Publications by authors named "Daniel R Gentry"

9 Publications

  • Page 1 of 1

A rapid microtiter plate assay for measuring the effect of compounds on Staphylococcus aureus membrane potential.

J Microbiol Methods 2010 Nov 27;83(2):254-6. Epub 2010 Aug 27.

Antibacterial Discovery Performance Unit, Infectious Diseases Center of Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, PA 19426, United States.

We developed a homogenous microtiter based assay using the cationic dye 3, 3'-Diethyloxacarbocyanine iodide, DiOC2(3), to measure the effect of compounds on membrane potential in Staphylococcus aureus. In a screen of 372 compounds from a synthetic compound collection with anti-Escherichia coli activity due to unknown modes of action at least 17% demonstrated potent membrane activity, enabling rapid discrimination of nuisance compounds.
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http://dx.doi.org/10.1016/j.mimet.2010.08.012DOI Listing
November 2010

Type IIA topoisomerase inhibition by a new class of antibacterial agents.

Nature 2010 Aug 4;466(7309):935-40. Epub 2010 Aug 4.

Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.

Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.
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http://dx.doi.org/10.1038/nature09197DOI Listing
August 2010

Genetic characterization of Vga ABC proteins conferring reduced susceptibility to pleuromutilins in Staphylococcus aureus.

Antimicrob Agents Chemother 2008 Dec 6;52(12):4507-9. Epub 2008 Oct 6.

Department of Microbiology, ID-CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

Retapamulin MICs of > or =2 microg/ml were noted for 6 of 5,676 S. aureus recent clinical isolates evaluated. The ABC proteins VgaAv and VgaA were found to be responsible for the reduced susceptibility to pleuromutilins exhibited by these six isolates.
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http://dx.doi.org/10.1128/AAC.00915-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592886PMC
December 2008

Selection for high-level telithromycin resistance in Staphylococcus aureus yields mutants resulting from an rplB-to-rplV gene conversion-like event.

Antimicrob Agents Chemother 2008 Mar 14;52(3):1156-8. Epub 2008 Jan 14.

Department of Microbiology, ID-CEDD, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

While most Staphylococcus aureus telithromycin-resistant mutants isolated in this study possessed duplications within rplV (encoding ribosomal protein L22), four isolates possessed insertions within rplV that were identical to a portion of the gene rplB (encoding ribosomal protein L2). This novel type of mutation is the result of an apparent gene conversion-like event.
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http://dx.doi.org/10.1128/AAC.00923-07DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2258494PMC
March 2008

Stepwise exposure of Staphylococcus aureus to pleuromutilins is associated with stepwise acquisition of mutations in rplC and minimally affects susceptibility to retapamulin.

Antimicrob Agents Chemother 2007 Jun 2;51(6):2048-52. Epub 2007 Apr 2.

UP1345, GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426, USA.

To assess their effects on susceptibility to retapamulin in Staphylococcus aureus, first-, second-, and third-step mutants with elevated MICs to tiamulin and other investigational pleuromutilin compounds were isolated and characterized through exposure to high drug concentrations. All first- and second-step mutations were in rplC, encoding ribosomal protein L3. Most third-step mutants acquired a third mutation in rplC. While first- and second-step mutations did cause an elevation in tiamulin and retapamulin MICs, a significant decrease in activity was not seen until a third mutation was acquired. All third-step mutants exhibited severe growth defects, and faster-growing variants arose at a high frequency from most isolates. These faster-growing variants were found to be more susceptible to pleuromutilins. In the case of a mutant with three alterations in rplC, the fast-growing variants acquired an additional mutation in rplC. In the case of fast-growing variants of isolates with two mutations in rplC and at least one mutation at an unmapped locus, one of the two rplC mutations reverted to wild type. These data indicate that mutations in rplC that lead to pleuromutilin resistance have a direct, negative effect on fitness. While reduction in activity of retapamulin against S. aureus can be seen through mutations in rplC, it is likely that target-specific resistance to retapamulin will be slow to emerge due to the need for three mutations for a significant effect on activity and the fitness cost of each mutational step.
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http://dx.doi.org/10.1128/AAC.01066-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891380PMC
June 2007

Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes.

Antimicrob Agents Chemother 2003 Jun;47(6):1784-9

Department of Microbiology, Microbial, Musculoskeletal, and Proliferative Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline, Collegeville, Pennsylvania 19426, USA.

