Chemical Modifications Reduce Auditory Cell Damage Induced by Aminoglycoside Antibiotics

Article abstract of DOI:10.1021/jacs.9b12420

Although aminoglycoside antibiotics are effective against Gram-negative infections, these drugs often cause irreversible hearing damage. Binding to the decoding site of the eukaryotic ribosomes appears to result in ototoxicity, but there is evidence that

Full text of DOI:10.1021/jacs.9b12420

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Article
Chemical Modifications Reduce Auditory Cell
Damage Induced by Aminoglycoside Antibiotics
Sivan Louzoun Zada, Bar Ben Baruch, Luba Simhaev, Hamutal Engel, and Micha Fridman
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12420 • Publication Date (Web): 20 Jan 2020
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Chemical Modifications Reduce Auditory Cell Damage Induced by
Aminoglycoside Antibiotics
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Sivan Louzoun Zada,a Bar Ben Baruch,a Luba Simhaev,b Hamutal Engel,b
and Micha Fridmana,*.
a School of Chemistry, Raymond and Beverley Sackler Faculty of Exact Sciences, Tel Aviv University, Tel Aviv, Israel,
6997801.
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b Blavatnik Center for Drug Discovery, Tel Aviv University, Tel Aviv, 6997801, Israel.
ABSTRACT: Although aminoglycoside antibiotics are effective against Gram-negative infections, these drugs often cause
irreversible hearing damage. Binding to the decoding site of eukaryotic ribosomes appears to result in ototoxicity, but there
is evidence that other effects are involved. Here we show how chemical modifications of apramycin and geneticin,
considered amongst the least and most toxic aminoglycosides, respectively, reduce auditory cell damage. Using molecular
dynamics simulations, we studied how modified aminoglycosides influence the essential freedom of movement of the
decoding site of the ribosome, the region targeted by aminoglycosides. By determining the ratio of a protein translated in
mitochondria to that of a protein translated in the cytoplasm, we showed that aminoglycosides can paradoxically elevate
rather than reduce protein levels. We showed that certain aminoglycosides induce rapid plasma membrane
permeabilization and that this non-ribosomal effect can also be reduced through chemical modifications. The results
presented suggest a new paradigm for the development of safer aminoglycoside antibiotics.
INTRODUCTION
dizziness. Some AGs are more toxic to the cochlea, the
inner ear part responsible for hearing, whereas others are
more toxic to the vestibular apparatus, responsible for
balance.9,10 Ototoxicity is dose dependent, and certain
patients are genetically more susceptible than others.11–15
The severity of AG-induced ototoxicity was analyzed in a
study of 153 adult cystic fibrosis patients who were treated
with AGs: About 42% suffered mild ototoxic side effects,
and about 9% had ototoxicity that ranged from moderate
to severe.16 In cystic fibrosis patients, life-threatening
infections and lack of suitable alternative antibiotics often
make it necessary to continue treatment with AG
antibiotics despite their toxicity.
For almost eight decades, aminoglycosides (AGs) have
been clinically useful antibiotics.1 First discovered in 1943,
the AG streptomycin is still used today in treatment of
tuberculosis including cases of infection with multidrug-
resistant Mycobacterium tuberculosis.2–4 AGs are key drugs
for treatment of neonatal sepsis5, a potentially fatal
infection. Cystic fibrosis patients often suffer from
reoccurring lung infections, and AGs markedly slow the
decline in lung function in these patients.6
Unfortunately, the efficacy of AGs is greatly overshadowed
by nephrotoxicity, which is largely reversible, and
irreversible ototoxicity caused by these antibiotics. AGs are
poorly metabolized drugs and reach the kidneys largely
intact, thereby retaining both their desired antibacterial
activity and undesired adverse effects.7 Treatment with
AGs can, therefore, result in acute kidney injury and
increase the risk of developing a chronic kidney disease. In
the kidney, AGs accumulate in proximal tubular cells
where they disrupt several intracellular processes, leading
to tubular epithelial cell death and tubular necrosis.8
Although the effects of AGs on the kidney proximal tubular
epithelial cells and inner ear sensory cells have been
extensively studied, whether the exact same molecular
mechanisms lead to ototoxic and nephrotoxic effects is
unclear.17,18 There has been some success in development
of AGs that are less prone to modifications that confer
resistance to these antibiotics, yet no AGs specifically
developed to reduce toxicity are available for clinical use so
far.19–22
AGs may also cause ototoxicity, which is inner ear
dysfunction with symptoms of hearing loss and/or
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AGs interfere with bacterial protein synthesis by binding
to the A-site of the bacterial ribosome; the A-site binds
tRNA during the mRNA decoding
process.23
AGs are cationic under physiological conditions, and this
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allows these drugs to displace natural bivalent cations
responsible for membrane integrity leading to cell
permeabilization. Interactions between bivalent cations
such as Ca2+ and Mg2+ and lipids facilitate the structural
integrity and function of the plasma membrane.39 It was
previously shown that AGs like streptomycin and
isepamicin markedly increase permeability of the
mammalian cell membrane, whereas other such as
gentamicin have little effect on mammalian cell
membranes.40 This indicates that both the cationic nature
and the specific structure of an AG are related to off-target
effects. The membrane-disrupting effects of AGs have been
enhanced in the past decade by development of AG-based
antimicrobial cationic amphiphiles that rapidly disrupt
cell-membrane integrity.41–46
Crystallographic studies of complexes between different
AGs and the bacterial A-site suggest that these antibiotics
stabilize an A-site conformation similar to that induced by
the binding of cognate tRNA to the bacterial 30S ribosome
subunit.24,25 Studies published over the years suggest that
perturbation of mammalian cell translation is responsible
for the toxicity of AG antibiotics: The relatively high
sequence similarity between the decoding A-site of
bacterial ribosomes and mammalian mitochondrial and
cytoplasmic ribosomes leads to limited selectivity, and
non-specific ionic interactions between the highly
positively charged AGs and negatively charged rRNA
backbone further limit the selectivity of these
antibiotics.26–29 Clear evidence for the effects of AGs on
mitochondrial translation were obtained by the Böttger
group through isolation of mitochondria from mammalian
cells pretreated with an AG and by development of in vitro
translation assays using hybrid ribosomes engineered to
have the A-site sequence of the human mitochondrial
ribosome in an otherwise bacterial ribosome context.30,31
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Depending on the AG and the administration regimen, the
maximal plasma concentrations of AGs range from about
10 to about 100 µg/mL.47–50 It remains an open question
whether AGs reach the auditory hair cells at
concentrations sufficient to perturb cytosolic and
mitochondrial translation. It should also be noted that
perturbation of mitochondrial translation requires that the
AG cross the cytoplasmic membrane as well as both the
outer and inner mitochondrial membranes.
Using an in vitro model, Francis and co-workers showed
that AGs' toxicity can also be correlated with the extent of
inhibition of cytoplasmic translation.27 In addition, several
clinically used AGs, including tobramycin and gentamicin,
display similar potencies as inhibitors of mitochondrial
and cytosolic eukaryotic translation in cell-free systems.32
These data suggest that perturbation of the fidelity of both
mammalian translation machineries contribute to the
toxicity of AG antibiotics.
To identify chemical modifications that improve the safety
profile of AGs while preserving antibacterial efficacy, the
relationships between specific structural motifs and
toxicity must first be established. With the goal of
identifying a molecular basis for the structure/ototoxicity
relationship of AGs, we investigated how chemical
modifications that do not significantly affect the
antibacterial activity affect key functions of immortalized
inner ear cells that express genes characteristic of inner-ear
sensory hair cells.
In search of strategies to selectively enhance inhibition of
bacterial translation by AGs we previously reported that
modifying the C-5 alcohol of certain AGs composed of a
4,6-disubstituted 2-deoxystreptamine with a β-O-linked
ribofuranose can improve selectivity for the bacterial
ribosome.33 Crich and coworkers recently introduced
RESULTS AND DISCUSSION
Ribosylation and N-demethylation of the AGs
apramycin and geneticin. We focused our investigation
on apramycin (APR) and geneticin (G-418) (Figure 1); these
two AGs are at the two ends of the spectrum of AG-induced
ototoxicity. APR induces only modest hair cell damage in
cultures of cochlear explants as well as in several animal
propylamycin,
a
4′-deoxy-4′-propyl
semisynthetic
derivative of paromomycin that has potent antibacterial
activity. Using in vitro translation assays with hybrid
ribosomes they showed that propylamycin has higher
affinity for the bacterial ribosome compared to human
mitochondrial or cytosolic ribosomes. Propylamycin is also
less ototoxic than paromomycin in guinea pigs.34
models including
a guinea pig model of chronic
ototoxicity.51–53 G-418, on the other hand, is considered the
most toxic AG and is not in clinical use.28 Several previous
reports provided evidence that APR inhibits translation
but does not enhance misreading errors in bacterial and
mammalian cytosolic ribosomes.51,53–56 In contrast,
investigations of G-418 bound to cytosolic A-site and to the
bacterial A-site revealed that rather than inhibiting
translation, this AG promotes missense errors during
protein synthesis.57,58 We examined the effects of two types
of modifications to these AGs (Figure 1): 5-O-ribosylation
(compounds 1, 3, 4, 7, and 9) and N-demethylation of the
secondary amine (compounds 2 and 5). To evaluate the
significance of ribosylation of the complete AG scaffold,
this modification was carried out on the truncated pseudo-
The mechanisms of AG-induced ototoxicity appear to
differ depending on the specific AG. An accumulation of
experimental evidence indicates that different AGs not
only have differences in binding to the A-site but also differ
in off-target activities associated with their toxicity to
mammalian cells in general and auditory cells in particular.
