Biomedical subjects

Michael N Gwynn

Publications and source records attributed to Michael N Gwynn.

7 recordsLinked to original sources

PubMed2006

Drugs for bad bugs: confronting the challenges of antibacterial discovery.

The sequencing of the first complete bacterial genome in 1995 heralded a new era of hope for antibacterial drug discoverers, who now had the tools to search entire genomes for new antibacterial targets. Several companies, including GlaxoSmithKline, moved back into the antibacterials area and embraced a genomics-derived, target-based approach to screen for new classes of drugs with novel modes of action. Here, we share our experience of evaluating more than 300 genes and 70 high-throughput screening campaigns over a period of 7 years, and look at what we learned and how that has influenced GlaxoSmithKline's antibacterials strategy going forward.

Animals↗

PubMed2005

Molecular evolution perspectives on intraspecific lateral DNA transfer of topoisomerase and gyrase loci in Streptococcus pneumoniae, with implications for fluoroquinolone resistance development and spread.

Fluoroquinolones are an important class of antibiotics for the treatment of infections arising from the gram-positive respiratory pathogen Streptococcus pneumoniae. Although there is evidence supporting interspecific lateral DNA transfer of fluoroquinolone target loci, no studies have specifically been designed to assess the role of intraspecific lateral transfer of these genes in the spread of fluoroquinolone resistance. This study involves a comparative evolutionary perspective, in which the evolutionary history of a diverse set of S. pneumoniae clinical isolates is reconstructed from an expanded multilocus sequence typing data set, with putative recombinants excluded. This control history is then assessed against networks of each of the four fluoroquinolone target loci from the same isolates. The results indicate that although the majority of fluoroquinolone target loci from this set of 60 isolates are consistent with a clonal dissemination hypothesis, 3 to 10% of the sequences are consistent with an intraspecific lateral transfer hypothesis. Also evident were examples of interspecific transfer, with two isolates possessing a parE-parC gene region arising from viridans group streptococci. The Spain 23F-1 clone is the most dominant fluoroquinolone-nonsusceptible clone in this set of isolates, and the analysis suggests that its members act as frequent donors of fluoroquinolone-nonsusceptible loci. Although the majority of fluoroquinolone target gene sequences in this set of isolates can be explained on the basis of clonal dissemination, a significant number are more parsimoniously explained by intraspecific lateral DNA transfer, and in situations of high S. pneumoniae population density, such events could be an important means of resistance spread.

Amino Acid Sequence↗

PubMed2004

Staphylococcus aureus 3-hydroxy-3-methylglutaryl-CoA synthase: crystal structure and mechanism.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a member of the family of acyl-condensing enzymes, catalyzes the first committed step in the mevalonate pathway and is a potential target for novel antibiotics and cholesterol-lowering agents. The Staphylococcus aureus mvaS gene product (43.2 kDa) was overexpressed in Escherichia coli, purified to homogeneity, and shown biochemically to be an HMG-CoA synthase. The crystal structure of the full-length enzyme was determined at 2.0-A resolution, representing the first structure of an HMG-CoA synthase from any organism. HMG-CoA synthase forms a homodimer. The monomer, however, contains an important core structure of two similar alpha/beta motifs, a fold that is completely conserved among acyl-condensing enzymes. This common fold provides a scaffold for a catalytic triad made up of Cys, His, and Asn required by these enzymes. In addition, a crystal structure of HMG-CoA synthase with acetoacetyl-CoA was determined at 2.5-A resolution. Together, these structures provide the structural basis for an understanding of the mechanism of HMG-CoA synthase.

Amino Acid Motifs↗

PubMed2004

Novel chromosomally encoded multidrug efflux transporter MdeA in Staphylococcus aureus.

