Targeting quinolone- and aminocoumarin-resistant bacteria with new gyramide analogs that inhibit DNA gyrase

Article abstract of DOI:10.1039/c7md00012j

Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological barriers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the characterization of three exciting new gyramide analogs - from a library of 183 derivatives - that are potent inhibitors of DNA gyrase and are active against clinical strains of Gram-negative bacteria (Escherichia coli, Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and Gram-positive bacteria (Bacillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent inhibitors of the DNA supercoiling activity of DNA gyrase (IC₅₀s of 47-170 nM) but do not alter the enzyme's ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations support the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other known inhibitors.

Full text of DOI:10.1039/c7md00012j

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RESEARCH ARTICLE
Targeting quinolone- and aminocoumarin-
resistant bacteria with new gyramide analogs that
Cite this: DOI: 10.1039/c7md00012j
inhibit DNA gyrase†‡§
Katherine A. Hurley,¶a Thiago M. A. Santos,¶a Molly R. Fensterwald,b
Madhusudan Rajendran,a Jared T. Moore,b Edward I. Balmond,b Brice J. Blahnik,a
Katherine C. Faulkner,a Marie H. Foss,a Victoria A. Heinrich,a Matthew G. Lammers,a
Lucas C. Moore,b Gregory D. Reynolds,a Galen P. Shearn-Nance,b Brian A. Stearns,c
b
b
ade
*
*
Zi W. Yao, Jared T. Shaw and Douglas B. Weibel
Bacterial DNA gyrase is an essential type II topoisomerase that enables cells to overcome topological bar-
riers encountered during replication, transcription, recombination, and repair. This enzyme is ubiquitous in
bacteria and represents an important clinical target for antibacterial therapy. In this paper we report the
characterization of three exciting new gyramide analogs—from a library of 183 derivatives—that are potent
inhibitors of DNA gyrase and are active against clinical strains of Gram-negative bacteria (Escherichia coli,
Shigella flexneri, and Salmonella enterica; 3 of 10 wild-type strains tested) and Gram-positive bacteria (Ba-
cillus spp., Enterococcus spp., Staphylococcus spp., and Streptococcus spp.; all 9 of the wild-type strains
tested). E. coli strains resistant to the DNA gyrase inhibitors ciprofloxacin and novobiocin display very little
cross-resistance to these new gyramides. In vitro studies demonstrate that the new analogs are potent in-
hibitors of the DNA supercoiling activity of DNA gyrase (IC50s of 47–170 nM) but do not alter the enzyme's
ATPase activity. Although mutations that confer bacterial cells resistant to these new gyramides map to the
genes encoding the subunits of the DNA gyrase (gyrA and gyrB genes), overexpression of GyrA, GyrB, or
GyrA and GyrB together does not suppress the inhibitory effect of the gyramides. These observations sup-
port the hypothesis that the gyramides inhibit DNA gyrase using a mechanism that is unique from other
known inhibitors.
Received 7th January 2017,
Accepted 21st February 2017
DOI: 10.1039/c7md00012j
rsc.li/medchemcomm
stranded DNA preceding the replication fork. The GyrA
subunit is involved in stabilizing the double-stranded DNA
Introduction
DNA gyrase is a validated antibiotic target that continues to
capture attention in drug discovery.1 DNA gyrase is a type II
topoisomerase consisting of two subunits that combine into a
heterotetrameric A2B2 holoenzyme complex and uses the en-
ergy from ATP hydrolysis to negatively supercoil double-
break, passing the second DNA strand through the break, and
religating the DNA strands. The GyrB subunit contains the
ATP binding site and is involved in ATP hydrolysis.2,3
Antibiotics that bind to different regions of DNA gyrase
have been reported. Ciprofloxacin (1) (Fig. 1A) is a clinical
fluoroquinolone antibiotic that binds DNA gyrase between the
DNA nucleotides on either side of the double stranded DNA
break, prevents DNA religation, and stabilizes double-
stranded breaks in the DNA bound to the protein.4 The forma-
tion of linear double stranded DNA triggers the SOS response
and inhibits cell division until the DNA is repaired.5 Novobio-
cin (2) (Fig. 1A) is an aminocoumarin that binds in the ATP
pocket of the GyrB subunit and competitively inhibits its
ATPase activity.6,7 In contrast to 1, 2 does not cause DNA dam-
age or activate the SOS response;5 however it causes many
species of cells to become filamentous, which is a hallmark of
the SOS response.8 Both 1 and 2 also inhibit topoisomerase
IV9–11—the bacterial topoisomerase involved in decatenation
of chromosomes during DNA replication. 1 also binds to the
a Department of Biochemistry, University of Wisconsin – Madison, Madison,
Wisconsin, USA. E-mail: douglas.weibel@wisc.edu
b Department of Chemistry, University of California – Davis, Davis, California,
USA. E-mail: jtshaw@ucdavis.edu
c Inception Sciences, Inc., San Diego, California, USA
d Department of Chemistry, University of Wisconsin – Madison, Madison,
Wisconsin, USA
e Department of Biomedical Engineering, University of Wisconsin – Madison,
Madison, Wisconsin, USA
† The authors declare no competing interests.
‡ The authors declare no competing financial interest.
§ Electronic supplementary information (ESI) available: Tables, figures, bacterial
strain information and compound purity data. See DOI: 10.1039/c7md00012j
¶ These authors contributed equally to this article.
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Fig. 1 A) The chemical structures of ciprofloxacin (1) and novobiocin (2). B) 3 was created from a MIC-guided SAR study of 6–8. 3 has activity
against wild-type E. coli and was used as a lead structure for further analog design and synthesis. C) Design of a chemical library of 183 gyramide
analogs. Two reactions were performed with 30 aldehydes (and 3 arylsulfonamides) and 31 sulfonyl chlorides (and 3 arylamines) to create 183
gyramide analogs.
eukaryotic homologue topoisomerase II (topo II) causing asso-
ciated side effects.12
non-pathogenic (biosafety level 1, BSL-1) bacteria and 13
pathogenic isolates (BSL-2) to identify compounds with re-
duced drug efflux, improved inhibitory activity, and extended
spectrum. We performed further characterization of three
new gyramide analogs [gyramide D (3), gyramide E (4), and
gyramide F (5)] (Fig. 1B and C) that fit these criteria, and
demonstrate that these compounds have several exciting
characteristics: 1) they are among the most potent inhibitors
of the supercoiling activity of DNA gyrase to-date; 2) they
have MICs in the single μg mL−1 range against a range of
wild-type, clinical Gram-negative and Gram-positive bacteria;
3) they have activity against E. coli strains resistant to 1 and
2; and 4) their activity is not antagonized by the
overexpression of DNA gyrase (akin to 1). Our data supports
the hypothesis that the improvement of the antibacterial ac-
tivity of these compounds arises from reducing efflux, sug-
gests chemical modifications for reducing the efflux of other
aryl-containing antibiotics, and moves these compounds one
step further along their development as a new family of
antibiotics.