Production of toxic concentrations of reactive oxygen
species is a well-documented effect of AGs on mammalian
cells that, in turn, triggers apoptosis.35–37 However, this
effect may be a consequence of perturbation of translation
that leads to elevated levels of dysfunctional mitochondrial
proteins, especially those involved in the respiratory chain
complexes that facilitate mitochondrial electron
transport.38
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disaccharide scaffolds of APR and G-418 (compounds 6 demethylation of G-418 had little to no effect on its
1
2
3
4
and 8) to afford the corresponding ribosylated derivatives
(7 and 9).
antibacterial activity. MIC values of 5, the N-demethylated
derivative of G-418, were the same or differed by no more
than 2 fold from those of the parent AG against most of the
strains tested.
OH
OH
O
H2N
HO
G-418
4
R1 = Me R2 = H
APR
OH
R1HN
O
HO
HO
HO
O
O
N
R
2 = ribose
R1 = Me
R1 = H
1
2
H2N
H2N
O
O
H2
N
5
NH2
H2
R2
O
O
R2 = H
5
NH2
O
R2
O
6
7
8
9
OH
O
R1 = H R2 = ribose
3
HO
R1HN
OH
HO
ribose
=
O
HN
HO
OH
OH
HO
HO
OH
H2
O
HO
HO
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O
N
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H
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N
H2
N
N
H2
H2
O
O
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R2 ribose
=
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NH2
NH2
R2
O
O
OH
OH
Figure 1: Structures of the AGs APR and G-418 and their
semisynthetic derivatives.
Briefly, N-demethylation products of APR and G-418 were
readily formed as separable products of an azido-transfer
reaction to protect the primary amines of the parent AGs.59
The formation of N-demethylated APR under the
conditions of the azido-transfer reaction was previously
reported by Crich and coworkers; an improved and
selective protocol for de-methylation of APR using iodine
under basic conditions was also reported by the same
group.60 Selective acetylation of all alcohols of the resultant
azido-protected AGs with the exception of the desired 5-
OH yielded AG-derived glycosyl acceptors; these were
glycosylated with the 2,3,5-tri-O-benzoyl-D-ribofuranosyl-
trichloroacetimidate followed by two to three deprotection
steps to afford the ribosylated derivatives. Detailed
synthetic schemes and procedures and compound
characterization data are described in section 1 in the
supporting information.
Figure 2. Minimal inhibitory concentrations of APR and
G-418 and their semisynthetic derivatives. Plots of MIC
values for indicated compounds. Blue dots represent Gram-
negative strains and red represent Gram-positive strains. MIC
values were determined using the broth double-dilution
method. Each concentration was tested in triplicate, and
results were reproduced in two independent experiments.
Ribosylation of C-5 alcohol of the pseudo-disaccharides
neamine and nebramine yields the natural AG
ribostamycin and its semi-synthetic derivative 3'-deoxy-
ribostamycin. Both have markedly improved antibacterial
activity compared to the corresponding pseudo-
disaccharides from which they are derived.33 However,
with MIC values of 64 µg/mL or higher against most of the
tested strains, APR-derived pseudo-disaccharide 6 and its
5-O-ribosylated derivative 7 were poor antibacterial agents
(Supporting Table S1). Similarly, G-418 derived pseudo-
disaccharide 8 and its ribosylated derivative 9 were not
toxic to bacterial cells. These results indicate that 5-O-
ribosylation cannot be applied as a general strategy to
restore or improve the antibacterial activity of pseudo-
disaccharide segments generated from AGs belonging to
the 4,6-disubstituted series.
Ribosylation and N-demethylation have opposite
effects on antibacterial activities of APR and G-418.
The antibacterial activities of the parental and
semisynthetic AGs were evaluated on a panel of Gram-
negative and Gram-positive bacterial pathogens. Minimal
inhibitory concentration (MIC) values were determined
using the broth double-dilution method. Results of the
antibacterial activity tests are summarized in Figure 2 and
Supporting Table S1. The MIC values of compound 1, the
ribosylated derivative of APR, were comparable or differed
by no more than 4-fold from those of the parent AG (Figure
2). These results are in agreement with those of Abe and
coworkers who reported an alternative synthesis of
compound 1 in 1981.61 In contrast, and in agreement with
previous observations made by Crich and coworkers, the
MIC values of 2, the N-demethylated derivative of APR,
were higher than those of APR by 2 to 8 fold.60
Interestingly, ribosylation of 2 resulted in enhancement of
antibacterial activity; the MIC values of ribosylated
derivative 3 were either comparable or 2-fold improved
than those of 2 (Figure 2).
In summary, N-demethylation and ribosylation had
opposite effects on APR and G-418. Whereas the APR
scaffold tolerated 5-O-ribosylation, N-demethylation
reduced its antibacterial efficacy. The reverse trend was
observed for G-418. Ribosylation of G-418 abrogated its
antibacterial activity, and N-demethylation of this AG had
little or no effect on its antibacterial activity.
Effects of ribosylation and N-demethylation on the
selectivity of inhibition of bacterial translation in
vitro. To determine how ribosylation and N-
demethylation affect bacterial and eukaryotic cytosolic
translation, we focused on APR and its derivatives 1 and 3
and on G-418 and its derivative 5; these were the most
potent antibacterials of the AGs tested. We measured
effects of these compounds on translation in cell-free
extracts containing ribosomes isolated from E. coli or from
the cytosol of rabbit reticulocytes. The concentrations at
which the tested compounds inhibited luciferase activity
In contrast to its effect on the APR scaffold, ribosylation of
G-418 led to a significant drop in antibacterial activity.
Compared to G-418, the MIC values of 4, the ribosylated
derivative of G-418, were higher by at least 8 fold against
the majority of the tested panel (Figure 2). N-
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by 50% (IC50) were determined, and the results are and A1493 adopt different conformations. In the “on” state,
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3
4
5
6
7
summarized in Figure 3. The IC50 values measured for APR
are in good agreement with those previously reported by
Böttger and coworkers.60 Selectivity for bacterial
translation was calculated as the ratio of the IC50 value for
cytosolic translation divided by the IC50 value for bacterial
translation, and the results are summarized in Figure 3.
the two adenines are bulged out from the A-site helix, and
they participate in the stabilization of the Watson-Crick
base pairs between the codon of the mRNA and the
anticodon of the tRNA.24,25,63 In the “off” state, the two
adenines are stacked in the A-site double helix.
Interactions between the AG and the A-site nucleotides
stabilize the on-state.
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9
To prevent decoding errors, the A-site must maintain a
conformational balance between on and off states. Upon
binding to an AG, the A-site rigidifies, and the freedom of
movement required to achieve optimal speed and accuracy
of translation is compromised. The movement of the A-site
in the presence and absence of AGs was explored by Pilch
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and coworkers using
a time-resolved fluorescence
anisotropy assay.64 Their results suggest that in designing
AGs intermolecular and intramolecular dynamics should
be addressed. Blanchard and coworkers investigated the
structural and dynamic impacts of 4,5-linked AG
antibiotics on the E. coli 70S ribosome via a single molecule
FRET system.65 By harnessing in silico molecular dynamics
(MD) simulations Trylska and coworkers studied the
effects of AGs on ribosome function and dynamics.66
Figure 3. Selectivity of APR, G-418, and compounds 1, 3,
and 5 for inhibition of cytosolic translation vs. bacterial
translation. Selectivity values, calculated as the cytosolic IC50
divided by the bacterial IC50 are plotted on a log10 scale with
the value above the bar. The IC50 values for the in vitro
inhibition of cytosolic and bacterial translation and their
standard deviations are given with units in µM below the plot.
Each concentration was tested in duplicate, and the results are
the averages of two independent experiments.
Here we used MD simulations to study how the derivatives
of APR and G-418 affect the flexibility of the bacterial and
eukaryotic A-sites. Binding of all AGs to the A-sites occurs
in the vicinity of adenines A1492 and A1493 in bacteria,
cytosolic, and mitochondrial A-sites.24,57,67,68 For docking
calculations and MD simulations, we used fragments
consisting of the 24 ribonucleotides surrounding these
adenines in each site (Figure 4A-C). For the bacterial A-site
structure, we cropped a sequence of 24 ribonucleotides
from a complex of the bacterial 30S ribosome subunit with
APR (PDB code: 4AQY).51
Ribosylation of APR enhanced selectivity to E. coli
ribosomes; the selectivity of the ribosylated derivative 1
was higher than that of APR (Figure 3). APR and its
ribosylated derivative 1 were recently evaluated by Böttger,
Crich, and coworkers as inhibitors of engineered bacterial
ribosomes in which the native A-site was replaced by a 30-
basepair sequence of the human mitochondrial A-site.62
Similar to the trend observed for inhibition of mammalian
cytosolic translation, ribosylation of APR decreased the
undesired inhibition of the hybrid ribosomes that
contained the mitochondrial A-site by approximately 4-
fold. This indicates that ribosylation of APR reduces its
undesired inhibition of both mitochondrial and cytosolic
mammalian translation.
