Derivatives of Ribosome-Inhibiting Antibiotic Chloramphenicol Inhibit the Biosynthesis of Bacterial Cell Wall

Article abstract of DOI:10.1021/acsinfecdis.8b00078

Here, we describe the preparation and evaluation of α,β-unsaturated carbonyl derivatives of the bacterial translation inhibiting antibiotic chloramphenicol (CAM). Compared to the parent antibiotic, two compounds containing α,β-unsaturated ketones (1 and 4) displayed a broader spectrum of activity against a panel of Gram-positive pathogens with a minimum inhibitory concentration range of 2-32 μg/mL. Interestingly, unlike the parent CAM, these compounds do not inhibit bacterial translation. Microscopic evidence and metabolic labeling of a cell wall peptidoglycan suggested that compounds 1 and 4 caused extensive damage to the envelope of Staphylococcus aureus cells by inhibition of the early stage of cell wall peptidoglycan biosynthesis. Unlike the effect of membrane-disrupting antimicrobial cationic amphiphiles, these compounds did not rapidly permeabilize the bacterial membrane. Like the parent antibiotic CAM, compounds 1 and 4 had a bacteriostatic effect on S. aureus. Both compounds 1 and 4 were cytotoxic to immortalized nucleated mammalian cells; however, neither caused measurable membrane damage to mammalian red blood cells. These data suggest that the reported CAM-derived antimicrobial agents offer a new molecular scaffold for development of novel bacterial cell wall biosynthesis inhibiting antibiotics.

Full text of DOI:10.1021/acsinfecdis.8b00078

Article
pubs.acs.org/journal/aidcbc
Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX
Derivatives of Ribosome-Inhibiting Antibiotic Chloramphenicol
Inhibit the Biosynthesis of Bacterial Cell Wall
Sivan Louzoun Zada,† Keith D. Green,‡ Sanjib K. Shrestha,‡ Ido M. Herzog,†
Sylvie Garneau-Tsodikova,*,‡ and Micha Fridman*,†
†Raymond and Beverly Sackler Faculty of Exact Sciences, School of Chemistry, Tel Aviv University, Tel Aviv, 6997801, Israel
‡Department of Pharmaceutical Sciences, University of Kentucky, Lexington, Kentucky 40536-0596, United States
S
* Supporting Information
ABSTRACT: Here, we describe the preparation and evalua-
tion of α,β-unsaturated carbonyl derivatives of the bacterial
translation inhibiting antibiotic chloramphenicol (CAM).
Compared to the parent antibiotic, two compounds containing
α,β-unsaturated ketones (1 and 4) displayed a broader
spectrum of activity against a panel of Gram-positive
pathogens with a minimum inhibitory concentration range of
2−32 μg/mL. Interestingly, unlike the parent CAM, these
compounds do not inhibit bacterial translation. Microscopic
evidence and metabolic labeling of a cell wall peptidoglycan
suggested that compounds 1 and 4 caused extensive damage to
the envelope of Staphylococcus aureus cells by inhibition of the
early stage of cell wall peptidoglycan biosynthesis. Unlike the
effect of membrane-disrupting antimicrobial cationic amphiphiles, these compounds did not rapidly permeabilize the bacterial
membrane. Like the parent antibiotic CAM, compounds 1 and 4 had a bacteriostatic effect on S. aureus. Both compounds 1 and 4
were cytotoxic to immortalized nucleated mammalian cells; however, neither caused measurable membrane damage to
mammalian red blood cells. These data suggest that the reported CAM-derived antimicrobial agents offer a new molecular
scaffold for development of novel bacterial cell wall biosynthesis inhibiting antibiotics.
KEYWORDS: antibiotics, bacterial resistance, chloramphenicol, cell envelope, in vitro translation
onsisting of the three ribosomal ribonucleic acids
(rRNAs), 16S, 23S, and 5S, and over 50 proteins, the
enzymatic modifications including O-acetylation by acetyl-
transferases or, in some cases, O-phosphorylation by CAM
phosphotransferases.13−15 Resistance to CAM is also acquired
by mutations or modifications of nucleotides in the 23S rRNA,
through decreased cell permeability, and through expression of
multidrug efflux proteins that reduce its intracellular concen-
tration.8 In addition to emergence of resistance, the potent
antibacterial activity of CAM is overshadowed by its reversible
bone marrow depressing effect that can, in severe cases, lead to
lethal aplastic anemia and is therefore a serious obstacle that
limits its use in cases of systemic infections.16
The search for phenicols with improved therapeutic
properties has so far yielded two clinically useful CAM
derivatives. TAM (Figure 1B), also known as thiophenicol, is
a p-methylsulfonylphenyl derivative of CAM that possesses a
similar spectrum of activity.17,18 To address the emerging
resistance to both CAM and TAM, conversion of the primary
alcohol of TAM to the corresponding fluorine functionality
resulted in florfenicol (Figure 1B), a semisynthetic phenicol
that is particularly effective against CAM-resistant bacteria and
C
bacterial ribosome, which carries out the translation process,
contains the target sites for many clinically used antibiotics.1−5
One such class of antibiotics is the phenicols, which includes
the natural product chloramphenicol (CAM) and its two
semisynthetic derivatives thiamphenicol (TAM) and florfenicol
(Figure 1).6
CAM was discovered through its isolation from Streptomyces
venezuelae and Streptomyces phleochromogenes var. chloromyceti-
cus in 1947.7 Notably, CAM is one of the few natural products
discovered to date that contains both a nitrophenyl ring and a
halogen functional group.8 CAM inhibits translation by binding
at the peptidyltransferase center of the 50S subunit of the
bacterial ribosome in a position that overlaps with the
aminoacyl moiety of the A-site tRNA.9,10 An X-ray crystal
structure of CAM bound to the 50S subunit of the bacterial
ribosome revealed that its primary and secondary alcohols as
well as the carbonyl of its dichloroacetamide functionality form
hydrogen bonds with nucleotides of the 23S rRNA (Figure
1A).11,12
As is the fate of all antibiotics, the percentage of bacteria that
have evolved resistance to CAM is on the rise. The most
common mechanism of resistance is deactivation through
Received: March 22, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
that compound 1 lacks the primary and secondary alcohols of
CAM that serve as the target for enzymatic inactivation by
acetyl- and phosphotransferases. Since the essential roles of the
primary and secondary alcohols of CAM in facilitating binding
to the peptidyltransferase center of the bacterial ribosome was
unraveled decades after the discovery of 1, we questioned its
function as an inhibitor of bacterial translation.11,12 Herein, we
report a structure−activity relationship (SAR) and a mode of
action study of this CAM derivative.