The gyramides are a family of synthetic, small molecules
that inhibit the type II topoisomerase DNA gyrase;13,14 our
current data supports these compounds binding to a new site
of DNA gyrase14 that differentiates them from the binding
site for 1, 2, and other families of DNA gyrase inhibi-
tors.4,15,16 Mapping amino acid mutations that convey
gyramide resistance to Escherichia coli cells and comparing to
related data for 1 and 2 reveals that these positions are spa-
tially distinct.14 Gyramides are specific inhibitors of DNA gyr-
ase and do not inhibit bacterial topoisomerase IV.17 We hy-
pothesize that DNA gyrase inhibition by the gyramides stalls
replication fork progression, impairs chromosome segrega-
tion, and initiates the SOS response through a non-canonical
pathway that ultimately blocks cell division.17 A challenge
with this family of compounds has been reducing drug efflux
out of cells, which has limited in vivo experiments to-date
and created a roadblock toward developing antimicrobial
agents that are effective against wild-type cells. Using insight
gleaned from an analysis of successful antibiotics proved
largely ineffective in our design of gyramide analogs.18
In this paper we describe a series of new gyramides with
potent activity against DNA gyrase and that have an apparent
reduction in efflux from different bacterial species. We tested
the compounds for antibiotic activity against 9 strains of
Results and discussion
Discovery and characterization of the antibacterial activity of 3
We previously altered the substituents of the benzyl group in
gyramide A (6)—discovered in a high-throughput screen—to
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create analogs with limited success, including gyramide B (7)
and gyramide C (8).13 In this paper we introduce different
functional groups to the arylsulfonamide ring to change its
size and polarity (Fig. 1B). In lieu of a co-crystal structure to
enable a rational optimization of the compounds, we ex-
panded the initial set of compounds reported previously by
exploring the diversity available within commercially avail-
able aldehydes and sulfonyl chlorides, while avoiding func-
tionality on the two aromatic rings with known metabolic lia-
bilities (e.g., nitro groups). We initially prepared 48 analogs
(structures not shown) and found that 47 had higher MICs
than the parent compounds (6–8) against E. coli strains. The
remaining compound (gyramide D, 3)—containing a 3-cyano
group on the arylsulfonamide ring—was remarkably potent
and killed wild-type E. coli strains (Fig. 1B). Based on this
compound, we performed a thorough and systematic varia-
tion of the two core appendages, i.e. the N-benzyl and
N-arylsulfonyl substituents. Although we initially envisioned a
matrix of four cores containing 50 appendages, the limited
availability of different benzaldehydes, sulfonyl chlorides,
and hetero-aromatic substrates created a hurdle. After scruti-
nizing available building blocks, we settled on an expanded
set of cores: a set of three 3-amino-1-benzyl pyrrolidines
gyramides (E and F; 4 and 5) that we re-synthesized on pre-
parative scale to perform additional experiments (Fig. S2§).
We found that inclusion of a 3-cyano group in 3 signifi-
cantly improved the MIC of the compounds against a wild-
type laboratory strain of Escherichia coli (16 μg mL−1) com-
pared to previously reported analogs 6–8 (activity of >160 μg
mL−1 against wild-type E. coli);14 3 also displayed activity
against an E. coli UPEC strain (16 μg mL−1), Shigella flexneri
(8 μg mL−1), Salmonella enterica subsp. enterica serovar
Typhimurium (S. enterica enterica Typhimurium) (2 μg mL−1),
and Edwardsiella tarda (16 μg mL−1) (Table 1).
3–5 are active against various strains of Gram-negative and
Gram-positive bacteria
The antibacterial activity of 3 against E. coli, S. flexneri, S.
enterica enterica Typhimurium, and E. tarda encouraged us
to explore additional changes to the substituents on the sul-
fonamide ring. We were interested in whether we could in-
crease the activity against E. coli, identify derivatives that
were potent and effective against other Gram-negative bacte-
ria, and discover compounds with a preference for Gram-
negative or Gram-positive bacteria. After introducing a series
of electron-withdrawing groups into the 3-position of the aryl-
sulfonamide ring (not shown), we noticed that a nitrile in-
creased activity and inhibited growth of wild-type E. coli cells.
Additional modifications eventually produced 4 and 5, both
of which have an MIC of 8 μg mL−1 against E. coli BW25113
(Fig. 1C and Table 1).
treated with 31 sulfonyl chlorides and
a set of three
3-sulfonamido pyrrolidines benzylated with 30 substituted
benzaldehydes and heteroaromatic analogs (Fig. S1§); the
combination of these two sets of intermediates yielded a li-
brary consisting of 183 analogs (Fig. 1C). Each gyramide ana-
log in the library was synthesized on >5 mg scale and puri-
fied to >95% purity by HPLC (see ESI§). Screening of these
183 compounds as described below revealed two new
Of the Gram-negative strains we tested, 3–5 were effective
against E. coli, Edwardsiella tarda, S. enterica enterica
Table 1 MICs of 3–5 against Gram-negative and Gram-positive bacterial strains. Italics text highlights the lowest MIC values
Compound (μg mL−1
)
Bacterial strains
3
4
5
Gram-negative bacteria
E. coli BW25113
16
8
8
E. coli JW5503
0.1
8
1
16
>16
16
>16
>16
>16
>16
2
0.4
16
1
>16
>16
8
>16
>16
>16
>16
4
0.1
8
0.5
E. coli MC4100
E. coli BAS849b
E. coli UPECa
>16
>16
8
>16
>16
>16
>16
2
Acinetobacter baumanniia
Edwardsiella tardaa
Enterobacter aerogenesa
Klebsiella pneumoniae
Morganella morganiia
Pseudomonas aeruginosa
Salmonella enterica
Shigella flexneri
8
4
4
Gram-positive bacteria
Bacillus subtilis
Bacillus cereus
>16
>16
>16
>16
>16
>16
>16
>16
>16
8
8
16
8
8
8
8
8
4
>16
>16
>16
>16
>16
>16
>16
>16
>16
Enterococcus faecalis
Enterococcus faecium
Staphylococcus aureusa
Staphylococcus saprophyticusa
Staphylococcus epidermidisa
Streptococcus agalactiae
Streptococcus pyogenes
a
Denotes clinical isolates. b Denotes hyperpermeable strain derivative of MC4100.