For simulations of the cytosolic A-site two structures were
used. The first was from the X-ray structure of a complex
between an oligo ribonucleotide mimic of the cytosolic A-
site and APR (PDB code: 2G5K).55 To facilitate
crystallization, this RNA sequence contained several
ribonucleotides that differ from those in the conserved
Homo sapiens cytosolic A-site sequence. For MD
simulations, these ribonucleotides were replaced with the
conserved residues (Supporting Figure S1). The second
structure was of the yeast 80S ribosome in complex with
G-418 (PDB code: 4U4O)69; the A-site sequence of this
rRNA is identical to that of the cytosolic H. sapiens A-site.
N-demethylation considerably improved the selectivity of
G-418 to inhibition of E. coli ribosomes. Compared to the
parent AG, the selectivity of compound 5 increased by over
an order of magnitude. Of note, the potencies of G-418 and
5 as inhibitors of bacterial translation were similar (Figure
3); the higher selectivity of 5 resulted from a considerably
lower inhibition potency in the mammalian cytosolic
translation system. This result is of particular interest since
N-demethylation of G-418 reduced the undesired
inhibition of cytosolic eukaryotic translation without
significantly affecting its antibacterial activity.
Since there are currently no available X-ray structures of
complexes of the mitochondrial A-site with APR or G-418,
the 24-nucleotide sequence of the mitochondrial apo A-
site rRNA was cropped from the cryogenic electron
microscopy structure of the mammalian mitochondrial
ribosome (PDB code: 5AJ3). In this structure, A1492 and
A1493 are bulged out of the helix.29
APR and G-418 were first docked into the selected
structures using rDock, which successfully reproduced the
binding modes of APR and G-418 observed in the crystal
structures of bacterial and human cytosolic A-sites.
The impact of chemical modifications on the
flexibility of bacterial and human A-sites. During the
bacterial translation process, A-site ribonucleotides A1492
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Figure 4. A-site secondary structures and the effects of APR, G-418, and compounds 1, 3, and 5 on the movement of
ribonucleotides in the different A-sites. (A-C) Secondary structures of the A) bacterial, B) H. sapiens cytosolic, and C) H.
sapiens mitochondrial A-sites. Ribonucleotides shown in red are those for which RMSF values were calculated. (D-F) RMSF values
for apo A-sites and the A-sites in the presence of APR and its derivatives 1 and 3. (G-I) RMSF values for apo A-sites and the A-sites
in the presence of G-418 and its derivative 5. RMSF values and standard deviations were determined from three independent
simulations.
Compared to the previously reported structures, the best
(lowest) docking scores were within 1.0 Å and 0.7 Å for APR
(PDB code: 4AQY) and G-418 (PDB code: 1MWL),
respectively, in the bacterial A-site and within 2.8 Å and 1.1
Å for APR (PDB code: 2G5K) and G-418 (PDB code: 4U4O),
respectively, in the human cytosolic A-site (Section 4 in the
Supporting Information). APR derivatives 1 and 3 and G-
418 derivative 5 were then docked into the bacterial,
cytosolic, and mitochondrial A-sites (Supporting Figure
S2). Computations indicated that in the bacterial A-site,
APR stabilized a conformation in which A1492 and A1493
are bulged out of the double helix; however, in the
cytosolic A-site APR stabilized a conformation in which
A1491 is bulged out, A1492 is intercalated into the helix, and
A1493 is slightly bulged out. Complexes with the lowest
docking scores were subjected to further analysis using MD
simulations. For each complex, three 20-ns simulations
were performed; 20 ns is the time required for the two
adenines in the bacterial apo A-site to complete the
movement between the bulged out and bulged in states.
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Each replica was started from a different random seed. difference in RMSF values calculated for the latter two
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5
6
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8
Simulations were probed every 10 ps to produce 2000
frames for each simulation. The stability of the resulting
trajectories was tested based on the root mean square
deviation (RMSD) values of the backbone atoms with
respect to the equilibrated structures of the A-sites
(Supporting Table S2).
adenines (Figure 4H). Notably, in agreement with the
results of the cytosolic in vitro translation assay, the N-
demethylated derivative 5 had a smaller effect on the
freedom of movement of ribonucleotides in the cytosolic
A-site than did G-418. The RMSF values for A1492, and
A1493 when bound to 5 were higher than for G-418.
Interestingly, G-418 and its N-demethylated derivative 5
had similar effects on the freedom of movement of A1493
in the mitochondrial A-site, but 5 reduced the freedom of
movement of A1492 and of adjacent ribonucleotides C1491
and A1490 more than did the parent antibiotic G-418
(Figure 4I). The important role of the phylogenetic
differences between ribonucleotides in proximity to A1492
and A1493, including ribonucleotide 1491, in facilitating the
A-site selectivity of AGs has been established by Böttger
and coworkers.32,70The different interactions of the AG
with ribonucleotide 1491, which is a guanine in the
bacterial A-site and a cytosine in the mitochondrial A-site
(Figure 4A and 4C, respectively), appear to underlie the
different effects of the AGs on the flexibility of the
mitochondrial and bacterial A-sites.
To determine the degree of freedom of each ribonucleotide
in each structure, the root mean square fluctuation (RMSF)
values were calculated and compared to the corresponding
values in the AG-bound A-sites (Figure 4D-I and
Supporting Tables S3-S5). The lower the difference
between the RMSF values of the ribonucleotides in the AG-
bound sequence and the values of the corresponding
ribonucleotides in the apo A-site, the lower the
perturbation to the motion of the A-site. An optimal AG
should significantly reduce the free movement of the
ribonucleotides of the bacterial A-site but should not
interfere with movement of the ribonucleotides in the
eukaryotic A-sites.
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The calculated RMSF values of A1492 and A1493 in the
presence of APR and its derivatives 1 and 3 were similar in
the bacterial A-site and lower than those in the bacterial
apo A-site. These results suggest that the core APR scaffold
confines the movement of A1492 and A1493 and that the
added ribose moiety in 1 and 3 does not further reduce the
movement of these two A-site ribonucleotides (Figure 4D).
In summary, MD simulations of the three A-sites indicated
that the predominant effect of all AGs in this study was to
reduce the freedom of movement of A1492 and A1493;
however, some of the AGs also significantly affected the
movement of ribonucleotides adjacent to these two
adenines. Since there is higher sequence identity between
the bacterial and human mitochondrial A-sites than
between the bacterial and human cytosolic ones, we had
expected that the AGs would have similar effects on the
ribonucleotides of the bacterial and mitochondrial A-sites:
The MD simulations suggested otherwise.
MD simulations of the H. sapiens cytosolic A-site in the
presence of APR and its derivatives 1 and 3 indicated that
compared to the apo A-site, APR slightly reduced the
freedom of movement of A1491 and A1492 yet, slightly
increased the movement of A1493 (Figure 4E). Compared
to the parent APR, compound 3, the N-demethylated and
ribosylated derivative, significantly constrained the
movement of A1493. Of all AGs evaluated, ribosylated APR
derivative 1 had the least effect on the movement of
ribonucleotides of the eukaryotic cytosolic A-site.
Ribosylation and N-demethylation reduce the effect
of AGs on the viability of HEI-OC1 cells. We next
evaluated the viability of immortalized cells derived from
inner ear auditory cells in the presence of the most potent
antibacterials of the AGs studied here; the results are
summarized in Figure 5. As an inner ear cell model, we
used HEI-OC1 cells, which are immortalized mouse inner
ear cells that express markers of auditory sensory cells.
This cell line, developed by Kalinec and co-workers, has
become a widely used model for the study of the damage
induced by AGs on auditory sensory cells.71,72
In the mitochondrial A-site the RMSF values of A1492 were
similar for APR and its derivatives 1 and 3 (Figure 4F). For
A1493, the average RMSF value in the presence of 1 was
higher than average RMSF values in the presence of APR
and of 3, suggesting that ribosylation of APR reduced the
effect of this AG on the movement of the eukaryotic
mitochondrial A-site ribonucleotides. Notably, the results
of the MD simulations of APR and its derivative 1 in
complex with the mitochondrial A-site sequence are
consistent with the IC50 values of these AGs recently
determined by analyses of bacterial ribosomes in which the
native A-site was replaced by the 30-basepair sequence of
the mitochondrial A-site; in this system, ribosylation of
APR reduced its undesired inhibitory effect on cytosolic
and mitochondrial A-sites.62
At the highest concentration tested, APR and its two
derivatives 1 and 3 did not measurably decrease the
viability of HEI-OC1 cells (IC50 >> 5 mg/mL, Figure 5). G-
418 was highly toxic to the cells with an IC50 value of about
0.2 mg/mL. Notably, the IC50 value of 5 was about 1.8
mg/mL, which is higher by close to an order of magnitude
compared to that of the parent antibiotic. The effects of the
tested AGs on the viability of HEI-OC1 cells correlated well
with their effects on cytosolic translation in vitro. Less
potent inhibitors of translation in the rabbit reticulocyte
lysates were also the less toxic to HEI-OC1 cells. For
example, G-418 inhibited eukaryotic cytosolic translation
over an order of magnitude more potently than its
demethylated derivative 5 (Figure 3); the parent AG was
G-418 and its N-demethylated derivative
5
also
significantly reduced the movement of adenines 1492 and
1493 of the bacterial A-site (Figure 4G). However, the
RMSF values of 5 were slightly higher than those of G-418.