RESULTS AND DISCUSSION
■
Synthesis. We first developed a robust synthetic route for
the generation of enone and enal analogues of compound 1 for
SAR studies. The enone and enal derivatives were generated
from commercially available CAM and TAM (compounds 1a
and 2a, respectively, Scheme 1A) or from the unnatural CAM
derivatives 3a−5a. Compounds 3a−5a were prepared via
acylation of commercially available (1R,2R)-(−)-2-amino-1-(4-
nitrophenyl)-1,3-propanediol (also known as CAM base). The
enone derivatives 1−5 were synthesized in two steps from
starting materials 1a−5a. Selective esterification of the primary
alcohol of 1a−5a afforded compounds 1b−5b with a primary
alcohol protected by a pivaloyl ester. Dess-Martin oxidation of
the secondary alcohol of 1b−5b (35−100% isolated yields)
followed by chromatography on silica gel, which catalyzed the
elimination of the O-pivaloyl ester, gave the desired enone
derivatives 1−5 in 81−100% isolated yields (Scheme 1A).
Enal derivatives 6−9 were prepared from CAM (1a), TAM
(1b), and their analogues 3a−4a in a four-step sequence
(Scheme 1B). The primary alcohols of compounds 1a−4a were
selectively protected with a tert-butyldimethylsilyl (TBDMS)
group to afford compounds 6a−9a in 34−100% isolated yields.
The secondary alcohol of compounds 6a−9a was protected
with a methoxymethyl (MOM) group to afford compounds
Figure 1. (A) Structure of CAM and its interactions with 23S rRNA
nucleotides. (B) Structures of synthetic members of the phenicol class
of antibiotics and of the antibacterial CAM derivative 1.
is used solely in veterinary medicine.19,20 Not surprisingly,
evolution of resistance did not skip florfenicol, and of the
known CAM resistance genes, a limited number also confer
resistance to florfenicol.21,22
Among the numerous CAM analogues generated to date that
have not reached the clinic, our attention was drawn to the
unique structure of the CAM derivative α-dichloroacetamido-p-
nitroacrylophenone (compound 1, Figure 1B) that was
reported in 1969 by Kono et al.23,24 This CAM derivative
displayed potent antibacterial activity against a CAM-resistant
Staphylococcus aureus. This activity was attributed to the fact
Scheme 1. Synthesis of CAM-Derived Enone and Enal Analogues
B
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
a
Table 1. Minimal Inhibitory Concentration (MIC) Values in μg/mL
b
bacterial strain
compound
1
2
3
4
5
6
7
8
9
10
CAM
TAM
Gram-positive
A
B
C
D
E
8
8
8
4
4
4
2
8
8
8
8
64
32
32
32
8
8
8
4
4
4
4
8
8
8
8
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
64
64
4
>64
>64
>64
32
16
8
64
16
16
16
64
16
>64
64
4
F
32
16
4
G
H
I
32
16
>64
>64
>64
>64
64
64
64
16
8
8
32
16
>64
>64
>64
64
>64
>64
>64
64
16
8
32
32
8
J
64
32
8
16
2
K
L
16
32
2
32
8
64
64
32
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
8
M
N
O
P
32
16
2
4
16
64
32
16
>64
4
>64
8
32
>64
>64
64
64
16
Gram-negative
>64
64
>64
32
>64
>64
>64
>64
>64
>64
>64
4
>64
32
32
>64
64
>64
>64
Q
R
S
4
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
>64
4
T
U
V
>64
8
>64
a
MIC values were determined using the double-dilution method. The MIC for each compound was determined in triplicate in two independent sets
b
of experiments. MIC values were determined against the following strains. Gram-positive: A = Staphylococcus aureus ATCC 33592; B = S. aureus
ATCC BAA-43; C = S. aureus ATCC 33591; D = S. aureus ATCC 43300; E = S. aureus USA600; F = S. aureus G1; G = S. aureus C2; H = S. aureus
ATCC 29213; I = S. aureus Cowan; J = Listeria monocytogenes ATCC 19115; K = Streptococcus pyogenes ATCC 14289; L = Enterococcus faecalis
ATCC 29212; M = Bacillus megaterium ATCC 14945; N = Enterococcus feacalis ATCC 51299; O = Enterococcus faecium ATCC 19434. Gram-
negative: P = Escherichia coli ATCC 25922; Q = E. coli NR698; R = Salmonella enterica ATCC 14028; S = Pseudomonas aeruginosa ATCC 27853; T =
Klebsiella pneumoniae ATCC 27736; U = Acinetobacter baumannii ATCC 19606; V = Enterobacter cloacae ATCC 13047.