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Typhimurium, and S. flexneri with MICs in the range of 2–16
μg mL−1 (Table 1). Of the three gyramide analogs, 4 was the
only compound that was effective against all 9 of the Gram-
positive strains tested (MIC values ranging from 4–16 μg
mL−1), including Bacillus subtilis, Bacillus cereus, Enterococcus
faecium, Staphylococcus aureus subsp. aureus, Staphylococcus
saprophyticus subsp. saprophyticus, Staphylococcus epi-
dermidis, Streptococcus agalactiae, Enterococcus faecalis, and
Streptococcus pyogenes (Table 1).
JW5503 were ∼10–100-fold higher than against E. coli
JW5503. GyrR strains remained susceptible to 1 and 2 and
the MIC values of these compounds were 1.6–3.2 μg mL−1
and 0.8 μg mL−1, respectively, and thus similar to E. coli
JW5503 (Table 2). MIC values of 3–5 against the cipR strains
derived from E. coli JW5503 were 2-4-fold higher than against
E. coli JW5503, and the MIC value of 2 against the cipR
strains was 0.8 μg mL−1 for all of the strains, including E. coli
JW5503 (Table 2). These results demonstrate very minor
cross-resistance of gyrR and cipR E. coli strains to the
gyramides.
Analogs 3–5 reduce gyramide efflux in bacteria
Compounds 3–5 are effective against novR strains in the
presence of 100 μM of the efflux pump inhibitor, phenylala-
nine arginine β-naphthylamide (PAβN).22 We used PAβN be-
cause, unlike the cipR and gyrR strains, the novR strains con-
tain the TolC protein and therefore are capable of pumping
the drug out of the cell.22,23 MIC values of 3–5 against novR
strains were 2–4-fold higher than the MIC values against E.
coli HB101 (the background strain used to make the novR
strains). These results indicate the absence of cross-
resistance between 2 and 3–5 in novR strains. We detected
some examples of cross-resistance to 1 and 2 in novR strains;
1 had MIC values of 13 ng mL−1 and 26 ng mL−1 against the
novR strains CC5 and LE316, respectively, which is 8-fold and
16-fold higher than the MIC observed for the background
strain E. coli HB101 (Table 2). The cross-resistance between 1
and 2 in novR strains is in contrast to a previous report.24
Based on the promising potent antibacterial activity of the
gyramides and the lack of cross-resistance between 3–5 and
the known DNA gyrase inhibitors 1 and 2, we characterized
the activity of these analogs against E. coli DNA gyrase
in vitro.
Drug efflux systems and membrane permeability are two fac-
tors that affect the activity of antimicrobials against Gram-
negative and Gram-positive bacteria.19 To gauge the extent of
drug efflux, we measured the MIC of 3–5 against an E. coli
strain lacking the outer membrane component of the AcrAB-
TolC efflux pump (TolC): strain JW5503 (derived from E. coli
BW25113). We used the relative ratio of the MICs of com-
pounds against E. coli BW25113 and E. coli JW5503 as an in-
dicator of compound efflux, where MICBW25113/MICJW5503 is 1
in the absence of efflux, and high values indicate more efflux.
As a point of reference, 8 [a previously reported analogue
structurally related to 3 (ref. 14)] had the same MIC (0.1 μg
mL−1) as 3 against E. coli strain JW5503, yet its MIC against
E. coli BW25113 was >160 μg mL−1 (a ratio of MICBW25113
/
MICJW5503 > 1600, indicating significant efflux). We found ra-
tios for 3, 4, and 5 of 160, 20, and 80, respectively, indicating
that the AcrAB-TolC efflux pump complex reduces the po-
tency of the gyramides (Table 1), albeit less so than for previ-
ous analogues. Modification of the gyramides to include a cy-
ano group (3 and 5) or a trifluoromethyl group (4) at the
3-position of the benzenesulfonamide ring reduced efflux sig-
nificantly; our data suggests that the trifluoromethyl modifi-
cation has the largest reduction on efflux. To study the role
of membrane permeability on the potency of gyramides in E.
coli, we tested the MIC of 3 against a strain with increased
membrane permeability, E. coli BAS849.20 This strain con-
tains multiple mutations, including changes in lptD that al-
ters outer membrane permeability to small organic mole-
cules; although this mutation does not change inner
membrane structure per se, it increases the concentration of
antibiotics presented at the inner membrane and thereby in-
creases the permeability of lipophilic molecules across this
barrier.20,21 We found that the MIC of 3 against E. coli
BAS849 is 1 μg mL−1 and is 8-fold lower than the MIC against
its parent strain E. coli MC4100 (8 μg mL−1), indicating that
poor membrane permeability also reduces the antibacterial
activity of the gyramides.
Compounds 3–5 inhibit the supercoiling activity of DNA
gyrase in vitro
We evaluated the effect of compounds 3–5 on the DNA super-
coiling activity of DNA gyrase as described previously.14 We
measured the 50% inhibitory concentration (IC50) of com-
pounds using E. coli DNA gyrase and relaxed pUC19 plasmid
as a substrate. IC50 values of 3, 4, and 5 were 52 9.2, 170
48, and 47 19 nM, respectively. The IC50 value of 1 and 2
was 170 63 nM and 144 20 nM, respectively (Fig. 2 and
Fig. S3§). Gyramide IC50s are ∼4-fold lower than the corre-
sponding MICs, suggesting that efflux systems and mem-
brane permeability are the primary barriers currently limiting
the activity of the gyramides.