By binding to the cytosolic A-site, G-418 reduced the
movement of A1491, A1492, and A1493 with the largest
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about an order of magnitude (~9-fold) more toxic to HEI- Chemical modifications affect the abilities of the AGs
1
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5
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OC1 cells than 5 (Figure 5).
to permeabilize HEI-OC1 cells. We next asked if the AGs
APR and G-418 induce rapid permeabilization of HEI-OC1
cells and if chemical modifications affect this
phenomenon. To evaluate the permeability of HEI-OC1
cells, we measured the uptake of 4′,6-diamidino-2-
phenylindole (DAPI), which is excluded from healthy cells
due to the integrity of the plasma membrane. Uptake of
DAPI was measured after a 3-hour incubation with the
tested AGs using flow cytometry. As a positive control we
chose a membrane-disrupting cationic amphiphile derived
from the AG tobramycin (S-14) previously developed by
our group; S-14 induces rapid and extensive plasma
membrane damage.73 As a negative control, cells were
exposed to the translation-inhibiting antibiotic
chloramphenicol (CAM).74 We quantified DAPI-free cells,
cells stained with DAPI, and dead cells. Results are
summarized in Figure 6 and in Figure S3.
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Figure 5. Effects of APR, G-418, and compounds 1, 3, and 5
on the viability of HEI-OC1 cells. Cells were treated with
different concentrations of each compound for 72 h, and
viability was determined using the methylthiazolyldiphenyl-
tetrazolium bromide (MTT) assay; log10 scale IC50 values
plotted are relative to untreated control cells. Each
concentration was tested in duplicate, and the results are
expressed as log10 of the IC50 values from two independent
experiments.
Compared to untreated cells, a dose-dependent increase in
the percentage of permeabilized cells was observed in
samples treated with APR. This increase was moderate
compared to that induced by G-418. Interestingly,
compared to G-418-treated cells, a substantially lower
percentage of cells were DAPI stained in samples treated
with N-demethylated derivative 5. Following a 3-hour
exposure to 300 g/mL of 5, the percentage of DAPI-
stained cells was about an order of magnitude lower than
that of cells treated with 300 g/mL of G-418 and slightly
lower than that of cells treated with 300 g/mL of APR
(Figure 6).
The maximal concentration of AGs in human plasma (Cmax
)
depends on numerous parameters such as the AG's
chemical structure and overall positive charge, the
administration regime, the dose, and the patient’s
physiology including weight and kidney function. Based on
several clinical reports, the Cmax values of clinically used
AGs in human plasma are in the range of 10 to 100 g/mL.47–
50
Interestingly, this suggests that, with an IC50 value of
about 200 g/mL, G-418 is the only AG in this study that
would reach the cochlea at a concentration sufficient to
induce significant toxicity to the auditory hair cells.
Figure 6. Effects of APR, G-418 and compounds 1, 3 and 5 on the permeabilization of HEI-OC1 cells. Cells were treated with
different concentrations of each compound for 3 h, and permeability was evaluated by flow cytometry analysis of DAPI staining.
The percentages of permeabilized and dead cells were determined relative to untreated cells. CAM was used as negative control.
Dead cell population was determined using the parameters measured for cells treated with cationic amphiphile S-14 that causes
rapid and extensive loss of membrane integrity. Results are expressed as means ± SD from two independent experiments.
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The results of the cell permeabilization experiments Briefly, HEI-OC1 cells were treated with AGs for 6 days at
1
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5
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8
indicate that, depending on the structure, AGs can induce
rapid plasma membrane damage. Importantly, AGs
enhanced DAPI permeability after a relatively short
incubation of 3 hours, suggesting that the enhanced
permeability is unlikely to result only from the
accumulation of dysfunctional proteins. Rather, we
speculate that permeabilization mainly results from direct
plasma membrane disruption by these highly positively
charged molecules. Thus, the results of the cell
permeabilization assay suggest that, depending on the AG,
the mechanism leading to auditory cells damage may vary
and that even minor chemical modifications affect the
mode of action leading to cell damage.
concentrations around the reported plasma Cmax values of
AGs (10, 50, 100, and 300 µg/mL). Cells were then
harvested, fixed, and permeabilized in suspension. COX-1
and SDHA were detected by flow cytometry with highly
specific monoclonal antibodies labeled with two different
fluorophores (Figure 7 and Figure S4). This protocol
minimizes sample preparation and handling to enable
evaluation of the effects of the tested AG on the
mitochondrial and cytosolic protein levels.
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In samples treated with 25 µg/mL of the control antibiotic
CAM, there was a reduction in the level of COX-1, which is
translated in mitochondria, as compared to untreated
control samples (Figure 7). This is in agreement with the
previous observation made by Kang et al. that COX-1 levels
are reduced in CAM-treated cells.76 In contrast to the effect
on COX-1, treatment with CAM led to a dose-dependent
increase in the level of SDHA, a mitochondrial protein
translated by cytosolic ribosomes (Figure 7). This
unexpected effect may be the result of activation of a
stress-response mechanism; it was previously shown that
shear stress in mammalian cells leads to elevated levels of
SDHA.77
AG structure influences the levels of mitochondrial
proteins translated by cytosolic and mitochondrial
ribosomes in HEI-OC1 cells. Current techniques for
analysis of in vitro translation are based on cell-free assays,
and it is technically difficult to generate extracts of
mitochondrial ribosomes devoid of contaminating
cytosolic ribosomes. We therefore developed an
experiment to study the effects of AGs on the levels of
mitochondrial proteins translated by mitochondrial and
cytosolic ribosomes in intact cells. This was accomplished
by repurposing a flow cytometry assay that was originally
designed to evaluate drug-induced effects on
mitochondrial biogenesis. This assay relies on the
statistical power of flow cytometry to evaluate the ratio
between two mitochondrial proteins, one encoded by
mitochondrial DNA and translated by mitochondrial
ribosomes (COX-1) and the second encoded by nuclear
DNA and translated by cytoplasmic ribosomes (SDHA). As
a positive control for the assay we used CAM, which
inhibits mitochondrial translation.75
Paradoxically, the AGs that effectively inhibited both
bacterial and mammalian translation in vitro increased
levels of both SDHA and COX-1 in HEI-OC1 cells; the
extent of elevation depended on the concentration and on
the AG. Only a modest dose-dependent increase in the
levels of COX-1 and of SDHA was measured in cells treated
APR or with its derivatives 1 or 3 (Figure 7). In cells treated
with G-418, the dose-dependent increase in COX-1 and
SDHA levels was higher compared to that measured for
APR and its derivatives. Compared to G-418, 100 µg/mL of
5, the N-demethylated derivative of G-418, had much less
effect.
Figure 7. Effects of APR, G-418, and compounds 1, 3, and 5 on levels of mitochondrial proteins translated in the cytoplasm
and the mitochondria of HEI-OC1 cells. Cells were treated with different concentrations of each compound for 6 days, and
levels of COX-1 (translated in the mitochondria, in green) and SDHA (translated in the cytoplasm, in magenta) were determined
by flow cytometry. The percentages of protein levels relative to untreated cells are plotted. CAM was used as a positive control for
inhibition of mitochondrial translation. The results are expressed as means ± SD from two independent experiments.
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Interestingly, the elevation of COX-1 and SDHA levels mammalian cell translation processes, thereby paving the
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induced by the different AGs correlated with their effects
on the viability of HEI-OC1 cells. For example, of the AGs
tested in this study, G-418 was the most toxic to HEI-OC1
cells (IC50 about 200 µg/mL, Figure 5), and it also induced
the largest increase in COX-1 and SDHA levels (Figure 7).
N-demethylated G-418 derivative 5 was about 9-fold less
toxic to HEI-OC1 cells than the parent AG, and the
increases it induced in levels of the proteins translated in
the mitochondria and the cytosol were considerably lower
than that of the parent AG. The results of these assays
counterintuitively suggest that, when introduced at
clinically relevant concentrations, AGs may enhance rather
than reduce the levels of certain proteins in mammalian
cells. This is likely the result of a stress response and does
not contradict the well-established paradigm that AGs
perturb with both bacterial and eukaryotic translation
processes.
way for rational design of additional AG derivatives.
Even at concentrations one to two orders of magnitude
higher than the reported maximal plasma concentrations
range of AGs in various mammals, including humans, APR
and its ribosylated derivatives 1 and 3 did not affect the
viability of HEI-OC1 cells. These immortalized mouse
inner ear cells express markers of auditory sensory cells.
Notably, although it is a relatively minor chemical
modification, N-demethylation of the highly toxic G-418
markedly reduced its toxicity to HEI-OC1 cells; the N-
demethylated derivative 5 inhibited viability of HEI-OC1
cells by 50% at a concentration close to an order of
magnitude higher than the IC50 of the parent AG G-418.
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All AGs in this study inhibited both the bacterial and
eukaryotic cytosolic ribosomes in cell-free experiments;
however, a different picture appeared when additional
effects of these antibiotics on HEI-OC1 cells were studied.
A dose-dependent enhancement in uptake of DAPI by
HEI-OC1 cells after only 3 hours of incubation with the AGs
CONCLUSIONS
was indicative of
a
rapid plasma membrane
Here we investigated how known, as well as novel chemical
modifications of APR, considered among the least toxic of
the AGs, and G-418, considered the most toxic, influenced
different aspects of their biological activity with a focus on
their effects on auditory cells. Interestingly, for APR and G-
418, N-demethylation and ribosylation resulted in opposite
effects on the antibacterial activity. Conversion of the
single secondary amine of APR to a primary amine through
N-demethylation reduced its antibacterial activity; the
effect of this modification on the antibacterial activity of
G-418 was significantly less pronounced. Ribosylation, in
contrast, did not considerably alter the antibacterial
activity of APR yet completely abrogated that of G-418.
