ACS Chemical Biology
Articles
as well as evaluation of activity in other Gram positive and
Gram negative bacteria.
ASSOCIATED CONTENT
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S
* Supporting Information
The Supporting Information is available free of charge on the
METHODS
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Chemical Synthesis. Detailed experimental procedures for all
reactions and compound characterization data can be found in the
General methods, materials, instrumentation, synthetic
procedures and compound characterization data, and
supplemental Figures 1−4 (PDF)
Pyrazole Synthesis. Route 1. To a stirred solution of terminal
alkyne (1 equiv) in THF (0.2 M) was added triethylamine (3 equiv),
bistriphenylphosphine palladium dichloride (0.05 equiv), copper(I)
iodide (0.1 equiv), and acid chloride (1.5 equiv) at RT. The reaction
was stirred until conversion of starting material was observed by TLC.
The reaction was diluted 1:1 with acetonitrile followed by the addition
of hydrazine hydrate (4 equiv). The reaction was stirred until complete
as determined by TLC. The reaction was filtered through Celite,
concentrated, and purified by preparative scale reverse phase HPLC.
Route 2. To a solution of crude diketone in ethanol (0.25 M) in a
microwave vial was added hydrazine hydrate (2 equiv). The vial was
sealed and heated to 150 °C in a microwave reactor for 5 min. The
reaction was concentrated, and the product was purified by preparative
scale reverse phase HPLC.
AUTHOR INFORMATION
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Corresponding Author
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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We thank G. M. Kurkis for compound preparation. We thank J.
E. Hempel and L. J. Lojek for critical reading of the manuscript.
We thank C. D. Weaver for important discussions. B.F.D. was
supported by National Institutes of Health Grant T32
GM065086. L.A.M. was supported by National Institutes of
Health Grant T32 HL069765. This work was funded by
National Institutes of Health R01 AI069233 and National
Institutes of Health R01 AI073843.
XylE Assay. Previously reported strains were used.11 Cultures
grown overnight in 5 mL of TSB with 10 μg mL−1 chloramphenicol
for 15−18 h were subcultured 1:100 into 0.5 mL TSB with 10 μg
mL−1 chloramphenicol containing compound and incubated at 37 °C,
at 180 rpm for 6 h. Cells were washed and lysed as previously
described. A total of 200 μL of a 200 μM catechol solution in 100 mM
potassium phosphate (pH 8.0) was added to 20 μL of lysate, and the
oxidation of catechol was followed by monitoring absorbance at 375
nm for 10 min. Samples were normalized to protein concentration as
determined by BCA assay (Pierce).
HssRS Activation Dose Response Curves and EC50 Determi-
nation. The above XylE procedure was followed using different
concentrations of compound. The data were then entered into
Graphpad Prism 6 and fit to a curve to determine EC50 values.
IC50 Determination. Cultures of wild type S. aureus strain
Newman and ΔmenB14 were grown in aeration tubes aerobically at
37 °C with shaking for 15−18 h. Anaerobic cultures were prepared by
growing bacteria at 37 °C without shaking in an anaerobic chamber for
15−18 h. Bacteria from each condition were subcultured 1:100 into
TSB containing various concentrations of compound in a 96 well plate.
Aerobic wild type and ΔmenB plates were incubated aerobically at 37
°C with shaking while anaerobic plates were grown in an anaerobic
chamber (Coy) at 37 °C without shaking. The absorbance at 600 nm
(OD600) was determined after 9 h of growth and the fraction of growth
at each compound concentration is determined by dividing the OD600
by the vehicle control (DMSO) value. IC50’s were calculated using
Graphpad Prism 6, and errors are reported as 95% confidence
intervals.
Heme Adaptation Assays. A total of 500 μL of TSB were
inoculated with a colony of S. aureus and incubated at 37 °C for 3 − 4
h. Bacteria were then subcultured 1:100 into 500 μL of TSB
containing a compound in 1.5 mL tubes and incubated at 37 °C with
shaking for 15 h. Bacteria from the compound treated cultures were
then subcultured 1:100 into 100 μL of TSB containing heme and
incubated at 37 °C with shaking for 8 h. Growth was monitored by
reading the OD600 on a Biotek microplate reader at the defined time
intervals.
Iron Chelation Assay. Iron chelation by 1 was characterized using
the CAS assay. Solutions were prepared as described.21 The clinical
iron chelator deferasirox (AK Scientific) was used as a control.
Samples were incubated in 1 mL cuvettes at RT for 30 min after
addition of compound. The maximum concentration of compound
used was 30 μM, which is a 4:1 stoichiometry of 1 to Chromeazural-S.
Absorbance at 630 nm was measured on a Varian UV/vis
spectrophotometer.
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