Antibiotic efflux is an important mechanism of resistance in pathogenic bacteria. Here we describe the identification and characterization of a novel chromosomally encoded multidrug resistance efflux protein in Staphylococcus aureus, MdeA (multidrug efflux A). MdeA was identified from screening an S. aureus open reading frame expression library for resistance to antibiotic compounds. When overexpressed, MdeA confers resistance on S. aureus to a range of quaternary ammonium compounds and antibiotics, but not fluoroquinolones. MdeA is a 52-kDa protein with 14 predicted transmembrane segments. It belongs to the major facilitator superfamily and is most closely related, among known efflux proteins, to LmrB of Bacillus subtilis and EmrB of Escherichia coli. Overexpression of mdeA in S. aureus reduced ethidium bromide uptake and enhanced its efflux, which could be inhibited by reserpine and abolished by an uncoupler. The mdeA promoter was identified by primer extension. Spontaneous mutants selected for increased resistance to an MdeA substrate had undergone mutations in the promoter for mdeA, and their mdeA transcription levels were increased by as much as 15-fold. The mdeA gene was present in the genomes of all six strains of S. aureus examined. Uncharacterized homologs of MdeA were present elsewhere in the S. aureus genome, but their overexpression did not mediate resistance to the antibacterials tested. However, MdeA homologs were identified in other bacteria, including Bacillus anthracis, some of which were shown to be functional orthologs of MdeA.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2004

Genomic approaches to antibacterial discovery.

This chapter describes two key strategies for the discovery of new antibacterial agents and illustrates the critical role played by genomics in each. The first approach is genomic target-based screening. Comparative genomics and bioinformatics are used to identify novel, selective antibacterial targets of the appropriate antibacterial spectrum. Genetic technologies integral for the success of this approach, such as essentiality testing, are also described. An unprecedented number of novel targets have been discovered via this approach, and a plethora of examples are discussed. This section concludes with the case history of a target successfully progressed from identification by genomics, to high-throughput screening, and onto proof of concept in curing experimental infections. The second approach is based on screening for compounds with antibacterial activity and then employing a broad variety of newer technologies to identify the molecular target of the antibacterial agent. The advantage of this approach is that compounds already possess antibacterial activity, which is a property often challenging to engineer into molecules obtained from enzyme-based screening approaches. The recent development of novel biochemical and genomic technologies that facilitate identification and characterization of the mode of action of these agents has made this approach as attractive as the genomic target-based screening strategy.

Anti-Bacterial Agents↗

PubMed2002

Staphylococcus aureus gyrase-quinolone-DNA ternary complexes fail to arrest replication fork progression in vitro. Effects of salt on the DNA binding mode and the catalytic activity of S. aureus gyrase.

Type II topoisomerases bind to DNA at the catalytic domain across the DNA gate. DNA gyrases also bind to DNA at the non-homologous C-terminal domain of the GyrA subunit, which causes the wrapping of DNA about itself. This unique mode of DNA binding allows gyrases to introduce the negative supercoils into DNA molecules. We have investigated the biochemical characteristics of Staphylococcus aureus (S. aureus) gyrase. S. aureus gyrase is known to require high concentrations of potassium glutamate (K-Glu) for its supercoiling activity. However, high concentrations of K-Glu are not required for its relaxation and decatenation activities. This is due to the requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results suggest that S. aureus gyrase can bind to DNA at the catalytic domain independent of K-Glu concentration, but high concentrations of K-Glu are required for the binding of the C-terminal domain of GyrA to DNA and the wrapping of DNA. Thus, salt modulates the DNA binding mode and the catalytic activity of S. aureus gyrase. Quinolone drugs can stimulate the formation of covalent S. aureus gyrase-DNA complexes, but high concentrations of K-Glu inhibit the formation of S. aureus gyrase-quinolone-DNA ternary complexes. In the absence of K-Glu, ternary complexes formed with S. aureus gyrase cannot arrest replication fork progression in vitro, demonstrating that the formation of a wrapped ternary complex is required for replication fork arrest by a S. aureus gyrase-quinolone-DNA ternary complex.

Catalysis↗

PubMed2002

Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis.

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.

Acyl Coenzyme A↗