Comparison of the effects of these AGs on inhibition of
bacterial versus eukaryotic cytosolic translation revealed
that N-demethylation increased the selectivity of G-418 by
more than an order of magnitude.
permeabilization effect. The results of the DAPI uptake
assay indicated that this effect could be significantly
reduced through chemical modifications. For example, a
considerably lower percentage of cells were permeable to
DAPI upon treatment with 5, the N-demethylated
derivative of G-418, than upon treatment with the parent
antibiotic.
By concomitant flow cytometry analyses of the levels of
COX-1,
a protein translated by the mitochondrial
ribosome, and SDHA, which is produced in the cytoplasm,
we observed that, at clinically relevant concentrations, the
AGs tested enhanced, rather than reduced, the levels of
these mitochondrial proteins in HEI-OC1 cells with the
most significant dose-dependent enhancement measured
in cells treated with G-418. This seemingly paradoxical
effect may be due to a stress response. Importantly, these
results emphasize the need for further investigation of the
role of the stress response in AG-induced auditory cell
damage.
MD simulations of complexes between the AGs and rRNA
fragments corresponding to bacterial, eukaryotic cytosolic,
and eukaryotic mitochondrial A-sites showed that binding
to AGs rigidified the A-site structures reducing the
freedom of movement necessary for accurate translation.
A correlation was found between the extent of restriction
of movement and the inhibition of cell-free translation.
AGs that effectively inhibited eukaryotic and/or bacterial
translation processes also effectively reduced the
movement of the corresponding A-sites. In the A-sites, the
positions of the adenines that define the on and off states
and those nucleotides in close proximity to these adenines
were affected more than other nucleotides in the A-sites by
AG binding. MD simulations indicated that even minor
modification to the AG scaffold, such as N-demethylation,
significantly altered the effect of the AG on the flexibility
of the A-site. Further, differences in A-site sequences, such
as those that differentiate between the mitochondrial and
bacterial A-sites, resulted in differences in interactions
with AGs. The in silico analyses proved a powerful tool for
prediction of the effects of AGs on bacterial and
To summarize, this study demonstrated that despite
common chemical features, the vast structural diversity
among different members of the AG class of antibiotics
results in major differences in desirable and undesirable
biological activities. All AGs tested inhibited the activities
of mammalian and bacterial ribosomes when tested in cell-
free assays, albeit with different efficacies; however, our
data suggest that, depending on the AG, toxicity to
auditory cells may stem from rapid plasma membrane
permeabilization. Moreover, although AGs are potent
inhibitors of translation in cell-free extracts, in intact cells
these drugs can cause an elevation in the levels of certain
proteins, presumably, due to stress responses to these
antibiotics. Importantly, this study demonstrated that
chemical modifications can improve selectivity of AGs as
well as reduce plasma membrane permeabilization of
auditory cells. The synthetic strategies and approaches for
biological and in silico evaluation described should lead to
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Induced Cochleotoxicity: A Review. Frontiers in Cellular
Neuroscience. 2017, 308.
https://doi.org/10.3389/fncel.2017.00308.
breakthroughs in development of AGs with reduced
toxicity to auditory cells.
p
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Huth, M. E.; Han, K. H.; Sotoudeh, K.; Hsieh, Y. J.; Effertz, T.;
Vu, A. A.; Verhoeven, S.; Hsieh, M. H.; Greenhouse, R.;
Cheng, A. G.; Ricci, A. J. Designer Aminoglycosides Prevent
Cochlear Hair Cell Loss and Hearing Loss. J. Clin. Invest.
2015, 125 (2), 583–592. https://doi.org/10.1172/JCI77424.
O’Sullivan, M. E.; Perez, A.; Lin, R.; Sajjadi, A.; Ricci, A. J.;
Cheng, A. G. Towards the Prevention of Aminoglycoside-
Related Hearing Loss. Front. Cell. Neurosci. 2017, 11, 325.
https://doi.org/10.3389/fncel.2017.00325.
Lacy, M. K.; Nicolau, D. P.; Nightingale, C. H.; Quintiliani, R.
The Pharmacodynamics of Aminoglycosides. Clin. Infect.
Dis. 1998, 27 (1), 23–27. https://doi.org/10.1086/514620.
Igumnova, V.; Veidemane, L.; Vīksna, A.; Capligina, V.; Zole,
E.; Ranka, R. The Prevalence of Mitochondrial Mutations
Associated with Aminoglycoside-Induced Deafness in
Ethnic Latvian Population: The Appraisal of the Evidence. J.
ASSOCIATED CONTENT
Supporting Information
Detailed synthetic procedures; MIC assay protocol and MIC
values (Table S1); in vitro translation protocols; docking and
MD calculation protocols; RMSD values (Table S2); RMSF
values(Tables S3-S5); cytosolic A-site sequence used for MD
simulations (Figure S1); docked structures of the AGs in the
different A-sites (Figure S2); HEI-OC1 cell viability assay
protocol; cell permeability assay protocol (Figure S3); protocol
for in-cell mitochondrial and cytosolic protein translation
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(Figure S4); H and 13C NMR assignments for intermediate
1
compounds and final compounds 1-9; MS data; H and 13C
NMR spectra of compounds 1-9 (Figures S5-S22).
This material is available free of charge via the Internet at
http://pubs.acs.org.
Hum.
Genet.
2019,
64
(3),
199–206.
https://doi.org/10.1038/s10038-018-0544-6.
(14)
Prezant, T. R.; Agapian, J. V.; Bohlman, M. C.; Bu, X.; Öztas,
S.; Qiu, W. Q.; Arnos, K. S.; Cortopassi, G. A.; Jaber, L.;
Rotter, J. I.; Shohat, M.; Fischel-Ghodsian, N. Mitochondrial
Ribosomal RNA Mutation Associated with Both Antibiotic-
Induced and Non-Syndromic Deafness. Nat. Genet. 1993, 4
(3), 289–294. https://doi.org/10.1038/ng0793-289.
Kros, C. J.; Desmonds, T. Drug-Induced Hearing Loss:
Infection Raises the Odds. Sci. Transl. Med. 2015, 7 (298),
298fs31. https://doi.org/10.1126/scitranslmed.aac9811.
AUTHOR INFORMATION
Corresponding Author
*mfridman@tauex.tau.ac.il
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ACKNOWLEDGMENT
We thank Dr. Federico Kalinec (House Ear Institute, Los Angeles,
California) for his generous gift of HEI-OC1 cells and for his
advice. We thank Dr. Reuven Stein (Department of Neurobiology,
George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel
Aviv, Israel) for his guidance and access to the mammalian cell
culture facility. This work was supported by Israel Science
Foundation Grant 6/14.
Prayle, A.; Watson, A.; Fortnum, H.; Smyth, A. Side Effects
of Aminoglycosides on the Kidney, Ear and Balance in Cystic
Fibrosis.
Thorax
2010,
65
(7),
654–658.
https://doi.org/10.1136/thx.2009.131532.
(17)
(18)
Mingeot-Leclercq, M. P.; Tulkens, P. M. Aminoglycosides:
Nephrotoxicity. Antimicrobial Agents and Chemotherapy.
1999, pp 1003–1012. https://doi.org/10.1128/aac.43.5.1003.
Paquette, F.; Bernier-Jean, A.; Brunette, V.; Ammann, H.;
Lavergne, V.; Pichette, V.; Troyanov, S.; Bouchard, J. Acute
Kidney Injury and Renal Recovery with the Use of
Aminoglycosides: A Large Retrospective Study. Nephron
2015, 131 (3), 153–160. https://doi.org/10.1159/000440867.
Zhang, J.; Chiang, F.-I.; Wu, L.; Czyryca, P. G.; Li, D.; Chang,
C.-W. T. Surprising Alteration of Antibacterial Activity of 5"-
Modified Neomycin against Resistant Bacteria. J. Med.
REFERENCES
(1)
Takahashi, Y.; Igarashi, M. Destination of Aminoglycoside
Antibiotics in the “Post-Antibiotic Era.” Journal of
Antibiotics. 2018, pp 4–14. https://doi.org/10.1038/ja.2017.117.
Schatz, A.; Bugle, E.; Waksman, S. A. Streptomycin, a
Substance Exhibiting Antibiotic Activity Against Gram-
Positive and Gram-Negative Bacteria. Exp. Biol. Med. 1944,
55 (1), 66–69. https://doi.org/10.3181/00379727-55-14461.
Hoagland, D. T.; Liu, J.; Lee, R. B.; Lee, R. E. New Agents for
the Treatment of Drug-Resistant Mycobacterium
Tuberculosis. Advanced Drug Delivery Reviews. 2016, pp 55–
72. https://doi.org/10.1016/j.addr.2016.04.026.
Dookie, N.; Rambaran, S.; Padayatchi, N.; Mahomed, S.;
Naidoo, K. Evolution of Drug Resistance in Mycobacterium
Tuberculosis: A Review on the Molecular Determinants of
Resistance and Implications for Personalized Care. J.
Antimicrob. Chemother. 2018, 73 (5), 1138–1151.
https://doi.org/10.1093/jac/dkx506.
(2)
(19)
Chem.