We attempted to measure IC50 values of ATPase activity of
the tested compounds; however, we were only reliably able to
measure an IC50 for 2 (245 42 nM). We measured the inhi-
bition of ATPase activity of 1, and 3–5 up to a concentration
of 100 μM and did not observe a decrease in ATPase activity,
which is expected for 1;27 compounds 3–5 precipitated at a
concentration >100 μM, which invalidates earlier measure-
ments suggesting that these compounds are ATPase
inhibitors.17
E. coli strains resistant to 1 and 2 are susceptible to 3–5
We evaluated the cross-resistance of gyramide-resistant
(gyrR),14 ciprofloxacin-resistant (cipR), and novobiocin-
resistant (novR) strains of E. coli to 1–5.22 We tested multiple
mutations conferring resistance to 1, 2, or 3–5 (Table 2). MIC
values of 3–5 against the five gyrR strains derived from E. coli
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Table 2 MIC of compounds 1–5 against E. coli and isogenic resistant mutants
Strainsa
1 (ng mL−1
)
2 (μg mL−1
)
3 (μg mL−1
)
4 (μg mL−1
)
5 (μg mL−1
)
Parent strain: E. coli JW5503
gyrR strains
1.6
1.6
1.6
3.2
3.2
3.2
6.4
16
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
0.8
>100
3.2
102
6.4
12.8
0.1
10
>10
10
10
10
0.2
0.4
0.4
0.2
>16
0.1
0.2
0.1
0.2
0.4
11
22
11
22
5.5
0.4
0.8
0.2
1.6
4
0.1
0.4
0.2
0.2
0.1
5.4
22
2.7
22
1.3
0.2
0.2
0.1
0.4
1
0.1
0.2
0.1
0.2
GyrA Pro35Thr
GyrA Ser97Pro
GyrA Phe96Leu
GyrA His45Tyr
GyrB Thr508Met
GyrA Ser83Leu
GyrA Asp87Gly
GyrA Asp87Tyr
GyrB Ser464Phe
cipR strainsb
32
16
Parent strain: E. coli HB101
E. coli HB101 + 100 μM PAβNc
novR strains + 100 μM PAβNc
1.6
1.6
13
3.2
26
CC5 (GyrB Arg136His)
CC7 (GyrB Arg136Ser)
LE316 (GyrB Arg136Val)
a
All the gyrR and cipR mutants are derived from E. coli JW5503 (ref. 14 and this study), while novR mutants are derived from E. coli HB101.22
We isolated the cipR strains from a spontaneous resistant mutant screen and determined that 3 of the 4 mutations in gyrA that conferred
b
resistance to 1 have been confirmed in other studies.25,26 c The MIC determination for novR strains was performed in LB broth supplemented
with 100 μM of the efflux pump inhibitor, phenylalanine arginine β-naphthylamide (PAβN). We used PAβN because these strains contain an in-
tact tolC gene.
Overexpression of GyrA and GyrB does not suppress the
biological activity of gyramides
a multicopy plasmid vector.30 Overexpression of gyrB—and
not gyrA—was sufficient to suppress the inhibitory effect of
2, as cultures of cells harboring pCA24N-gyrB grew at concen-
trations of 2 as high as 51 μg mL−1, which corresponds to a
>10-fold increase in the MIC observed for E. coli JW5503 (Ta-
ble S1§). The result we observed for 2 is consistent with the
model for it binding to DNA gyrase and its inhibition of the
ATP hydrolysis activity of the GyrB subunit.6,7,31
Overexpression of gyrA or gyrB was not sufficient to suppress
the susceptibility of cells to 1 or 3 (Table 3). Although we are
unsure of the DNA gyrase subunit to which the gyramides
bind, we anticipated that overexpression of GyrA would re-
duce the susceptibility of cells to 1.
To obtain more insight into the potency of 3 and the region
of DNA gyrase to which it binds, we performed a target-
multicopy suppression experiment. In this experiment, we
tested whether an abundance of DNA gyrase outcompetes the
toxicity of an inhibitor and its suppression of cell growth.28,29
We used two E. coli JW5503 strains for these experiments,
each containing a gyrA or gyrB operational reading frame on
We hypothesized that the results of the target-multicopy
suppression experiment we observed for 1 and 3 may be due
to the requirement of an excess of the DNA gyrase holoen-
zyme to titrate the effect of the drug. To test whether the
DNA gyrase complex suppressed the biological activity of 1
and 3, we cloned gyrA and gyrB into compatible vectors to si-
multaneously overexpress GyrA and GyrB in E. coli cells. To
Table 3 Target overexpression confers resistance to 2, but not to 1 or 3
Fig. 2 Dose-response curves for DNA gyrase-dependent supercoiling
activity in the presence of 1, 2, and 3–5. We performed DNA super-
coiling reactions with E. coli DNA gyrase, relaxed pUC19 plasmid sub-
strate, and various concentrations of 1, 2 and 3–5. Reactions were run
for 30 min and the DNA was separated on a 0.8% (w/v) agarose gel for
3 h. We labeled the DNA by immersing the gel in a solution of 0.5 mg
mL−1 ethidium bromide for 1 h. The fluorescence intensity of the
supercoiled DNA band was normalized to the DMSO solvent control
and plotted for each concentration of compound tested. See the Ma-
terial and methods section for experimental details. Each reaction was
performed in triplicate. Error bars indicate the standard deviation.
Overexpression
Compound
(gyrA/gyrB)
(−/−)
Induction
1 (ng mL−1
)
2 (μg mL−1
)
3 (μg mL−1
)
+
—
2.1
2.1
2.1
2.1
2.1
2.1
2.1
2.1
0.4
0.4
0.8
0.8
6.3
0.8
3.2
0.4
0.2
0.2
0.1
0.2
0.1
0.2
0.1
0.2
(+/−)
(−/+)
(+/+)
+
—
+
—
+
—
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confirm the overexpression of GyrA and GyrB and the forma-
tion of the functional DNA gyrase holoenzyme, we performed
controls using E. coli mutant strains containing temperature
sensitive GyrA or GyrB (Fig. S4§). Simultaneous
overexpression of gyrA and gyrB did not suppress the biologi-
cal activity of 1 or 3. The results for 1 are different from re-
cent studies in which overexpression of DNA gyrase increased
the susceptibility of E. coli cells to 1 from 9 ng mL−1 to 2 ng
mL−1,32 which is attributed to a drug-bound DNA gyrase cre-
ating additional DNA damage rather than conferring drug re-
sistance. Our experiments using a single concentration of
IPTG for GyrA and GyrB induction from different plasmids
and a different nutrient media—from that reported in32—
yielded an MIC of 2 ng mL−1. Small differences in MICs may
be due to differences between the two studies: e.g., growth
conditions, strains, and methods for performing measure-
ments. Our results similarly show that overexpression of DNA
gyrase subunits in cells did not reduce their susceptibility to
the gyramides toxicity. If the gyramides inhibit DNA gyrase
partway through its catalytic cycle as demonstrated for 1,4,32
overexpression of DNA gyrase subunits may exacerbate cellu-
lar toxicity.
that the extended α4-helix domain of S. aureus GyrA is par-
tially responsible for the increased resistance of S. aureus gyr-
ase to quinolones compared to E. coli gyrase, and the higher
concentration of potassium glutamate in the cytoplasm of S.
aureus modulates this effect.35 The differences in the activity
that we observed for the gyramide analogs against different
organisms could similarly connect back to the DNA-gyrase-
gyramide ternary complex, the intracellular environment in
which these compounds are active, or a combination of these
factors.