6b−9b in 50−100% isolated yields, followed by removal of the
TBDMS group to afford compounds 6c−9c (83−100% isolated
yields). Using a strategy similar to that developed for the
generation of the enone derivatives 1−5, Dess-Martin oxidation
of the primary alcohol of 6c−9c followed by elimination of the
resulting oxidation product during flash chromatography on
silica gel afforded the desired enal derivatives 6−9 in 58−100%
isolated yields.
resistant Gram-positive pathogens for which new antibiotics are
needed. To investigate the antimicrobial activity of compounds
1−10 against Gram-negative bacteria, we chose a panel of seven
representative bacterial pathogens (P−V). To exclude the
possibility of poor anti-Gram-negative activity due to
permeability limitations, activity was evaluated against the
Escherichia coli (E. coli) NR698 (LptD4213 mutant, strain Q),
which has an outer membrane with increased permeability.26
The minimum inhibitory concentration (MIC) values of the
parent CAM and TAM and their derivatives 1−10 are
presented in Table 1. Of the tested enones 1−5, compounds
1 and 4 exhibited antibacterial activity against all Gram-positive
strains in the panel with MIC values in the range of 2−32 μg/
mL. Notably, these two compounds were potent against Gram-
positive strains B, C, and N that were highly tolerant to the
parents CAM and TAM (MICs ≥ 64 μg/mL). All of the tested
Gram-negative strains, including LptD4213 mutant E. coli strain
that has a more permeable outer membrane, were tolerant to all
derivatives, including compounds 1 and 4 (MICs ≥ 64 μg/
mL). These data suggest that limited outer membrane
permeability is unlikely to account for the lack of anti-Gram-
negative activity of compounds 1 and 4 and that it is possible
that the target(s) of these antibacterial agents may exist only in
Gram-positive bacteria or differ in structure from the
corresponding target(s) in Gram-negative bacteria.
Finally, enal 10 was generated in three steps from CAM base
(Scheme 1C). Selective removal of the acetyl ester from the
primary hydroxyl of the di-O-acetyl-N-phthalimide CAM
derivative 10a under acidic conditions afforded the mono-
acetylated compound 10b in 50% isolated yield. Dess-Martin
oxidation of 10b followed by elimination of the resulting
oxidation product during flash chromatography on silica gel
afforded enal 10 in 66% isolated yield. The structures of all final
1
compounds were confirmed and characterized by H and 13C
NMR (Figures S1−S20) as well as high-resolution mass
spectrometry.
Antibacterial Activity. The antibacterial activity of
compounds 1−10 against a panel of 22 bacterial strains was
evaluated using the broth double-dilution method.25 As control
antibiotics, we tested the phenicols CAM and TAM from which
compounds 1−10 were derived. The chosen panel of Gram-
positive pathogens was composed of 15 bacterial strains (A−
O). Of nine strains of S. aureus (strains A−I), seven (strains A−
G) are methicillin-resistant S. aureus (MRSA). Notably, the
panel of Gram-positive bacteria included vancomycin-resistant
E. faecalis and E. faecium (strains N and O, respectively), drug-
Analysis of the antibacterial activity experiments provided
several interesting SAR insights. None of the tested enals 6−10
displayed antibacterial activity. More specifically, unlike enones
1 and 4, which displayed the most potent antibacterial activity,
C
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
the corresponding enals 6 and 9, respectively, were inactive,
indicating that the enone structure is necessary for antibacterial
activity. Enones 1 and 2 differ structurally solely by the
substitution on their phenyl ring (p-nitro and p-methylsulfonyl,
respectively); however, whereas 1 displayed the highest
antibacterial potency against the tested panel of bacteria, 2
exhibited modest to poor antibacterial activity. These data
indicate that the para-positioned nitro group contributes
significantly to the interactions of compounds 1 and 4 with
their target(s). Enones 1 and 3 differ structurally only by the
substitution on the amide moiety (dichloroacetamide and
acetamide, respectively). The antimicrobial activity of 3 was
modest compared to that of 1 indicating that the dichlor-
oacetamide group is important for the antibacterial activity of
compound 1. Nevertheless, enones 4 and 1 that differ solely by
their amide substitution (p-nitrobenzamide and dichloroaceta-
mide, respectively) displayed a similar antibacterial activity
potency and spectrum, suggesting that there is some tolerance
to structural changes at the amide substituent.
with compounds 1, 4, or CAM. These results suggest that,
similarly to CAM and as opposed to the rapidly acting
membrane disrupting cation amphiphile S-14, compounds 1
and 4 exert a bacteriostatic antibacterial effect even at 2-fold
over their MIC.
In Vitro Resistance Selection Experiment. Bacteria
evolve resistance to antimicrobial agents, and it is desirable
that resistance does not evolve rapidly. To study the potential
of compounds 1 and 4 to induce resistance compared to CAM,
we exposed S. aureus ATCC 29213 (strain H) to subinhibitory
concentrations of these agents during 12 successive sub-
cultures.31 Briefly, the tested bacterium was exposed to
subinhibitory concentrations (0.5× MIC) of compounds 1, 4,
and CAM. The ratios of the measured MIC values for each of
the 12 passages were then determined. Data are presented in
Figure 3.
Time-Kill Kinetic Test. To investigate whether compounds
1 and 4 exert a bactericidal or bacteriostatic effect, we studied
growth kinetics in an assay with S. aureus ATCC 29213 (strain
H, a quality control strain). Briefly, bacterial cultures were
treated with CAM, compound 1, compound 4, and the cationic
amphiphile 6′-S-tetradecyl tobramycin (S-14)27−30 at concen-
trations 2-fold higher than their MICs. S-14 is known to be
bactericidal and served as a control. The culture was incubated
at 35 °C. After 0, 1, 2, 4, 6, and 24 h, 20 μL of the suspension
was serially diluted and inoculated on agar plates. After 24 h of
incubation at 35 °C, the numbers of viable bacteria colonies
were counted, and the results are summarized in Figure 2.
