but extensive comparison with the classical procedure for determining PAEs has not been carried out. We have now compared PAE values obtained using a rapid microplate method with those achieved by the classical viable count procedure. Methods: We determined PAE values for a variety of antibiotics against Staphylococcus aureus and Escherichia coli following exposure to 5 × MIC drug concentrations for 60 min in Mueller–Hinton Broth (MHB). The duration of the PAE was obtained by following the recovery of bacterial growth in antibiotic-free MHB measured either as colony forming units on Mueller–Hinton agar, or as culture absorbance (600 nm) in a microplate reader. Results: For bacteriolytic agents there was poor correlation between the two methods for both S. aureus (R2=0.096) and E. coli (R2=0.5456). However, when PAEs for bacteriostatic agents and non-lytic bactericidal agents were compared, correlation between the two methods was high for both S. aureus (R2=0.7529) and E. coli (R2=0.7687). Conclusions: The spectrophotometric microplate method for determining PAEs may be a suitable alternative to the classical method for those antibiotics that do not induce bacterial cell lysis.
Blueprints for an inhibitor: The de novo molecular design program SPROUT was used in conjunction with the X‐ray crystal structures of the bacterial enzymes DdlB and VanA to produce a novel ...enzyme‐selective inhibitor template. Following short and efficient synthesis and in keeping with the design predictions, the resulting inhibitor showed useful levels of enzyme‐selective inhibition.
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