Many antibiotics are transported out of Gram-negative or-
ganisms by a bacterial efflux transporter in the resistance-
nodulation-division (RND) protein family that spans the cell
envelope;36,37 specifically the AcrAB-TolC tripartite pump is
found in E. coli, Salmonella spp., and Shigella spp. and con-
tributes to multidrug resistance.37–39 Our observation that
3–5 are most active against strains of the Gram-negative bac-
teria E. coli, S. enterica, and S. flexneri—and very potent
against strains harboring the ΔtolC mutation—supports the
hypothesis that the cyano and trifluoromethyl modifications
in the 3-position of the benzenesulfonamide ring reduce drug
efflux against this family of drug pumps.
Five efflux drug pump families [ATP binding cassette
(ABC), major facilitator superfamily (MFS), multidrug and
toxin extrusion (MATE), RND, and small multidrug resistance
(SMR)] pump antibiotics out of Gram-positive cells.40 Surpris-
ingly, 4—and not 3 and 5—is also active against Gram-
positive bacteria, suggesting that the trifluoromethyl modifi-
cation to the benzenesulfonamide ring may play a unique
role in reducing its specificity for pumping by these five ef-
flux protein families. Lacking a definitive set of molecular de-
sign rules for drug reducing efflux by bacteria, and taking
into consideration that many clinical antibiotics contain an
arylfluoro group (e.g., the entire family of fluoroquinolones),
replacing this group with a trifluoromethyl moiety may re-
duce efflux further, expand the activity of drugs to Gram-
positive organisms, contribute to understanding bacterial ef-
flux mechanisms, and contribute to antibiotic development.
Importantly, the gyramides are still effective against E. coli
strains resistant to the known gyrase inhibitors 1 and 2, indi-
cating no cross-resistance when bacteria with altered suscep-
tibility to 1 or 2 are treated with compounds 3–5. Our results
imply that the mutations that confer resistance to 1 and 2 oc-
cur in a region of DNA gyrase that does not affect the binding
of gyramides to DNA gyrase. Further structural biology data
can provide insight into the region of DNA gyrase to which
the gyramides bind. We observed that two of the novR mu-
tants confer resistance to 1, which may arise from
uncharacterized mutations.
Conclusions
DNA gyrase inhibitors are one of the most successful classes
of clinical antibiotics currently used.33 Nevertheless, the suc-
cess of these therapeutic agents has been compromised by
the development of drug tolerance and resistance among
clinical strains of bacteria.34 Here we report the characteriza-
tion of three new analogs of the gyramides, a family of syn-
thetic small molecules that inhibit bacterial DNA gyrase.
Compounds 3 and 5 both contain a 3-cyano group on the
benzenesulfonamide ring (and are identical with the excep-
tion of a 6-methyl group on the benzenesulfonamide ring in
5), display narrow spectrum activity against only Gram-
negative bacteria, and are only active against ∼30% of the
Gram-negative strains that we tested. The effectiveness of 4
against both Gram-negative and Gram-positive bacteria is
likely due to the inclusion of the trifluoromethyl group at the
3-position on the benzenesulfonamide ring. It is tempting to
point out that 4 also differs from 3 and 5 by incorporation of
an arylisopropylether at the other end of the molecule (com-
pared to a phenyl group in 3 and 5), however this difference
has no obvious effect on gyramide efflux. We support this hy-
pothesis with the observation that gyramide A (6) is very simi-
lar in structure to 4 with the exception of a 3-fluoro group (6)
compared to a 3-trifluoromethyl group (4), and yet the com-
pounds have extremely different efflux profiles from a com-
parison of their MICs against E. coli BW25113 and JW5503. It
may also be possible that 4 also targets DNA gyrase from dif-
ferent organisms effectively.
Compounds 3 and 5 are more potent inhibitors of the
in vitro DNA supercoiling activity of E. coli DNA gyrase than
1, 2, 4, and the gyramide analogs that we reported previ-
ously.14 Importantly, we previously demonstrated that the
gyramides do not affect the activity of other type II
topoisomerases.17 When testing the inhibition of the ATPase
activity of DNA gyrase, we observed 3–5 precipitate at a
Although we do not yet understand the structural and bio-
chemical basis for the difference in the sensitivity of gyrase
from different organisms (i.e., Gram-negative versus Gram-
positive bacteria), previous studies of quinolones suggested
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concentration >100 μM and therefore obfuscate absorbance
measurement, which previously led us to the incorrect con-
clusion that 6 competitively inhibited the ATPase activity of
DNA gyrase at millimolar concentrations;17 it does not. We
do not currently have an explanation for the reduction in the
activity of 4 (3-trifluoromethyl modification) against E. coli
DNA gyrase compared to 3 and 5 (3-cyano modification).
Our investigation of the inhibition of ATPase activity and
overexpression studies of different gyrase subunits suggests
that 3 does not compete directly for the ATP binding site. Un-
like 2, 3 did not inhibit the ATPase activity of DNA gyrase
and overexpression of GyrB did not promote resistance to 3.
Overexpression of both GyrA and GyrB in the same cell did
not affect the antibacterial activity of either 1 or 3. However,
it has been recently demonstrated that simultaneous
overexpression of GyrA and GyrB exacerbates the antibacterial
activity of 1. This effect could be caused by the covalent at-
tachment of 1 to the GyrA-DNA bound state as the amount of
GyrA-DNA bound complexes increases, thus blocking key cel-
lular processes.32 Compound 3 may also create DNA gyrase-
DNA bound complexes that are toxic to cells and
overexpression may not reduce susceptibility to the com-
pounds. We have previously shown the mutations that confer
resistance to gyramides do not map to the same region as
mutations that confer resistance to quinolones.14
was accomplished with UV light or acidic ceric ammonium
molybdate (CAM) followed by heating.
1H NMR spectra and proton-decoupled 13C NMR spectra
were obtained on a 400 MHz Bruker NMR spectrometer.