2008,
51
(23),
7563–7573.
(3)
(4)
https://doi.org/10.1021/jm800997s.
(20)
Maianti, J. P.; Kanazawa, H.; Dozzo, P.; Matias, R. D.; Feeney,
L. A.; Armstrong, E. S.; Hildebrandt, D. J.; Kane, T. R.; Gliedt,
M. J.; Goldblum, A. A.; Linsell, M. S.; Aggen, J. B.; Kondo, J.;
Hanessian, S. Toxicity Modulation, Resistance Enzyme
Evasion, and A-Site X-Ray Structure of Broad-Spectrum
Antibacterial Neomycin Analogs. ACS Chem. Biol. 2014, 9
(9), 2067–2073. https://doi.org/10.1021/cb5003416.
Maianti, J. P.; Hanessian, S. Structural Hybridization of
Three Aminoglycoside Antibiotics Yields a Potent Broad-
Spectrum Bactericide That Eludes Bacterial Resistance
(21)
(5)
(6)
Simonsen, K. A.; Anderson-Berry, A. L.; Delair, S. F.; Dele
Davies, H. Early-Onset Neonatal Sepsis. Clin. Microbiol. Rev.
2014, 27 (1), 21–47. https://doi.org/10.1128/CMR.00031-13.
Howard, M.; Frizzell, R. A.; Bedwell, D. M. Aminoglycoside
Antibiotics Restore CFTR Function by Overcoming
Premature Stop Mutations. Nat. Med. 1996, 2 (4), 467–469.
https://doi.org/10.1038/nm0496-467.
Enzymes. Med. Chem. Commun. 2016,
https://doi.org/10.1039/c5md00429b.
7 (1), 170–176.
(22)
Garzan, A.; Willby, M. J.; Green, K. D.; Gajadeera, C. S.; Hou,
C.; Tsodikov, O. V; Posey, J. E.; Garneau-Tsodikova, S.
Sulfonamide-Based
Inhibitors
of
Aminoglycoside
Acetyltransferase Eis Abolish Resistance to Kanamycin in
Mycobacterium Tuberculosis. J. Med. Chem. 2016, 59 (23),
10619–10628. https://doi.org/10.1021/acs.jmedchem.6b01161.
Greenberg, W. A.; Priestley, E. S.; Sears, P. S.; Alper, P. B.;
Rosenbohm, C.; Hendrix, M.; Hung, S. C.; Wong, C. H.
Design and Synthesis of New Aminoglycoside Antibiotics
Containing Neamine as an Optimal Core Structure:
Correlation of Antibiotic Activity with in Vitro Inhibition of
Translation. J. Am. Chem. Soc. 1999, 121 (28), 6527–6541.
(7)
(8)
Lortholary, O.; Tod, M.; Cohen, Y.; Petitjean, O.
Aminoglycosides. Med. Clin. North Am. 1995, 79 (4), 761–
787. https://doi.org/10.1016/S0025-7125(16)30038-4.
Martínez-Salgado, C.; López-Hernández, F. J.; López-Novoa,
J. M. Glomerular Nephrotoxicity of Aminoglycosides.
Toxicology and Applied Pharmacology. August 15, 2007, pp
86–98. https://doi.org/10.1016/j.taap.2007.05.004.
(23)
(9)
Jiang, M.; Karasawa, T.; Steyger, P. S. Aminoglycoside-
ACS Paragon Plus Environment

Page 11 of 21
Journal of the American Chemical Society
https://doi.org/10.1021/ja9910356.
(36)
(37)
Forge, A.; Schacht, J. Aminoglycoside Antibiotics. Audiol.
Neuro-Otology 2000, (1), 3–22.
https://doi.org/10.1159/000013861.
(24)
François, B.; Russell, R. J. M.; Murray, J. B.; Aboul-ela, F.;
Masquida, B.; Vicens, Q.; Westhof, E. Crystal Structures of
Complexes between Aminoglycosides and Decoding A Site
Oligonucleotides: Role of the Number of Rings and Positive
Charges in the Specific Binding Leading to Miscoding.
Nucleic Acids Res. 2005, 33 (17), 5677–5690.
https://doi.org/10.1093/nar/gki862.
Pape, T.; Wintermeyer, W.; Rodnina, M. V. Conformational
Switch in the Decoding Region of 16S RRNA during
Aminoacyl-TRNA Selection on the Ribosome. Nat. Struct.
Biol. 2000, 7 (2), 104–107. https://doi.org/10.1038/72364.
Hutchin, T.; Haworth, L.; Higashi, K.; Fischel-ghodsian, N.;
5
1
2
3
4
5
Desa, D. E.; Nichols, M. G.; Smith, H. J. Aminoglycosides
Rapidly Inhibit NAD(P)H Metabolism Increasing Reactive
Oxygen Species and Cochlear Cell Demise. J. Biomed. Opt.
2018, 24 (5), 1–14. https://doi.org/10.1117/1.JBO.24.5.051403.
Topf, U.; Uszczynska-Ratajczak, B.; Chacinska, A.
Mitochondrial Stress-Dependent Regulation of Cellular
Protein Synthesis. Journal of cell science. 2019.
https://doi.org/10.1242/jcs.226258.
(38)
(39)
6
7
8
9
(25)
(26)
Au, S.; Weiner, N. D.; Schacht, J. Aminoglycoside Antibiotics
Preferentially Increase Permeability in Phosphoinositide-
Containing Membranes: A Study with Carboxyfluorescein in
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Stoneking, M.; Saha, N.; Arnos, C.; Cortopassi, G.
A
Molecular Basis for Human Hypersensitivity of
Aminoglyscoside Antibiotics. Nucleic Acids Res. 1993, 21 (18),
4174–4179. https://doi.org/10.1093/nar/21.18.4174.
Liposomes. BBA - Biomembr. 1987, 902 (1), 80–86.
https://doi.org/10.1016/0005-2736(87)90137-4.
Van Bambeke, F.; Mingeot-Leclercq, M. P.; Schanck, A.;
Brasseur, R.; Tulkens, P. M. Alterations in Membrane
Permeability Induced by Aminoglycoside Antibiotics:
Studies on Liposomes and Cultured Cells. Eur. J. Pharmacol.
(40)
(27)
(28)
Francis, S. P.; Katz, J.; Fanning, K. D.; Harris, K. A.; Nicholas,
B. D.; Lacy, M.; Pagana, J.; Agris, P. F.; Shin, J.-B. A Novel
Role of Cytosolic Protein Synthesis Inhibition in
Aminoglycoside Ototoxicity. J. Neurosci. 2013, 33 (7), 3079–
3093. https://doi.org/10.1523/JNEUROSCI.3430-12.2013.
Shulman, E.; Belakhov, V.; Wei, G.; Kendall, A.; Meyron-
Holtz, E. G.; Ben-Shachar, D.; Schacht, J.; Baasov, T. Designer
Aminoglycosides That Selectively Inhibit Cytoplasmic
Rather than Mitochondrial Ribosomes Show Decreased
Mol.
Pharmacol.
1993,
247
(2),
155–168.
https://doi.org/10.1016/0922-4106(93)90073-I.
(41)
Herzog, I. M.; Feldman, M.; Eldar-Boock, A.; Satchi-Fainaro,
R.; Fridman, M. Design of Membrane Targeting Tobramycin-
Based Cationic Amphiphiles with Reduced Hemolytic
Activity. Med. Chem. Commun. 2013,
https://doi.org/10.1039/c2md20162c.
4 (1), 120–124.
Ototoxicity:
A Strategy for the Treatment of Genetic
Diseases. J. Biol. Chem. 2014, 289 (4), 2318–2330.
https://doi.org/10.1074/jbc.M113.533588.
(42)
Berkov-Zrihen, Y.; Herzog, I. M.; Benhamou, R. I.; Feldman,
M.; Steinbuch, K. B.; Shaul, P.; Lerer, S.; Eldar, A.; Fridman,
M. Tobramycin and Nebramine as Pseudo-Oligosaccharide
Scaffolds for the Development of Antimicrobial Cationic
Amphiphiles. Chem. - A Eur. J. 2015, 21 (11), 4340–4349.
https://doi.org/10.1002/chem.201406404.
Zhang, Q.; Alfindee, M. N.; Shrestha, J. P.; Nziko, V. D. P. N.;
Kawasaki, Y.; Peng, X.; Takemoto, J. Y.; Chang, C. W. T.
Divergent Synthesis of Three Classes of Antifungal
Amphiphilic Kanamycin Derivatives. J. Org. Chem. 2016, 81
(22), 10651–10663. https://doi.org/10.1021/acs.joc.6b01189.
Subedi, Y. P.; Alfindee, M. N.; Takemoto, J. Y.; Chang, C. W.
T. Antifungal Amphiphilic Kanamycins: New Life for an Old
Drug. Med. Chem. Commun. 2018, pp 909–919.
https://doi.org/10.1039/c8md00155c.
Jaber, Q. Z.; Benhamou, R. I.; Herzog, I. M.; Ben Baruch, B.;
Fridman, M. Cationic Amphiphiles Induce Macromolecule
Denaturation and Organelle Decomposition in Pathogenic
Yeast. Angew. Chemie - Int. Ed. 2018, 57 (50), 16391–16395.
https://doi.org/10.1002/anie.201809410.