Figure 3. Comparative study on the emergence of resistance in
S. aureus ATCC 29213 (strain H) after 12 serial passages in the
presence of compound 1, compound 4, and CAM. Relative (Δ) MIC
is the normalized ratio of MIC obtained for a given subculture to MIC
obtained upon first exposure.
The bacteria developed resistance to the parent antibiotic
CAM: After 12 passages, the MIC value for CAM was 16-fold
higher than it was after the first passage. In contrast, this
bacterium showed low propensity to develop resistance to
compounds 1 and 4 with no increase in MIC value for 4 and
only a 2-fold increase for 1 after 12 passages. These results
suggest that compounds 1 and 4 are less prone than CAM to
induce the evolution of resistance in S. aureus.
Inhibition of Bacterial Translation. To study the mode of
action of the CAM-derived enones, we first tested the
possibility that, like the parent phenicol CAM, these
compounds act by binding to the bacterial ribosome, thereby
inhibiting bacterial translation. We evaluated the effect of CAM
as well as that of enones 1 and 4 on translation in commercially
available cell-free extracts from E. coli32 (Table 2 and Figure
S21). Enones 1 and 4 were more than 2 orders of magnitude
less potent than the parent CAM as inhibitors of in vitro
prokaryotic translation. These results suggest that, whereas the
parent antibiotic CAM inhibits bacterial translation, neither
compound 1 nor 4 does so efficiently. Thus, these CAM
derivatives likely exert their antibacterial activity via a different
mode of action.
Figure 2. Time-kill kinetics of S. aureus ATCC 29213 (strain H) with
compound 1, compound 4, CAM, and S-14 at 2× MIC. After 0, 1, 2, 4,
6, and 24 h, an aliquot (20 μL) was diluted (102-, 103-, 104-, and 105-
fold) into saline and 10 μL of each dilution was plated in duplicate on
Mueller-Hinton agar plates. After 24 h (starting from each tested time
point) of incubation at 35 °C, the number of colonies on each plate
was counted. Time-kill assays were analyzed by determining the
reductions in viable count (CFU/mL).
No viable bacteria were detected in the sample treated with
the membrane disrupting antimicrobial cationic amphiphile S-
14 after 2 h. In contrast, no significant changes in numbers of
viable colonies of compounds 1 and 4 and of the parent
antibiotic CAM were observed over the first 6 h of incubation.
Moreover, less than 1 order of magnitude decreases were
observed in number of viable colonies after 24 h of incubation
D
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
dramatic reduction in the inhibition of translation compared to
the parent CAM, evidence from the TEM images suggest that,
unlike the parent antibiotic CAM, compounds 1 and 4 exert
their antibacterial activity through perturbation of the bacterial
cell envelope biosynthesis or assembly.
Table 2. Prokaryotic in Vitro Translation 50% Inhibitory
Concentration (IC50) Values
a
compound
IC50 (μg/mL)
1
168 57
86 10
4
Evaluation of Bacterial Cell Membrane Perturbation
Effects. Unlike membrane disrupting antibiotics such as
lipopeptides34 and several classes of synthetic antimicrobial
cationic amphiphiles derived from amino sugars that have been
reported to date,35−37 compounds 1 and 4 are not chemically
defined as cationic amphiphiles. Nevertheless, the extensive
deformation that these compounds caused to the tested Gram-
positive cells after a short 1 h incubation at concentrations 2-
fold higher than their respective MIC values suggested that they
may act by perturbation of the bacterial membrane integrity.
To investigate the possible effects of compounds 1 and 4 on
bacterial membrane integrity, we performed a propidium iodide
(PI) test with S. aureus ATCC 29213 (strain H) treated with
these CAM derivatives.38 Cells with damaged membranes
become permeable to PI and are stained with this red
fluorescent dye. For the PI cell permability experiments,
bacterial cells were incubated with either compound 1 or 4 at
MIC for 1 h, after which the samples were stained with PI and
visualized by fluorescence microscopy (Figure 5). As controls,
we tested the effects of CAM, S-14, and oxacillin on bacterial
cell permeability.
CAM
0.80 0.09
a
Inhibition of luciferase translation was quantified in a coupled
transcription/translation assay using E. coli S30 extracts. Experiments
were performed in duplicate.
Transmission Electron Microscopy-Based Study of
Effects on Bacterial Cell Shape. A large percentage of the
antibiotics in clinical use, including CAM, target the bacterial
ribosome. Since in vitro translation experiments suggested that
neither compound 1 nor 4 likely target the bacterial ribosome,
we next explored the possibility that they may affect bacterial
cell envelope assembly. The complex multilayered structure
bacterial cell envelope and the biosynthetic pathways involved
in its biogenesis are targets for numerous antibiotics, including
β-lactams, glycopeptides, and lipopeptides. Transmission
electron microscopy (TEM) allows visualization of morpho-
logical changes of the membrane and cell wall ultrastructure
under native conditions that are indicative of perturbation of
cell envelope biosynthesis or assembly. We obtained TEM
images of S. aureus ATCC 29213 (strain H) cells that were
untreated or treated with either compound 1 or 4 (Figures 4
Untreated and CAM-treated S. aureus ATCC 29213 cells
(strain H) were not stained with PI under the experimental
conditions (Figure 5A−C,D−F, respectively). This result can
be rationalized by the fact that CAM, which inhibits bacterial
protein synthesis, has no direct effect on the permeability of the
bacterial cell within the incubation period used here. In
contrast, as expected, almost all of the bacteria in the sample
that was treated with the membrane disrupting antimicrobial
cationic amphiphile S-14 were stained with PI (Figure 5G−I).
