Structure-based design of new DHFR-based antibacterial agents: 7-aryl-2,4-diaminoquinazolines.

Dihydrofolate reductase (DHFR) inhibitors such as trimethoprim (TMP) have long played a significant role in the treatment of bacterial infections. Not surprisingly, after decades of use there is now bacterial resistance to TMP and therefore a need to develop novel antibacterial agents with expanded spectrum including these resistant strains. In this study, we investigated the optimization of 2,4-diamnoquinazolines for antibacterial potency and selectivity.

Regulation of mprF by antisense RNA restores daptomycin susceptibility to daptomycin-resistant isolates of Staphylococcus aureus.

Mutations in mprF have been shown to result in reduced susceptibility to daptomycin and other cationic antibacterials. An mprF antisense-inducible plasmid was constructed and used to demonstrate that depletion of mprF can reestablish susceptibility to daptomycin. Inducing antisense to mprF also resulted in increased susceptibility to vancomycin and gentamicin but, paradoxically, decreased susceptibility to oxacillin.

Elevated linezolid resistance in clinical cfr-positive Staphylococcus aureus isolates is associated with co-occurring mutations in ribosomal protein L3.

Resistance to linezolid (LZD) occurs through mutations in 23S rRNA and ribosomal proteins L3 and L4 or through methylation of 23S rRNA by Cfr. Here we report novel L3 mutations, ΔSer145/His146Tyr and ΔMet169-Gly174, co-occurring with cfr in LZD-resistant Staphylococcus aureus isolates recovered from a hospital outbreak in Madrid, Spain. LZD MIC values (16, 32, or 64 μg/ml) correlated with the presence and severity of the L3 mutation.

Structure-activity relationships of diverse oxazolidinones for linezolid-resistant Staphylococcus aureus strains possessing the cfr methyltransferase gene or ribosomal mutations.

Staphylococcal resistance to linezolid (LZD) is mediated through ribosomal mutations (23S rRNA or ribosomal proteins L3 and L4) or through methylation of 23S rRNA by the horizontally transferred Cfr methyltransferase. To investigate the structural basis for oxazolidinone activity against LZD-resistant (LZD(r)) strains, we compared structurally diverse, clinically relevant oxazolidinones, including LZD, radezolid (RX-1741), TR-700 (torezolid), and a set of TR-700 analogs (including novel CD-rings and various A-ring C-5 substituents), against a panel of laboratory-derived and clinical LZD(r) Staphylococcus aureus strains possessing a variety of resistance mechanisms. Potency against all strains was correlated with optimization of C- and D-rings, which interact with more highly conserved regions of the peptidyl transferase center binding site.

Staphylococcus aureus TargetArray: comprehensive differential essential gene expression as a mechanistic tool to profile antibacterials.

The widespread emergence of antibiotic-resistant bacteria and a lack of new pharmaceutical development have catalyzed a need for new and innovative approaches for antibiotic drug discovery. One bottleneck in antibiotic discovery is the lack of a rapid and comprehensive method to identify compound mode of action (MOA). Since a hallmark of antibiotic action is as an inhibitor of essential cellular targets and processes, we identify a set of 308 essential genes in the clinically important pathogen Staphylococcus aureus.

Identification of novel inhibitors of methionyl-tRNA synthetase (MetRS) by virtual screening.

Multiple inhibitors of the antibacterial target, Staphylococcus aureus MetRS, were identified by virtual screening. The process consisted of building a Catalyst pharmacophore from a ligand-S. aureus MetRS structure and using this pharmacophore to screen a commercial database.

Comparison of the essential cellular functions of the two murA genes of Bacillus anthracis.

Targeted antisense and gene replacement mutagenesis experiments demonstrate that only the murA1 gene and not the murA2 gene is required for the normal cellular growth of Bacillus anthracis. Antisense-based modulation of murA1 gene expression hypersensitizes cells to the MurA-specific antibiotic fosfomycin despite the normally high resistance of B. anthracis to this drug.

Evaluation of the metS and murB loci for antibiotic discovery using targeted antisense RNA expression analysis in Bacillus anthracis.

The biowarfare-relevant bacterial pathogen Bacillus anthracis contains two paralogs each of the metS and murB genes, which encode the important antibiotic target functions methionyl-tRNA synthetase and UDP-N-acetylenolpyruvoylglucosamine reductase, respectively. Empirical screens were conducted to detect and characterize gene fragments of each of these four genes that could cause growth reduction of B. anthracis when inducibly expressed from a plasmid-borne promoter.

A genome-wide strategy for the identification of essential genes in Staphylococcus aureus.

To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus.

Role of DNA methylation at GATC sites in the dnaA promoter, dnaAp2.

DnaA protein is required for the initiation of DNA replication at the bacterial chromosomal origin, oriC, and at the origins of many plasmids. The concentration of DnaA protein is an important factor in determining when initiation occurs during the cell cycle. Methylation of GATC sites in the dnaAp2 promoter, two of which are in the -35 and -10 sequences, has been predicted to play an important role in regulating dnaA gene expression during the cell cycle because the promoter is sequestered from methylation immediately following replication.