Bera, S.; Dhondikubeer, R.; Findlay, B.; Zhanel, G. G.;
Schweizer, F. Synthesis and Antibacterial Activities of
Amphiphilic Neomycin B-Based Bilipid Conjugates and
Fluorinated Neomycin B-Based Lipids. Molecules 2012, 17
(8), 9129–9141. https://doi.org/10.3390/molecules17089129.
Dahlgren, J. G.; Anderson, E. T.; Hewitt, W. L. Gentamicin
(29)
(30)
Greber, B. J.; Bieri, P.; Leibundgut, M.; Leitner, A.; Aebersold,
R.; Boehringer, D.; Ban, N. The Complete Structure of the 55S
Mammalian Mitochondrial Ribosome. Science (80-. ). 2015,
348 (6232), 303–308. https://doi.org/10.1126/science.aaa3872.
Hobbie, S. N.; Akshay, S.; Kalapala, S. K.; Bruell, C. M.;
Shcherbakov, D.; Böttger, E. C. Genetic Analysis of
Interactions with Eukaryotic RRNA Identify the
Mitoribosome as Target in Aminoglycoside Ototoxicity.
Proc. Natl. Acad. Sci. U. S. A. 2008, 105 (52), 20888–20893.
https://doi.org/10.1073/pnas.0811258106.
Hobbie, S. N.; Bruell, C. M.; Akshay, S.; Kalapala, S. K.;
Shcherbakov, D.; Böttger, E. C. Mitochondrial Deafness
Alleles Confer Misreading of the Genetic Code. Proc. Natl.
Acad. Sci. U. S. A. 2008, 105 (9), 3244–3249.
https://doi.org/10.1073/pnas.0707265105.
Perez-Fernandez, D.; Shcherbakov, D.; Matt, T.; Leong, N.
C.; Kudyba, I.; Duscha, S.; Boukari, H.; Patak, R.; Dubbaka,
S. R.; Lang, K.; Meyer, M.; Akbergenov, R.; Freihofer, P.;
Vaddi, S.; Thommes, P.; Ramakrishnan, V.; Vasella, A.;
Böttger, E. C. 4′-O-Substitutions Determine Selectivity of
Aminoglycoside Antibiotics. Nat. Commun. 2014, 5.
https://doi.org/10.1038/ncomms4112.
Herzog, I. M.; Louzoun Zada, S.; Fridman, M. Effects of 5- O
-Ribosylation of Aminoglycosides on Antimicrobial Activity
and Selective Perturbation of Bacterial Translation. J. Med.
(43)
(44)
(45)
(31)
(32)
(46)
(33)
(34)
(47)
(48)
(49)
(50)
Blood Levels:
Agents Chemother.
https://doi.org/10.1128/AAC.8.1.58.
A
Guide to Nephrotoxicity. Antimicrob.
Chem.
2016,
59
(17),
8008–8018.
1975, (1), 58–62.
8
https://doi.org/10.1021/acs.jmedchem.6b00793.
Matsushita, T.; Sati, G. C.; Kondasinghe, N.; Pirrone, M. G.;
Kato, T.; Waduge, P.; Kumar, H. S.; Sanchon, A. C.; Dobosz-
Bartoszek, M.; Shcherbakov, D.; Juhas, M.; Hobbie, S. N.;
Schrepfer, T.; Chow, C.; Polikanov, Y. S.; Schacht, J.; Vasella,
A.; Böttger, E. C.; Crich, D. Design, Multigram Synthesis, and
Black, R. E.; Lau, W. K.; Weinstein, R. J.; Young, L. S.; Hewitt,
W. L. Ototoxicity of Amikacin. Antimicrob. Agents
Chemother.
1976,
9
(6),
956–961.
https://doi.org/10.1128/aac.9.6.956.
Bock, B. V.; Edelstein, P. H.; Meyer, R. D. Prospective
Comparative Study of Efficacy and Toxicity of Netilmicin
and Amikacin. Antimicrob. Agents Chemother. 1980, 17 (2),
217–225. https://doi.org/10.1128/AAC.17.2.217.
Kashuba, A. D.; Bertino, J. S.; Nafziger, A. N. Dosing of
Aminoglycosides to Rapidly Attain Pharmacodynamic Goals
and Hasten Therapeutic Response by Using Individualized
Pharmacokinetic Monitoring of Patients with Pneumonia
Caused by Gram-Negative Organisms. Antimicrob. Agents
Chemother. 1998, 42 (7), 1842–1844.
in Vitro and in Vivo Evaluation of Propylamycin:
A
Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside
for the Treatment of Drug-Resistant Enterobacteriaceae and
Other Gram-Negative Pathogens. J. Am. Chem. Soc. 2019, 141
(12), 5051–5061. https://doi.org/10.1021/jacs.9b01693.
Lopez-Gonzalez, M. A.; Delgado, F.; Lucas, M.
Aminoglycosides Activate Oxygen Metabolites Production
in the Cochlea of Mature and Developing Rats. Hear. Res.
1999, 136 (1–2), 165–168. https://doi.org/10.1016/s0378-
5955(99)00122-7.
(35)
(51)
Matt, T.; Ng, C. L.; Lang, K.; Sha, S. H.; Akbergenov, R.;
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 12 of 21
Shcherbakov, D.; Meyer, M.; Duscha, S.; Xie, J.; Dubbaka, S.
Hermann, T.; Pilch, D. S. Defining the Molecular Forces That
R.; Perez-Fernandez, D.; Vasella, A.; Ramakrishnan, V.;
Schacht, J.; Böttger, E. C. Dissociation of Antibacterial
Activity and Aminoglycoside Ototoxicity in the 4-
Monosubstituted 2-Deoxystreptamine Apramycin. Proc.
Natl. Acad. Sci. U. S. A. 2012, 109 (27), 10984–10989.
https://doi.org/10.1073/pnas.1204073109.
Ishikawa, M.; García-Mateo, N.; Čusak, A.; López-
Hernández, I.; Fernández-Martínez, M.; Müller, M.;
Rüttiger, L.; Singer, W.; Löwenheim, H.; Kosec, G.; Fujs, Š.;
Martinez-Martinez, L.; Schimmang, T.; Petković, H.;
Knipper, M.; Durán-Alonso, M. B. Lower Ototoxicity and
Absence of Hidden Hearing Loss Point to Gentamicin C1a
and Apramycin as Promising Antibiotics for Clinical Use. Sci.
Rep. 2019, 9 (1), 2410. https://doi.org/10.1038/s41598-019-
38634-3.
Determine the Impact of Neomycin on Bacterial Protein
Synthesis: Importance of the 2′-Amino Functionality.
Antimicrob. Agents Chemother. 2007, 51 (5), 1760–1769.
https://doi.org/10.1128/AAC.01267-06.
Wasserman, M. R.; Pulk, A.; Zhou, Z.; Altman, R. B.; Zinder,
J. C.; Green, K. D.; Garneau-Tsodikova, S.; Doudna Cate, J.
H.; Blanchard, S. C. Chemically Related 4,5-Linked
Aminoglycoside Antibiotics Drive Subunit Rotation in
Opposite Directions. Nat. Commun. 2015, 6, 7896.
https://doi.org/10.1038/ncomms8896.
Długosz, M.; Trylska, J. Aminoglycoside Association
Pathways with the 30s Ribosomal Subunit. J. Phys. Chem. B
2009, 113 (20), 7322–7330. https://doi.org/10.1021/jp8112914.
Amunts, A.; Brown, A.; Toots, J.; Scheres, S. H. W.;
Ramakrishnan, V. The Structure of the Human
Mitochondrial Ribosome. Science. 2015, 348 (6230), 95–98.
https://doi.org/10.1126/science.aaa1193.
Qian, Y.; Guan, M. X. Interaction of Aminoglycosides with
Human Mitochondrial 12S RRNA Carrying the Deafness-
Associated Mutation. Antimicrob. Agents Chemother. 2009,
53 (11), 4612–4618. https://doi.org/10.1128/AAC.00965-08.
Garreau De Loubresse, N.; Prokhorova, I.; Holtkamp, W.;
Rodnina, M. V.; Yusupova, G.; Yusupov, M. Structural Basis
for the Inhibition of the Eukaryotic Ribosome. Nature 2014,
513 (7519), 517–522. https://doi.org/10.1038/nature13737.
Duscha, S.; Boukari, H.; Shcherbakov, D.; Salian, S.; Silva, S.;
Kendall, A.; Kato, T.; Akbergenov, R.; Perez-Fernandez, D.;
Bernet, B.; Vaddi, S.; Thommes, P.; Schacht, J.; Crich. D.;
Vasella, A.; Böttger, E. C. Identification and Evaluation of
1
2
3
4
5
6
7
8
(65)
(52)
9
(66)
(67)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(53)
(54)
Toxicological Evaluation of Certain Veterinary Drug
Residues in Food; 2012, 39-63.
Kondo, J.; Urzhumtsev, A.; Westhof, E. Two Conformational
States in the Crystal Structure of the Homo Sapiens
Cytoplasmic Ribosomal Decoding A Site. Nucleic Acids Res.
2006, 34 (2), 676–685. https://doi.org/10.1093/nar/gkj467.
Kondo, J.; François, B.; Urzhumtsev, A.; Westhof, E. Crystal
Structure of the Homo Sapiens Cytoplasmic Ribosomal
Decoding Site Complexed with Apramycin. Angew. Chemie
(68)
(69)
(70)
(55)
(56)
(57)
-
Int.
Ed.
2006,
45
(20),
3310–3314.
https://doi.org/10.1002/anie.200600354.