Compared to the untreated bacteria, no significant increase in
PI stained cells was observed in the sample treated with the cell
wall biosynthesis inhibitor oxacillin (Figure 5J−L), indicating
that the inhibition of cell wall biosynthesis did not cause an
increase in the bacterial membrane permeability under the
experiment conditions. Like oxacillin, neither compound 1 nor
4 significantly increased the permeability of the bacterial cells to
PI (Figure 5M−O,P−R, respectively). Taking into account the
observed bacteriostatic effect of compounds 1 and 4 on
S. aureus ATCC 29213 cells, the results of the PI assay further
support that these compounds do not directly interfere with the
bacterial membrane integrity and likely cause the cell shape
damage observed in the TEM images through perturbation of
bacterial cell wall biosynthesis.
Figure 4. TEM images of S. aureus ATCC 29213 (strain H) incubated
for 1 h at 37 °C with compounds at 2× MIC. (A) Untreated cells. (B)
Cells treated with CAM. (C) Cells treated with S-14. (D) Cells treated
with oxacillin. (E) Cells treated with compound 1. (F) Cells treated
with compound 4.
and S22). As positive controls, cells were treated with the
membrane-disrupting antimicrobial cationic amphiphile S-14
and oxacillin, a β-lactam that interferes with the synthesis of
peptidoglycan.33
Inhibition of Peptidoglycan Biosynthesis. To test if the
bacterial cell wall is targeted by compounds 1 and 4, we
investigated whether or not these compounds alter peptido-
glycan biosynthesis. It was previously demonstrated that
metabolic incorporation of R-2-amino-3-azidopropanoic acid,
an azido-D-alanine derivative, followed by attachment of a
fluorescent dye using a copper-free click reaction enables in vivo
visualization of peptidoglycans. This method was successfully
implemented for visualization of the effects of different cell wall
biosynthesis inhibiting antibiotics.39 Treatment of bacteria with
fosfomycin, a drug that inhibits one of the early stages of
peptidoglycan biosynthesis, completely abrogates the incorpo-
ration of the azido-D-alanine derivative. In contrast, penicillin,
which inhibits transpeptidation of the peptidoglycan chains in
The cell envelopes of untreated cells and CAM-treated cells
were intact (Figure 4A,B, respectively). This observation is in
agreement with the fact that the antimicrobial activity of CAM
results from its bacterial translation inhibiting activity and not
from direct perturbation of the bacterial cell wall or cell
membrane assembly. As expected, the bacterial cells treated
with the membrane disrupting cationic amphiphile S-14 (Figure
4C) appeared damaged with extensive ruptures. Finally,
significant deformations and decomposition were evident in
the cell envelope of bacteria treated with the peptidoglycan
transpeptidase inhibitor oxacillin (Figure 4D). Interestingly,
unlike the parent antibiotic CAM, the surfaces of cells treated
with CAM-derived compounds 1 and 4 were deformed and
ruptured (Figure 4E,F, respectively). Taken together with the
E
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
Figure 5. Bright field (top), fluorescence images of PI (537/26 nm excitation and 607/36 nm emission, red; middle), and merged images (bottom)
of cultures of S. aureus ATCC 29213 (strain H) incubated for 1 h at 37 °C and 1× MIC. (A−C) Untreated cells. (D−F) Cells treated with CAM.
(G−I) Cells treated with S-14. (J−L) Cells treated with oxacillin. (M−O) Cells treated with compound 1. (P−R) Cells treated with compound 4.
Figure 6. Bright field (top), fluorescence images of TAMRA-DBCO (537/26 nm excitation and 607/36 nm emission, magenta; middle), and
merged images (bottom) of cultures of S. aureus ATCC 29213 (strain H) incubated for 1 h at 37 °C without or with compounds at 2× their MIC
values. (A−C) Untreated cells. (D−F) Cells treated only with azido-D-alanine. (G−I) Cells treated with CAM. (J−L) Cells treated with S-14. (M−
O) Cells treated with oxacillin. (P−R) Cells treated with compound 1. (S−U) Cells treated with compound 4.
the later stage of cell wall biosynthesis, has a modest effect on
the incorporation of azido-D-alanine, presumably since the
incorporation occurs during the early stage of the peptidogly-
can biosynthesis.
On the basis of this labeling method, we evaluated the
incorporation of azido-D-alanine into the cell wall of S. aureus
ATCC 29213 (strain H). We compared the incorporation in
untreated bacteria to cultures treated with oxacillin, the
membrane disrupting cationic amphiphile S-14, CAM,
compound 1, and compound 4 (Figure 6). No fluorescent
staining was observed for bacteria that were not preincubated
with azido-D-alanine and then incubated with TAMRA-DBCO
dye ruling out the possibility of nonspecific staining of
untreated bacteria (Figure 6A−C). In contrast, bacteria that
were preincubated with azido-D-alanine and untreated with an
antibacterial agent were effectively stained by the fluorescent
dye (Figure 6D−F). Similar to the untreated bacteria that were
preincubated with azido-D-alanine, the samples that were
treated with CAM (Figure 6G−I) were labeled. This was
expected since this antibiotic inhibits translation and does not
directly affect the cell wall biosynthesis process. In cultures
treated with the membrane disrupting cationic amphiphile S-14,
all of the cells were fluorescently labeled (Figure 6J−L). Most
of the cells treated with oxacillin were labeled by the fluorescent
dye presumably because this antibiotic inhibits a late stage of
the peptidoglycan biosynthesis and therefore does not prevent
F
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
the incorporation of azido-D-alanine. Interestingly, in samples
treated with either compound 1 or 4 (Figure 6P−R and S−U,
respectively) most of the cells were unlabeled by the fluorescent
dye. These results support our hypothesis that, unlike the
parent antibiotic CAM, compounds 1 and 4 perturb the
integrity of the bacterial cell wall by inhibiting early stages of
peptidoglycan biosynthesis.