Chemical shifts (δ) are reported in parts per million (ppm)
relative to residual solvent. Multiplicities are given as: s (sin-
glet), d (doublet), t (triplet), q (quartet), dd (doublet of dou-
blets), td (triplet of doublet), ddd (doublet of doublet of dou-
blets), m (multiplet), br s (broad singlet) and sep (septet).
For AMM analysis, samples were analyzed by flow-
injection analysis into
a Thermo Fisher Scientific LTQ
Orbitrap XL (San Jose, CA) operated in the centroided mode.
Samples were injected into a mixture of 50% MeOH and
0.1% formic acid/H2O at a flow of 200 μl min−1. Source pa-
rameters were 5 kV spray voltage, capillary temperature of
275 °C and sheath gas setting of 20. Spectral data were ac-
quired at a resolution setting of 100 000 FWHM with the
lockmass feature, which typically results in a mass accuracy
<2 ppm.
tert-Butyl (R)-(1-([1,1′-biphenyl]-4-ylmethyl)pyrrolidin-3-yl)-
carbamate (15). To a solution of 4-phenylbenzaldehyde (323
mg, 1.1 equiv.) and tert-butyl (R)-pyrrolidin-3-ylcarbamate (300
mg,
1 equiv.) in anhydrous THF (16 mL) was added
NaBHIJOAc)3 (854 mg, 2.5 equiv.) at ambient temperature. Af-
ter stirring for 24 hours, the solvent was removed in vacuo,
and the remaining material was partitioned between 1 M HCl
and EtOAc. The aqueous portion was quickly neutralized with
saturated aqueous NaHCO3, then extracted with EtOAc. The
combined organic portions were dried over Na2SO4 and con-
centrated in vacuo. The crude product mixture was purified
by flash chromatography (95 : 5 to 90 : 10 CH2Cl2 : CH3OH, Rf
= 0.36 in 90 : 10 CH2Cl2 : CH3OH) to produce the title com-
pound as a white amorphous solid (535 mg, 94%). 1H NMR
(400 MHz, CD3OD) δ 7.67–7.57 (m, 4H), 7.50 (d, J = 8.0 Hz,
2H), 7.43 (t, J = 7.7 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 4.19 (td, J
= 7.6, 3.9 Hz, 1H), 3.23 (dd, J = 11.1, 7.3 Hz, 1H), 3.19–3.06
(m, 1H), 3.06–2.92 (m, 1H), 2.87 (dd, J = 11.5, 4.8 Hz, 1H),
2.31 (dtd, J = 14.3, 8.2, 6.3 Hz, 1H), 1.96 (s, 2H), 1.83 (dq, J =
13.4, 6.9 Hz, 1H), 1.43 (s, 9H). Carbamate NH not observed.
13C NMR (101 MHz, CD3OD) δ 157.8, 142.9, 141.6, 134.2,
131.5, 129.9128.6, 128.4, 128.0, 80.5, 60.1, 59.9, 53.6, 50.4,
Our previous and current data support the hypothesis that
the gyramides may bind to a new site on DNA gyrase and op-
erate through a new mechanism of inhibiting DNA replica-
tion and, consequently, bacterial cell growth. We hypothesize
that gyramides inhibit DNA gyrase during its catalytic cycle
resulting in stalled DNA-bound DNA gyrase and are currently
working on the structural biology of the gyramide/DNA gyrase
complex to test this hypothesis.
Experimental section
General synthetic chemistry methods and compound
characterization
We synthesized a library of 183 gyramide analogs using a pre-
viously reported strategy13 and varying the aldehyde and sul-
fonyl chloride building blocks (Fig. S1§); all 183 analogs that
we tested were >95% pure. After determining activities
against bacteria in minimum inhibitory concentration (MIC)
experiments, we resynthesized the most potent analogs (com-
pounds 3–5) and characterized them exhaustively using
HPLC, NMR, and MS (ESI§).
Unless otherwise specified, all commercially available re-
agents were used as received. All reactions using dried sol-
vents were carried out under an atmosphere of argon in
flame-dried glassware with magnetic stirring. Purification of
reaction products was carried out by flash chromatography
using SiliCycle Reagent silica gel F60 (230–400 mesh) and Dy-
namic Absorbents, Inc. Reagent silica gel. Analytical thin
layer chromatography was performed on Dynamic Absorbant
Inc. Reagent 0.25 mm silica gel F-254 plates. Visualization
+
31.6, 28.7. AMM (ESI) calculated for C22H29N2O2 (M + H)+
353.2224, found 353.2228.
(R)-1-([1,1′-Biphenyl]-4-ylmethyl)pyrrolidin-3-amine
(14).
To a solution of 15 (120 mg) in CH3OH (3.7 mL) was added
dropwise 4 M HCl in dioxane (3.7 mL). After 17 hours, the
mixture was concentrated in vacuo to produce the title com-
pound as the hydrochloride salt. The material was used with-
out further purification.
(R)-N-(1-([1,1′-Biphenyl]-4-ylmethyl)pyrrolidin-3-yl)-3-cyano-
benzenesulfonamide (3). To a solution of 14 (assumed to be
monohydrochloride salt, 65 mg,
1
equiv.) and
3-cyanobenzenesulfonyl chloride (52 mg, 1.15 equiv.) in
anhydrous CH2Cl2 (1.75 mL), Et3N (95 μL, 3 equiv.) was
added. After stirring for 21 hours, the mixture was diluted
with CH2Cl2 (3 mL) and washed with H2O (7 mL). The
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+
aqueous portion was made basic with saturated aqueous
NaHCO3, then extracted with CH2Cl2. The combined organic
portions were dried over Na2SO4 and concentrated in vacuo.