Tsai, A.; Uemura, S.; Johansson, M.; Puglisi, E. V.; Marshall,
R. A.; Aitken, C. E.; Korlach, J.; Ehrenberg, M.; Puglisi, J. D.
The Impact of Aminoglycosides on the Dynamics of
Translation Elongation. Cell Rep. 2013, 3 (2), 497–508.
https://doi.org/10.1016/j.celrep.2013.01.027.
Prokhorova, I.; Altman, R. B.; Djumagulov, M.; Shrestha, J.
P.; Urzhumtsev, A.; Ferguson, A.; Chang, C. W. T.; Yusupov,
M.; Blanchard, S. C.; Yusupova, G.; Puglisi, J. D.
Aminoglycoside Interactions and Impacts on the Eukaryotic
Ribosome. Proc. Natl. Acad. Sci. U. S. A. 2017, 114 (51),
E10899–E10908. https://doi.org/10.1073/pnas.1715501114.
Vicens, Q.; Westhof, E. Crystal Structure of Geneticin Bound
to a Bacterial 16 S Ribosomal RNA A Site Oligonucleotide. J.
Improved
Deoxystreptamines as next-Generation Aminoglycoside
Antibiotics. MBio 2014, (5).
https://doi.org/10.1128/mBio.01827-14.
4'-O-(Alkyl)
4,5-
Disubstituted
2-
5
(71)
Kalinec, G.; Thein, P.; Park, C.; Kalinec, F. HEI-OC1 Cells as
a Model for Investigating Drug Cytotoxicity. Hear. Res. 2016,
335, 105–117. https://doi.org/10.1016/j.heares.2016.02.019.
Kalinec, G. M.; Fernandez-Zapico, M. E.; Urrutia, R.;
Esteban-Cruciani, N.; Chen, S.; Kalinec, F. Pivotal Role of
Harakiri in the Induction and Prevention of Gentamicin-
Induced Hearing Loss. Proc. Natl. Acad. Sci. U. S. A. 2005,
(72)
(58)
(59)
Mol.
Biol.
2003,
326
(4),
1175–1188.
https://doi.org/10.1016/S0022-2836(02)01435-3.
102
(44),
16019–16024.
Nyffeler, P. T.; Liang, C. H.; Koeller, K. M.; Wong, C. H. The
Chemistry of Amine-Azide Interconversion: Catalytic
Diazotransfer and Regioselective Azide Reduction. J. Am.
https://doi.org/10.1073/pnas.0508053102.
(73)
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.; Fridman, M. 6’’-Thioether
Tobramycin Analogues: Towards Selective Targeting of
Bacterial Membranes. Angew. Chemie - Int. Ed. 2012, 51 (23),
5652–5656. https://doi.org/10.1002/anie.201200761.
Louzoun Zada, S.; Green, K. D.; Shrestha, S. K.; Herzog, I. M.;
Garneau-Tsodikova, S.; Fridman, M. Derivatives of
Ribosome-Inhibiting Antibiotic Chloramphenicol Inhibit
the Biosynthesis of Bacterial Cell Wall. ACS Infect. Dis. 2018,
4 (7), 1121–1129. https://doi.org/10.1021/acsinfecdis.8b00078.
Richter, U.; Lahtinen, T.; Marttinen, P.; Myöhänen, M.;
Greco, D.; Cannino, G.; Jacobs, H. T.; Lietzén, N.; Nyman, T.
A.; Battersby, B. J. A Mitochondrial Ribosomal and RNA
Decay Pathway Blocks Cell Proliferation. Curr. Biol. 2013, 23
(6), 535–541. https://doi.org/10.1016/j.cub.2013.02.019.
Li, C. H.; Cheng, Y. W.; Liao, P. L.; Yang, Y. T.; Kang, J. J.
Chloramphenicol Causes Mitochondrial Stress, Decreases
ATP Biosynthesis, Induces Matrix Metalloproteinase-13
Expression, and Solid-Tumor Cell Invasion. Toxicol. Sci.
2010, 116 (1), 140–150. https://doi.org/10.1093/toxsci/kfq085.
Wu, L. H.; Chang, H. C.; Ting, P. C.; Wang, D. L. Laminar
Shear Stress Promotes Mitochondrial Homeostasis in
Endothelial Cells. J. Cell. Physiol. 2018, 233 (6), 5058–5069.
https://doi.org/10.1002/jcp.26375.
Chem.
Soc.
2002,
124
(36),
10773–10778.
https://doi.org/10.1021/ja0264605.
Mandhapati, A. R.; Shcherbakov, D.; Duscha, S.; Vasella, A.;
Böttger, E. C.; Crich, D. Importance of the 6’-Hydroxy Group
(60)
(61)
(62)
and
Its
Configuration
2014,
for
9
Apramycin
(9),
Activity.
2074–2083.
(74)
(75)
(76)
(77)
ChemMedChem
https://doi.org/10.1002/cmdc.201402146.
Abe, Y.; Nakagawa, S.; Naito, T.; Kawaguchi, H.
Aminoglycoside Antibiotics. XIV Synthesis and Activity of 6-
O-(3-Amino-3-Deoxy-α-d-Glucopyranosyl)-and 5-o-(β-D-
Ribofuranosyl)Apramycins. J. Antibiot. (Tokyo). 1981, 34 (11),
1434–1446. https://doi.org/10.7164/antibiotics.34.1434.
Quirke, J. C. K.; Rajasekaran, P.; Sarpe, V. A.; Sonousi, A.;
Osinnii, I.; Gysin, M.; Haldimann, K.; Fang, Q. J.;
Shcherbakov, D.; Hobbie, S. N.; Sha, S. H.; Vasella, A.;
Böttger, E. C.; Crich. D. Apralogs: Apramycin 5- O-
Glycosides and Ethers with Improved Antibacterial Activity
and Ribosomal Selectivity and Reduced Susceptibility to the
Aminoacyltranserferase (3)-IV Resistance Determinant. J.
Am. Chem. Soc. 2019, 142(1), 530-544.
https://doi.org/10.1021/jacs.9b11601.
jacs.9b11601.
(63)
(64)
Ogle, J. M.; Murphy IV, F. V.; Tarry, M. J.; Ramakrishnan, V.
Selection of TRNA by the Ribosome Requires a Transition
from an Open to a Closed Form. Cell 2002, 111 (5), 721–732.
https://doi.org/10.1016/S0092-8674(02)01086-3.
Barbieri, C. M.; Kaul, M.; Bozza-Hingos, M.; Zhao, F.; Tor, Y.;
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Cytosolic IC50
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G
C
1495
1495
1495
U
U
U
G
G
G
C
C
G
C
U
A
C
U
C
A
A
A
A
A
A
A
A
G
C
A
G
G
C
A
G
A
C
G
U
C
A
A
A
U
G
A
C
C
C
U
C
1490
1490
1490
G
9
10
11
12
13
14
15
D. Bacterial A-site – APR derivatives
E. Cytosolic A-site – APR derivatives
F. Mitochondrial A-site – APR derivatives
4.5
4.5
4.5
4
3.5
3
2.5
2
1.5
1
4
3.5
3
2.5
2
4
3.5
3
16 2.5
2
17
1.5
1
1.5
1
0.5
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
0.5
0
0.5
0
0
C1490 G1491 A1492 A1493 G1494 U1495
A1490 A1491 A1492 A1493 G1494 U1495
A1490 C1491 A1492 A1493 G1494 U1495
Ribonucleotides
Ribonucleotides
Ribonucleotides
G. Bacterial A-site – G-418 derivatives
H. Cytosolic A-site – G-418 derivatives
I. Mitochondrial A-site – G-418 derivatives
4.5
4.5
4.5
4
3.5
3
2.5
2
1.5
1
4
3.5
3
2.5
2
4
3.5
3
2.5
2
1.5
1
1.5
1
0.5
0
0.5
0
0.5
0
C1490 G1491 A1492 A1493 G1494 U1495
A1490 A1491 A1492 A1493 G1494 U1495
A1490 C1491 A1492 A1493 G1494 U1495
Ribonucleotides
Ribonucleotides
Ribonucleotides
apo A-site
Compound 1
G-418
ACS Paragon Plus Environment
APR
Compound 3
Compound 5

4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
Journal of the American Chemical SPoacgiet1y8 of 21
>3.70
>3.70
>3.70
3.25
1
2
3
4
5
6
7
2.29
ACS Paragon Plus Environment
APR
1
3
G-418
5

1,600
1,400
Page 19Jofu2rn1al of the American Chemical Society
1,200
1,000
1800
2600
3400
4200
5
6
7
8
ACS Paragon Plus Environment
0
CTRL CAM S-14 APR APR
250 40 50 300
1
50
1
300
3
50
3
300
G-418 G-418
50 300
5
50
5
300
μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL
% permeabilized cells
% dead cells

350
300
250
200
Journal of the American Chemical SoPcaigeety20 of 21
1
150
2
100
3
4
50
5 0
6
ACS Paragon Plus Environment
CTRL CAM APR
25 10
APR
100
1
10
1
100
3
10
3
100
G-418 G-418
10 100
5
10
5
100
μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL μg/mL
7
% mitochondrial translation
% cytosolic translation
8

Page 21 of 21
Journal of the American Chemical Society
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Art-work for Table of Content
82x44mm (600 x 600 DPI)
ACS Paragon Plus Environment