Mammalian Cell Plasma Membrane and Cytotoxic
Effects. The observed bacterial cell deformation effects of
CAM-derived 1 and 4 raised the question of whether these
compounds might also disrupt mammalian cell membranes. We
therefore evaluated the effect of 1 and 4 on membranes of rat
red blood cells using a hemolysis experiment (Figure 7), a
Figure 8. Mammalian cell toxicity of CAM as well as compounds 1, 3,
and 4 against (A) A549 cell line and (B) BEAS-2B cell line. Note:
DMSO (1.25% final concentration) and Triton X were used as
negative and positive controls, respectively.
Figure 7. Dose-dependent rat erythrocytes hemolysis of compounds 1
and 4. CAM and S-14 were used as negative and positive control,
respectively.
antibacterial agents are toxic to mammalian cells, but that
their toxicity does not result from direct perturbation of
mammalian cell membrane as is evident from the results of the
hemolysis assay.
commonly used assay to evaluate mammalian plasma
membrane damage.27 Notably, up to a concentration of 128
μg/mL, which is 4- to 32-fold higher than the MIC values of
these compounds against the tested panel of Gram-positive
bacteria, no to very limited hemolysis was measured.
CONCLUSIONS
■
We developed a short synthetic route for the generation of a
unique type of derivative of the bacterial translation inhibiting
antibiotic CAM. Two of the derivatives, compounds 1 and 4,
which contain an enone group, displayed broad spectrum anti-
Gram-positive activity including that against strains with high
tolerance to the parent antibiotic CAM. Interestingly, through
bacterial in vitro translation experiments, we showed that, unlike
the parent antibiotic CAM, compounds 1 and 4 are poor
inhibitors of translation and therefore exert their antibacterial
activity via a different mechanism. Like CAM, compounds 1
and 4 exerted a bacteriostatic effect on S. aureus cells.
Propidium iodide-based cell permability experiments indicated
that compounds 1 and 4 did not rapidly increase the membrane
permeability of bacterial cells further indicative of the
bacteriostatic nature of these antibacterial agents. TEM images
of bacteria that were pretreated with compounds 1 and 4
showed that they caused extensive damage to the bacterial cell
envelope. Fluorescent labeling of S. aureus cells that were
preincubated with azido-D-alanine, which is metabolically
incorporated into the cell wall peptidoglycan, indicated that
compounds 1 and 4 likely exert their antibacterial activity by
inhibiting the early stage of cell wall biosynthesis. As there is an
alarming increase in resistance to the currently available
repertoire of cell wall biosynthesis inhibiting antibiotics
including β-lactams, glycopeptides, and fosfomycin, the
reported CAM-derived antibacterial enones offer a novel
Since compounds 1 and 4 contain an electrophilic α,β-
unsaturated carbonyl that can pose an inherent risk of
nonspecific reactions with numerous nucleophilic residues in
mammalian cells,40 we evaluated the potential mammalian cell
toxicity41 of these antibacterials. We used compound 3, which
displayed less potent antibacterial activity (Table 1), and CAM
for comparison. Two nucleated immortalized mammalian cells
lines were evaluated: the human lung carcinoma epithelial cells
A549 and the normal human bronchial epithelial cells BEAS-
2B. The cells were incubated with the test compounds for 24 h,
after which cell viability was evaluated using a colorimetric assay
with resazurin (Figure 8). The parent antibiotic CAM did not
have any measurable effect on the tested mammalian cell lines
up to 32 μg/mL. However, incubation of the cells with the
antibacterial CAM-derived 1 and 4 at a low concentration (2
μg/mL) resulted in significant reduction in cell viability. The
toxicity of compounds 1 and 4 to mammalian cell lines at the
same concentration range as that needed for inhibition of
bacterial growth limits their therapeutic value. Therefore,
chemical modification of the CAM-derived α,β-unsaturated
carbonyl scaffold of these compounds is necessary to reduce
toxicity and develop novel and clinically useful cell wall
targeting antibiotics. Similar data were observed with the less
active compound 3. These results indicate that these
G
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
(5) Watkins, D., Norris, F. A., Kumar, S., and Arya, D. P. (2013) A
fluorescence-based screen for ribosome binding antibiotics. Anal.
Biochem. 434 (2), 300−307.
molecular scaffold for development of new cell wall targeting
antibiotics.
(6) Dowling, P. M. (2013) Chloramphenicol, thiamphenicol, and
florfenicol. In Antimicrobial therapy in veterinary medicine, pp 269−277,
John Wiley & Sons, Inc., Hoboken, NJ, DOI 10.1002/
9781118675014.ch16.
(7) Ehrlich, J., Bartz, Q. R., Smith, R. M., Joslyn, D. A., and
Burkholder, P. R. (1947) Chloromycetin, a new antibiotic from a soil
actinomycete. Science 106 (2757), 417.
(8) Schwarz, S., Kehrenberg, C., Doublet, B., and Cloeckaert, A.
(2004) Molecular basis of bacterial resistance to chloramphenicol and
florfenicol. FEMS Microbiol. Rev. 28 (5), 519−542.
(9) Bulkley, D., Innis, C. A., Blaha, G., and Steitz, T. A. (2010)
Revisiting the structures of several antibiotics bound to the bacterial
ribosome. Proc. Natl. Acad. Sci. U. S. A. 107 (40), 17158−17163.
(10) Wilson, D. N. (2014) Ribosome-targeting antibiotics and
mechanisms of bacterial resistance. Nat. Rev. Microbiol. 12 (1), 35−48.
