Journal of Medicinal Chemistry
Article
were detected at the highest possible bacterial start concentration,
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resulting in a resistance frequency <4.5 × 10−11
.
Macromolecular Biosynthesis Assay. E. coli TolC was cultured in
3
lysogeny broth (LB) medium. H labeled precursors (1−1.25 μCi/
mL) were added during the logarithmic growth phase and several min
(3 min for uridine and thymidine, 5 min for acetic acid, 12 min for
glutamine) before the addition of compound 32 and the controls
chloramphenicol (Cam), cerulenin (Cer), ciprofloxacin (Cipro), and
rifampicin (Rif) at four times their MICs. For DNA, RNA, and protein
synthesis, 300 μL of the cultured bacteria were harvested 0 and 30 min
after addition of the inhibitors and supplemented with 2 volumes of
10% TCA. After 45 min at 4 °C, the precipitates were collected and
washed using 96-well glass fiber filter plates (Multiscreen GFB)
(Millipore, Billerica, MA). After adding Optiphase Supermix (Perkin-
Elmer, Waltham, MA), the quantification of radioactivity was
performed using a Wallac MicroBeta TriLux system (Perkin-Elmer).
For determination of lipid synthesis, cells were treated with CHCl3/
MeOH (1:1) and water, subsequently. The organic phase was
collected, evaporated, redissolved in cyclohexane, and supplemented
with Opti-Fluor O (Perkin-Elmer) before measuring the radioactivity
in the MicroBeta TriLux.
(7) Chopra, I. Bacterial RNA polymerase: a promising target for the
discovery of new antimicrobial agents. Curr. Opin. Invest. Dr. 2007, 8,
600−607.
́
(8) Arhin, F.; Belanger, O.; Ciblat, S.; Dehbi, M.; Delorme, D.;
Dietrich, E.; Dixit, D.; Lafontaine, Y.; Lehoux, D.; Liu, J.; McKay, G.
A.; Moeck, G.; Reddy, R.; Rose, Y.; Srikumar, R.; Tanaka, K. S. E.;
Williams, D. M.; Gros, P.; Pelletier, J.; Parr, T. R., Jr.; Far, A. R. A new
class of small molecule RNA polymerase inhibitors with activity against
rifampicin-resistant Staphylococcus aureus. Bioorg. Med. Chem. 2006, 14,
5812−5832.
(9) Artsimovitch, I.; Chu, C.; Lynch, A. S.; Landick, R. A new class of
bacterial RNA polymerase inhibitor affects nucleotide addition. Science
2003, 302, 650−654.
(10) Andre,
́
E.; Bastide, L.; Michaux-Charachon, S.; Gouby, A.;
i, M.; Leonetti, J.-
ELISA-Based RNAP Assembly Assay. The procedure was performed
as described by Husecken et al.14
Villain-Guillot, P.; Latouche, J.; Bouchet, A.; Gualtier
P. Novel synthetic molecules targeting the bacterial RNA polymerase
assembly. J. Antimicrob. Chemother. 2006, 57, 245−251.
́
̈
Core/Holo Transcription Assay. The procedure was performed as
described by Husecken et al.14
̈
(11) Villain-Guillot, P.; Gualtieri, M.; Bastide, L.; Roquet, F. o.;
Martinez, J.; Amblard, M.; Pugniere, M.; Leonetti, J.-P. Structure−
Activity Relationships of phenyl-furanyl-rhodanines as inhibitors of
RNA polymerase with antibacterial activity on biofilms. J. Med. Chem.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis, compound characterization, and computational
chemistry. This material is available free of charge via the
(12) Larsen, S. D.; Hester, M. R.; Craig Ruble, J.; Kamilar, G. M.;
Romero, D. L.; Wakefield, B.; Melchior, E. P.; Sweeney, M. T.;
Marotti, K. R. Discovery and initial development of a novel class of
antibacterials: Inhibitors of Staphylococcus aureus transcription/trans-
lation. Bioorg. Med. Chem. Lett. 2006, 16, 6173−6177.
AUTHOR INFORMATION
Corresponding Author
*Phone: +49 681 302 70300. Fax: +49 681 302 70308. E-mail:
■
(13) Haupenthal, J.; Husecken, K.; Negri, M.; Maurer, C. K.;
̈
Hartmann, R. W. Influence of DNA template choice on transcription
and inhibition of Escherichia coli RNA polymerase. Antimicrob. Agents
Chemother. 2012, 56, 4536−4539.
Author Contributions
The manuscript was written through contributions of all
authors.
(14) Husecken, K.; Negri, M.; Fruth, M.; Boettcher, S.; Hartmann, R.
̈
W.; Haupenthal, J. Peptide-based investigation of the Escherichia coli
RNA polymerase σ70: core interface as target site. ACS Chem. Biol.
2013, 8, 758−766.
Notes
The authors declare no competing financial interest.
(15) Thornsberry, C.; Hill, B. C.; Swenson, J. M.; McDougal, L. K.
Rifampin: spectrum of antibacterial activity. Rev. Infect. Dis. 1983, 5,
412−417.
ACKNOWLEDGMENTS
We thank Jeannine Jung for determination of RNAP IC50
values and Jannine Ludwig for determination of MIC values.
■
(16) Bandow, J. E.; Brotz, H.; Hecker, M. Bacillus subtilis tolerance of
̈
moderate concentrations of Rifampin involves the sigma(B)-depend-
ent general and multiple stress response. J. Bacteriol. 2002, 184, 459−
467.
ABBREVIATIONS USED
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(17) Fowler, C. E.; Soothill, J. S.; Oakes, L. MICs of rifampicin and
chloramphenicol for mucoid Pseudomonas aeruginosa strains are lower
when human lactoferrin is present. J. Antimicrob. Chemother. 1997, 40,
877−879.
CC, column chromatography; FabH, β-ketoacyl-acyl carrier
protein synthase III; LB, lysogeny broth; PPI, protein−protein
interaction; RNAP, RNA polymerase; SD, standard deviation;
TEA, triethylamine; TT, transcription/translation
(18) Williams, K. J.; Piddock, L. J. Accumulation of rifampicin by
Escherichia coli and Staphylococcus aureus. J. Antimicrob. Chemother.
1998, 42, 597−603.
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