The crude product mixture was purified by flash chromatog-
raphy (100 : 0 to 93 : 7 CH2Cl2 : CH3OH, Rf = 0.36 in 93 : 7
CH2Cl2 : CH3OH) to produce the title compound as a slightly
yellow amorphous solid (64 mg, 68%). 1H NMR (400 MHz,
CD3OD) δ 8.18 (t, J = 1.8 Hz, 1H), 8.10 (dt, J = 8.0, 1.5 Hz,
1H), 7.94–7.88 (m, 1H), 7.71 (t, J = 7.9 Hz, 1H), 7.64–7.58 (m,
2H), 7.58–7.53 (m, 2H), 7.43 (dd, J = 8.4, 6.9 Hz, 2H), 7.36–
7.29 (m, 3H), 3.82 (ddt, J = 9.2, 7.0, 5.3 Hz, 1H), 3.63 (d, J =
12.7 Hz, 1H), 3.55 (d, J = 12.6 Hz, 1H), 2.63 (td, J = 9.1, 8.3,
6.4 Hz, 2H), 2.52 (ddd, J = 9.4, 8.0, 6.0 Hz, 1H), 2.26 (dd, J =
10.0, 5.5 Hz, 1H), 2.18–1.98 (m, 1H), 1.56 (ddt, J = 13.7, 8.5,
5.7 Hz, 1H). Sulfonamide NH not observed. 13C NMR (101
MHz, CD3OD) δ 144.5, 142.1, 141.6, 138.2, 136.9, 132.2,
131.61, 131.59, 130.7, 129.9, 128.3, 127.92, 127.90, 118.4,
114.6, 60.9, 60.6, 53.41, 53.36, 32.6. AMM (ESI) calculated for
C24H24N3O2S+ (M + H)+ 418.1584, found 418.1583.
32.8, 28.4, 6.2. AMM (ESI) calculated for C19H29N2O3 (M +
H)+ 333.2173, found 333.2174.
(R)-1-(4-Cyclopropoxybenzyl)pyrrolidin-3-amine (13). To a
solution of 17 (70 mg, 1 equiv.) in CH3OH (2.1 mL), 4 M HCl
in dioxane (2.1 mL) was added. After stirring for 1 hour, the
mixture was concentrated in vacuo. The crude product mix-
ture was used without further purification.
2-Methyl-5-(trifluoromethyl)benzenesulfonyl chloride (18).
To chlorosulfonic acid (1 mL) cooled to
0
°C,
4-methylbenzotrifluoride (160 mg) was added dropwise. The
mixture was warmed to ambient temperature, then stirred for
2 hours. The mixture was then carefully added dropwise to
15 g ice, then neutralized with solid NaHCO3. After allowing
the ice to melt, the aqueous slurry was extracted with CHCl3.
The combined organic portions were dried over Na2SO4 and
concentrated in vacuo. The crude product mixture was puri-
fied by flash chromatography (100 : 0 to 50 : 50 hexanes :
CH2Cl2, Rf = 0.28 in 90 : 10 hexanes: CH2Cl2) to produce the ti-
tle compound as a clear oil (115 mg, 44%). 1H NMR (400
MHz, CDCl3) δ 8.33 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.59 (d, J
= 8.0 Hz, 1H), 2.87 (s, 3H). 1H NMR matched previously
reported values.
4-Cyclopropoxybenzaldehyde (16). In a sealed microwave
vial, 4-hydroxybenzaldehyde (120 mg,
1 equiv.), bromo-
cyclopropane (315 μL, 4 equiv.) and Cs2CO3 (960 mg, 3 equiv.)
were combined in anhydrous DMF (1.6 mL). The mixture was
heated using microwave irradiation to 200 °C for 1 hour. The
resulting mixture was diluted with EtOAc, filtered, and con-
centrated in vacuo. The crude product mixture was purified
by flash chromatography (100 : 0 to 80 : 20 hexanes : EtOAc, Rf
= 0.51 in 80 : 20 hexanes : EtOAc) to produce the title com-
pound as a clear oil (145 mg, 91%). A minimal inseparable
impurity remained, but did not negatively impact further re-
(R)-N-(1-(4-Cyclopropoxybenzyl)pyrrolidin-3-yl)-2-methyl-5-
(trifluoromethyl)benzenesulfonamide (4). To a solution of 13
(57 mg, 1 equiv., assumed to be monohydrochloride) and 18
(71 mg, 1.3 equiv.) in anhydrous CH2Cl2 (1.6 mL), Et3N (90
μL, 3 equiv.) was added. After stirring at ambient tempera-
ture for 14 hours, the mixture was diluted with CH2Cl2 and
washed with 50 : 50 H2O : NaHCO3 (saturated aq.). The or-
ganic portion was extracted with CH2Cl2, and the combined
organics were dried over Na2SO4 and concentrated in vacuo.
The crude product mixture was purified by flash chromatog-
raphy (100 : 0 to 98 : 2 CH2Cl2 : CH3OH, Rf = 0.56 in 92 :
8 CH2Cl2 : CH3OH) to produce the title compound as a pale
yellow oil (77 mg, 81%). 1H NMR (400 MHz, CD3OD) δ 8.14
(d, J = 1.9 Hz, 1H), 7.81–7.74 (m, 1H), 7.55 (d, J = 8.0 Hz, 1H),
7.14 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 8.6 Hz, 2H), 3.81–3.69 (m,
2H), 3.51 (d, J = 12.5 Hz, 1H), 3.44 (d, J = 12.5 Hz, 1H), 2.68
(s, 3H), 2.58 (ddd, J = 13.5, 9.5, 6.6 Hz, 2H), 2.48 (ddd, J = 9.5,
8.0, 6.0 Hz, 1H), 2.20 (dd, J = 10.0, 5.7 Hz, 1H), 2.06 (dddd, J
= 13.5, 9.2, 8.0, 5.9 Hz, 1H), 1.60 (ddt, J = 13.9, 8.6, 5.7 Hz,
1H), 0.85–0.72 (m, 2H), 0.72–0.61 (m, 2H). Sulfonamide NH
not observed. 13C NMR (101 MHz, CD3OD) δ 159.8, 143.2,
141.7, 134.8, 131.3, 130.1, 126.8, 123.7, 115.8, 115.6, 79.5,
60.6, 60.3, 53.18, 53.15, 51.7, 32.4, 20.4, 6.6. AMM (ESI) calcu-
lated for C22H26F3N2O3S+ (M + H)+ 455.1611, found 455.1610.