(11) Dunkle, J. A., Xiong, L., Mankin, A. S., and Cate, J. H. (2010)
Structures of the Escherichia coli ribosome with antibiotics bound near
the peptidyl transferase center explain spectra of drug action. Proc.
Natl. Acad. Sci. U. S. A. 107 (40), 17152−17157.
(12) Schlunzen, F., Zarivach, R., Harms, J., Bashan, A., Tocilj, A.,
Albrecht, R., Yonath, A., and Franceschi, F. (2001) Structural basis for
the interaction of antibiotics with the peptidyl transferase centre in
eubacteria. Nature 413 (6858), 814−821.
(13) Murray, I. A., and Shaw, W. V. (1997) O-Acetyltransferases for
chloramphenicol and other natural products. Antimicrob. Agents
Chemother. 41 (1), 1−6.
(14) Shaw, W. V., and Leslie, A. G. (1991) Chloramphenicol
acetyltransferase. Annu. Rev. Biophys. Biophys. Chem. 20, 363−386.
(15) Izard, T., and Ellis, J. (2000) The crystal structures of
chloramphenicol phosphotransferase reveal a novel inactivation
mechanism. EMBO J. 19 (11), 2690−2700.
(16) Turton, J. A., Andrews, C. M., Havard, A. C., and Williams, T. C.
(2002) Studies on the haemotoxicity of chloramphenicol succinate in
the Dunkin Hartley guinea pig. Int. J. Exp. Pathol. 83 (5), 225−238.
(17) Cutler, R. A., Stenger, R. J., and Suter, C. M. (1952) New
antibacterial agents. 2-Acylamino-1-(4-hydrocarbonylsulfonylphenyl)-
1,3-propanediols and related compounds. J. Am. Chem. Soc. 74 (21),
5475−5481.
(18) Suter, C. M., Schalit, S., and Cutler, R. A. (1953) New
antibacterial agents. II. An alternate synthesis of D,L-threo-2-dichloro-
acetamido-1-(4-methylsulfonylphenyl)-1,3-propanediol 1. J. Am. Chem.
Soc. 75 (17), 4330−4333.
(19) Neu, H. C., and Fu, K. P. (1980) In vitro activity of
chloramphenicol and thiamphenicol analogs. Antimicrob. Agents
Chemother. 18 (2), 311−316.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsinfec-
dis.8b00078.
■
S
Detailed synthetic procedures, 1H and 13C NMR
assignments for all of the intermediates and final
compounds 1−10, MS data, H and 13C NMR spectra
1
of compounds 1−10 (Figures S1−S20), detailed
biochemical and biological experimental procedures, a
figure with IC50 values (Figure S21), and a figure with
TEM images (Figure S22) (PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: sylviegtsodikova@uky.edu (S.G.-T.).
*E-mail: mfridman@post.tau.ac.il (M.F.).
ORCID
Sylvie Garneau-Tsodikova: 0000-0002-7961-5555
Micha Fridman: 0000-0002-2009-7490
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a BSF grant 2012007 (to S.G.-T.
and M.F.), by a National Institutes of Health (NIH) grant
AI090048 (to S.G.-T.), by the Israel Science Foundation Grant
6/14 (to M.F.), by the Israel Ministry of Science Technology &
Space, Grant 48966 (to M.F.), by the Israel Ministry of Science
Technology & Space, Scholarship 3-13550 (to S.L.Z.), and by
startup funds from the College of Pharmacy at the University of
Kentucky (to S.G.-T.). We thank Mr. Raphael I. Benhamou and
Mr. Kfir B. Steinbuch for their help with PI and hemolysis
experiments. We thank Dr. Vered Holdengreber for her
professional assistance in obtaining transmission electron
microscopy images. We thank Prof. Daniel E. Kahne (Harvard
University, USA) for the generous gift of the E. coli NR698
strain used for determination of MIC values.
(20) Syriopoulou, V. P., Harding, A. L., Goldmann, D. A., and Smith,
A. L. (1981) In vitro antibacterial activity of fluorinated analogs of
chloramphenicol and thiamphenicol. Antimicrob. Agents Chemother. 19
(2), 294−297.
ABBREVIATIONS
■
CAM, chloramphenicol; E. coli, Escherichia coli; MIC, minimum
inhibitory concentration; MOM, methoxymethyl; PI, propi-
dium iodide; rRNA, ribosomal ribonucleic acid; SAR,
structure−activity relationship; S. aureus, Staphylococcus aureus;
TAM, thiamphenicol; TEM, transmission electron microscopy;
TBDMS, tert-butyldimethylsilyl
(21) Kehrenberg, C., and Schwarz, S. (2004) fexA, a novel
Staphylococcus lentus gene encoding resistance to florfenicol and
chloramphenicol. Antimicrob. Agents Chemother. 48 (2), 615−618.
(22) Doublet, B., Schwarz, S., Nussbeck, E., Baucheron, S., Martel, J.
L., Chaslus-Dancla, E., and Cloeckaert, A. (2002) Molecular analysis of
chromosomally florfenicol-resistant Escherichia coli isolates from
France and Germany. J. Antimicrob. Chemother. 49 (1), 49−54.
(23) Kono, M., O'hara, K., Honda, M., and Mitsuhashi, S. (1969)
Drug resistance of staphylococci. XI. Induction of chloramphenicol
resistance by its derivatives and analogues. J. Antibiot. 22 (12), 603−
607.
(24) Kono, M., O’Hara, K., Nagawa, M., and Mitsuhashi, S. (1972)
Antibacterial activity of chloramphenicol-related compounds toward a
chloramphenicol-resistant strains of Staphylococcus aureus. Jpn. J.
Microbiol. 16 (6), 461−467.