As reported previously (J. R. Jarvest et al., J. Med. Chem. 45:1952-1962, 2002), potent inhibitors (at nanomolar concentrations) of Staphylococcus aureus methionyl-tRNA synthetase (MetS; encoded by metS1) have been derived from a high-throughput screening assay hit. Optimized compounds showed excellent activities against staphylococcal and enterococcal pathogens. We report on the bimodal susceptibilities of S. pneumoniae strains, a significant fraction of which was found to be resistant (MIC, > or =8 mg/liter) to these inhibitors. Using molecular genetic techniques, we have found that the mechanism of resistance is the presence of a second, distantly related MetS enzyme, MetS2, encoded by metS2. We present evidence that the metS2 gene is necessary and sufficient for resistance to MetS inhibitors. PCR analysis for the presence of metS2 among a large sample (n = 315) of S. pneumoniae isolates revealed that it is widespread geographically and chronologically, occurring at a frequency of about 46%. All isolates tested also contained the metS1 gene. Searches of public sequence databases revealed that S. pneumoniae MetS2 was most similar to MetS in Bacillus anthracis, followed by MetS in various non-gram-positive bacterial, archaeal, and eukaryotic species, with streptococcal MetS being considerably less similar. We propose that the presence of metS2 in specific strains of S. pneumoniae is the result of horizontal gene transfer which has been driven by selection for resistance to some unknown class of naturally occurring antibiotics with similarities to recently reported synthetic MetS inhibitors.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155832PMC
http://dx.doi.org/10.1128/aac.47.6.1784-1789.2003DOI Listing
June 2003

Confirmation of the antibacterial mode of action of SB-219383, a novel tyrosyl tRNA synthetase inhibitor from a Micromonospora sp.

J Antibiot (Tokyo) 2002 Apr;55(4):423-6

Department of Microbiology, GlaxoSmithKline, Collegeville, PA 19426, USA.

The compound designated SB-219383 is a potent and selective inhibitor of bacterial tyrosyl tRNA synthetases. It exhibits an IC50 of < 1 nM against Staphylococcus aureus tyrosyl tRNA synthetase and weak in vitro activity against Staphylococci and Streptococci. Here we present data consistent with SB-219383 eliciting an amino acid starvation in both S. aureus and Streptococcus pneumoniae, supporting the conclusion that the antibacterial activity of SB-219383 is due to tyrosyl tRNA synthetase inhibition.
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http://dx.doi.org/10.7164/antibiotics.55.423DOI Listing
April 2002

Nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase with potent antibacterial activity against gram-positive pathogens.

J Med Chem 2002 May;45(10):1959-62

Potent nanomolar inhibitors of Staphylococcus aureus methionyl tRNA synthetase have been derived from a file compound high throughput screening hit. Optimized compounds show excellent antibacterial activity against staphylococcal and enterococcal pathogens, including strains resistant to clinical antibiotics. Compound 11 demonstrated in vivo efficacy in an S. aureus rat abscess infection model.
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http://dx.doi.org/10.1021/jm025502xDOI Listing
May 2002

The effect of antibiotic treatment on the intracellular nucleotide pools of Staphylococcus aureus.

FEMS Microbiol Lett 2002 Mar;208(2):203-6

GlaxoSmithKline, 1250 S. Collegeville Road, Collegeville, PA 19426-0989, USA.

In an assessment of antibiotic action on Staphylococcus aureus, we found that distinct changes in intracellular nucleotide pools occur depending on the antibiotic mode of action. In particular, we have quantitated the effect of antibiotics on pools of the nucleotide guanosine 3'-diphosphate, 5'-triphosphate (pppGpp). Intracellular pppGpp levels increased in response to treatment with the isoleucyl tRNA synthetase inhibitor mupirocin, the uncoupler carbonyl cyanide-m-chlorophenylhydrazone, and rifampicin. These compounds were distinguishable by the degree in which they increased the pppGpp pool and by their differential effect on the pools of other nucleotides. This technique has been used to confirm and to refute the expected mode of action of several compounds identified as possible inhibitors of tRNA synthetases. Our results provide the framework for using nucleotide analysis in the assessment of novel antimicrobial compounds with unknown modes of action.
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http://dx.doi.org/10.1111/j.1574-6968.2002.tb11082.xDOI Listing
March 2002