(R)-N-(1-([1,1′-Biphenyl]-4-ylmethyl)pyrrolidin-3-yl)-5-cyano-
2-methylbenzenesulfonamide (5). To a solution of 14 (400
mg, 1.2 equiv.) in anhydrous CH2Cl2 (11 mL), Et3N (485 mL,
equiv.) was added. Then, 5-cyano-2-methylbenzenesulfonyl
chloride (250 mg, 1 equiv.) was added. The resulting mixture
was stirred for 3 hours at ambient temperature. The mixture
was diluted with CH2Cl2 and washed with H2O. The aqueous
portion was back-extracted with CH2Cl2. The combined or-
ganic portions were dried over Na2SO4 and concentrated in
vacuo. The crude product mixture was purified by flash
1
actions. H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 7.84 (d, J =
8.7 Hz, 2H), 7.16 (d, J = 8.7 Hz, 2H), 3.82 (tt, J = 6.1, 3.2 Hz,
1H), 0.90–0.78 (m, 4H). 13C NMR (101 MHz, CDCl3) δ 190.9,
164.2, 131.8, 130.3, 115.4, 51.4, 6.3. AMM (ESI) calculated for
C10H11O2+ (M + H)+ 163.0754, found 163.0751.
tert-Butyl
(R)-(1-(4-cyclopropoxybenzyl)pyrrolidin-3-yl)-
carbamate (17). To a solution of 16 (52 mg, 1.05 equiv.),
tert-butyl (R)-pyrrolidin-3-ylcarbamate (57 mg, 1 equiv.), and
NaOAc (25 mg, 1 equiv.) in anhydrous THF (3 mL), AcOH (20
μL, 1.15 equiv.) was added. After stirring at ambient tempera-
ture for 1 hour, NaBHIJOAc)3 (160 mg, 2.5 equiv.) was added.
The mixture was stirred at ambient temperature for 22 hours,
then concentrated in vacuo. The remaining solids were
partitioned between EtOAc and 50 : 50 H2O : NaHCO3 (sat.
aq.). The aqueous portion was extracted with EtOAc, and the
combined organics were dried over Na2SO4 and concentrated
in vacuo. The crude product mixture was purified by flash
chromatography (100 : 0 to 93 : 7 CH2Cl2 : CH3OH, Rf = 0.29 in
93 : 7 CH2Cl2 : CH3OH) to produce the title compound as a
1
clear oil (84 mg, 84%). H NMR (400 MHz, CDCl3) δ 7.20 (d, J
= 8.6 Hz, 2H), 6.99 (d, J = 8.6 Hz, 2H), 4.84 (s, 1H), 4.24–4.07
(m, 1H), 3.79–3.66 (m, 1H), 3.53 (s, 2H), 2.77 (d, J = 11.7 Hz,
1H), 2.59 (dd, J = 9.8, 6.4 Hz, 1H), 2.50 (d, J = 9.8 Hz, 1H),
2.40–2.18 (m, 2H), 1.43 (s, 10H), 0.84–0.69 (m, 4H). Carba-
mate NH not observed. 13C NMR (101 MHz, CDCl3) δ 158.0,
155.4, 131.2, 129.8, 114.7, 79.2, 60.8, 59.5, 52.5, 50.7, 49.8,
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chromatography (100 : 0 to 99.5 : 0.5 CH2Cl2 : CH3OH, Rf = 0.23
12 A. Bredberg, M. Brant and M. Jaszyk, Antimicrob. Agents
Chemother., 1991, 35, 448–450.
in 97 : 3 CH2Cl2 : CH3OH) to produce the title compound as a
1
slightly yellow amorphous solid (324 mg, 54%). H NMR (400
13 S. Mukherjee, C. A. Robinson, A. G. Howe, T. Mazor, P. A.
Wood, S. Urgaonkar, A. M. Hebert, D. Raychaudhuri and
J. T. Shaw, Bioorg. Med. Chem. Lett., 2007, 17, 6651–6655.
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Lett., 2011, 2, 289–292.
15 M. Edwards, R. H. Flatman, L. A. Mitchenall, C. E. M.
Stevenson, T. B. K. Le, T. A. Clarke, A. R. McKay, H.-P.
Fiedler, M. J. Buttner, D. M. Lawson and A. Maxwell, Science,
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MHz, CD3OD) δ 8.21 (d, J = 1.8 Hz, 1H), 7.77 (dd, J = 7.9, 1.8
Hz, 1H), 7.64–7.60 (m, 2H), 7.56 (d, J = 8.2 Hz, 2H), 7.49 (d, J
= 7.9 Hz, 1H), 7.43 (dd, J = 8.5, 7.0 Hz, 2H), 7.37–7.29 (m,
3H), 3.79 (ddt, J = 9.2, 6.9, 5.3 Hz, 1H), 3.64 (d, J = 12.6 Hz,
1H), 3.53 (d, J = 12.6 Hz, 1H), 2.71–2.62 (overlapping s and
m, 4H), 2.59–2.52 (m, 1H), 2.52–2.46 (m, 1H), 2.24 (dd, J =
10.0, 5.4 Hz, 1H), 2.10 (dtd, J = 13.6, 8.5, 5.7 Hz, 1H), 1.63
(ddt, J = 13.7, 8.6, 5.9 Hz, 1H). Sulfonamide NH not observed.
13C NMR (101 MHz, CD3OD) δ 144.3, 142.2, 142.1, 141.6,
138.3, 136.7, 134.9, 133.6, 130.7, 129.9, 128.4, 127.93, 127.90,
118.6, 111.5, 60.7, 60.6, 53.4, 53.3, 32.5, 20.7. AMM (ESI) cal-
culated for C25H26N3O2S+ (M + H)+ 432.1740, found 432.1739.
16 M. T. Black, T. Stachyra, D. Platel, A. Girard, M. Claudon, J.
Bruneau and C. Miossec, Antimicrob. Agents Chemother.,
2008, 52, 3339–3349.
17 M. Rajendram, K. A. Hurley, M. H. Foss, K. M. Thornton,
J. T. Moore, J. T. Shaw and D. B. Weibel, ACS Chem. Biol.,
2014, 9, 1312–1319.
Acknowledgements
We acknowledge materials from Dr. Tony Maxwell (E. coli
HB101, E. coli CC5, E. coli CC7, and E. coli LE316) and Dr.
Kenneth Kreuzer (E. coli KNK453).
18 R. O'Shea and H. E. Moser, J. Med. Chem., 2008, 51,
2871–2878.
19 H. Nikaido, Science, 1994, 264, 382–388.
20 B. A. Sampson, R. Misra and S. A. Benson, Genetics,
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22 A. Contreras and A. Maxwell, Mol. Microbiol., 1992, 6,
1617–1624.
This research was supported by the NIH (through award
1DP2OD008735 to D. B. W.) and the Human Frontiers Sci-
ence Program (through award RGY0076/2013 to D. B. W.).
The research described was partially funded by Venture Cata-
lyst in the UC Davis Office of Research through a Science
Translation and Innovative Research (STAIR™) Grant. M. R.
F. and Z. W. Y. thank the Arnold and Mabel Beckman
Foundation.
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