(25) Berkov-Zrihen, Y., Green, K. D., Labby, K. J., Feldman, M.,
Garneau-Tsodikova, S., and Fridman, M. (2013) Synthesis and
REFERENCES
■
(1) Poehlsgaard, J., and Douthwaite, S. (2005) The bacterial
ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3 (11),
870−881.
(2) Yonath, A. (2005) Antibiotics targeting ribosomes: resistance,
selectivity, synergism and cellular regulation. Annu. Rev. Biochem. 74,
649−679.
(3) Sutcliffe, J. A. (2011) Antibiotics in development targeting
protein synthesis. Ann. N. Y. Acad. Sci. 1241, 122−152.
(4) Tenson, T., and Mankin, A. (2006) Antibiotics and the ribosome.
Mol. Microbiol. 59 (6), 1664−1677.
H
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
evaluation of hetero- and homodimers of ribosome-targeting anti-
biotics: antimicrobial activity, in vitro inhibition of translation, and drug
resistance. J. Med. Chem. 56 (13), 5613−5625.
(26) Chimalakonda, G., Ruiz, N., Chng, S. S., Garner, R. A., Kahne,
D., and Silhavy, T. J. (2011) Lipoprotein LptE is required for the
assembly of LptD by the beta-barrel assembly machine in the outer
membrane of Escherichia coli. Proc. Natl. Acad. Sci. U. S. A. 108 (6),
2492−2497.
(27) Herzog, I. M., Green, K. D., Berkov-Zrihen, Y., Feldman, M.,
Vidavski, R. R., Eldar-Boock, A., Satchi-Fainaro, R., Eldar, A., Garneau-
Tsodikova, S., and Fridman, M. (2012) 6″-Thioether tobramycin
analogues: Towards selective targeting of bacterial membranes. Angew.
Chem., Int. Ed. 51 (23), 5652−5656.
(28) Fosso, M. Y., Zhu, H., Green, K. D., Garneau-Tsodikova, S., and
Fredrick, K. (2015) Tobramycin variants with enhanced ribosome-
targeting activity. ChemBioChem 16 (11), 1565−1570.
(29) Shrestha, S. K., Fosso, M. Y., Green, K. D., and Garneau-
Tsodikova, S. (2015) Amphiphilic tobramycin analogues as anti-
bacterial and antifungal agents. Antimicrob. Agents Chemother. 59 (8),
4861−4869.
(30) Herzog, I. M., Feldman, M., Eldar-Boock, A., Satchi-Fainaro, R.,
and Fridman, M. (2013) Design of membrane targeting tobramycin-
based cationic amphiphiles with reduced hemolytic activity.
MedChemComm 4 (1), 120−124.
(31) Pokrovskaya, V., Belakhov, V., Hainrichson, M., Yaron, S., and
Baasov, T. (2009) Design, synthesis, and evaluation of novel
fluoroquinolone-aminoglycoside hybrid antibiotics. J. Med. Chem. 52
(8), 2243−2254.
(32) Herzog, I. M., Louzoun Zada, S., and Fridman, M. (2016)
Effects of 5-O-ribosylation of aminoglycosides on antimicrobial activity
and selective perturbation of bacterial translation. J. Med. Chem. 59
(17), 8008−8018.
(33) Kong, K. F., Schneper, L., and Mathee, K. (2010) Beta-lactam
antibiotics: from antibiosis to resistance and bacteriology. APMIS 118
(1), 1−36.
(34) D’Costa, V. M., Mukhtar, T. A., Patel, T., Koteva, K.,
Waglechner, N., Hughes, D. W., Wright, G. D., and De Pascale, G.
(2012) Inactivation of the lipopeptide antibiotic daptomycin by
hydrolytic mechanisms. Antimicrob. Agents Chemother. 56 (2), 757−
764.
(35) Bera, S., Dhondikubeer, R., Findlay, B., Zhanel, G. G., and
Schweizer, F. (2012) Synthesis and antibacterial activities of
amphiphilic neomycin B-based bilipid conjugates and fluorinated
neomycin B-based lipids. Molecules 17 (8), 9129−9141.
(36) Guchhait, G., Altieri, A., Gorityala, B., Yang, X., Findlay, B.,
Zhanel, G. G., Mookherjee, N., and Schweizer, F. (2015) Amphiphilic
tobramycins with immunomodulatory properties. Angew. Chem., Int.
Ed. 54 (21), 6278−6282.
(37) Herzog, I. M., and Fridman, M. (2014) Design and synthesis of
membrane-targeting antibiotics: from peptides- to aminosugar-based
antimicrobial cationic amphiphiles. MedChemComm 5 (8), 1014−
1026.
(38) Salta, J., Benhamou, R. I., Herzog, I. M., and Fridman, M.
(2017) Tuning the effects of bacterial membrane permeability through
photo-isomerization of antimicrobial cationic amphiphiles. Chem. - Eur.
J. 23 (52), 12724−12728.
(39) Siegrist, M. S., Whiteside, S., Jewett, J. C., Aditham, A., Cava, F.,
and Bertozzi, C. R. (2013) (D)-Amino acid chemical reporters reveal
peptidoglycan dynamics of an intracellular pathogen. ACS Chem. Biol.
8 (3), 500−505.
(40) Amslinger, S. (2010) The tunable functionality of alpha,beta-
unsaturated carbonyl compounds enables their differential application
in biological systems. ChemMedChem 5 (3), 351−356.
(41) Shrestha, S. K., Garzan, A., and Garneau-Tsodikova, S. (2017)
Novel alkylated azoles as potent antifungals. Eur. J. Med. Chem. 133,
309−318.
I
DOI: 10.1021/acsinfecdis.8b00078
ACS Infect. Dis. XXXX, XXX, XXX−XXX