Tobramycin Variants with Enhanced Ribosome-Targeting Activity

Article abstract of DOI:10.1002/cbic.201500256

With the increased evolution of aminoglycoside (AG)-resistant bacterial strains, the need to develop AGs with 1) enhanced antimicrobial activity, 2) the ability to evade resistance mechanisms, and 3) the capability of targeting the ribosome with higher ef

Full text of DOI:10.1002/cbic.201500256

DOI: 10.1002/cbic.201500256
Communications
Tobramycin Variants with Enhanced Ribosome-Targeting
Activity
Marina Y. Fosso,[a] Hongkun Zhu,[b] Keith D. Green,[a] Sylvie Garneau-Tsodikova,*[a] and
Kurt Fredrick*[b]
With the increased evolution of aminoglycoside (AG)-resistant
bacterial strains, the need to develop AGs with 1) enhanced
antimicrobial activity, 2) the ability to evade resistance mecha-
nisms, and 3) the capability of targeting the ribosome with
higher efficiency is more and more pressing. The chemical deri-
vatization of the naturally occurring tobramycin (TOB) by at-
tachment of 37 different thioether groups at the 6’’-position
led to the identification of generally poorer substrates of TOB-
targeting AG-modifying enzymes (AMEs). Thirteen of these dis-
played better antibacterial activity than the parent TOB while
retaining ribosome-targeting specificity. Analysis of these com-
pounds in vitro shed light on the mechanism by which they
act and revealed three with clearly enhanced ribosome-target-
ing activity.
is also stabilized.[2] This also inhibits translocation, because A-
tRNA cannot readily move from the A to the P site.[3,7]
The clinical usefulness of AGs has been seriously compro-
mised by the growing prevalence of various resistance mecha-
nisms among pathogenic bacteria. These mechanisms include
the decrease in AG uptake into the bacteria, the alteration of
the bacterial ribosome, and the acquisition of AG-modifying
enzymes (AMEs), which represent the major cause of resistance
to AGs.[8] With more than 100 AMEs identified, these enzymes
pose a serious health threat as they chemically alter the
structures of AGs by N-acetylation (AACs), O-phosphorylation
(APHs), or O-nucleotidylation (ANTs). To overcome this issue,
AGs that could evade the action of AMEs while still targeting
bacterial ribosomal RNA have been investigated. This has led
to the development of structurally constrained AGs that would
mimic the ribosome-bound AG conformation,[9] guanidinylated
AGs,[10] and AG dimers,[11] which have been shown to also bind
viral and human RNAs.[12]
Aminoglycosides (AGs) represent one of the major groups of
antibiotics that target the bacterial ribosome. They interfere
with translation,[1] promote errors during decoding,[2] and in-
hibit translocation[3] and ribosome recycling.[4] AGs have been
shown to target the decoding site of the 30S subunit, interact-
ing at the internal loop of helix h44 in which A1408 lies across
from A1492 and A1493.[5] The most structurally conserved por-
tion of the AGs (rings I and II) forms most of the contacts to
h44. Ring I intercalates into h44, stacking on G1491 and form-
ing hydrogen bonds with A1408. This occludes A1492 and
A1493 from within h44 and hence stabilizes a “flipped out”
conformation of these nucleotides. An analogous rearrange-
ment occurs upon codon recognition during decoding: A1492
and A1493 flip out of h44 and dock into the minor groove of
the codon–anticodon helix.[6] The ability of AGs to stabilize this
conformation of h44 thus appears to disturb translational fidel-
ity during protein synthesis. AG binding pays the energetic
cost for the rearrangement and thereby stabilizes tRNA in the
A site. This leads to miscoding, because near-cognate aa-tRNA
We previously synthesized a number of 6’’-thioether tobra-
mycin (TOB) variants and assayed their antimicrobial activi-
ties.[13] Many of these compounds exhibited bacteriolytic activi-
ty, raising the possibility that the mode of action in these cases
involves membrane disruption rather than translation inhibi-
tion. Herein, we present the synthesis of 18 additional TOB var-
iants (Scheme 1), establish their antibacterial activity profile,
and investigate the mechanism by which the 18 new and the
19 previously reported 6’’-thioether TOB variants inhibit bacte-
rial growth, by using AG-resistant ribosomes with mutations in
the primary helix h44 site.
TOB was modified at the 6’’-position with various thioether
groups (Scheme 1). Whereas compounds 3a–j, 3l, 3m, 3p–t,
3jj, and 3kk have been described previously,[13–14] compounds
3k, 3n, 3o, and 3u–ii are new. Our collection encompasses
a diverse set of 6’’-substituents, including linear, branched, and
cyclic alkyl groups, and substituted aromatic rings.
Compounds 3a–f, 3k–p, and 3u–jj were screened for their
antibacterial activity against 19 diverse bacterial strains, and
their minimum inhibitory concentrations (MICs) were deter-
mined (Table S1 in the Supporting Information). Among the
variants with aliphatic substituents, compound 3 f (with a C14
chain) was the most potent against several of the TOB-resistant
bacterial strains, including the newly tested Enterococcus faeci-
um (C) and Streptococcus pyogenes (L). Compounds bearing ar-
omatic substituents generally exhibited promising antibacterial
activity (MICꢀ9.4 mgmLÀ1) against Bacillus anthracis (A), Bacil-
lus subtilis (B), Listeria monocytogenes (E), Mycobacterium smeg-
matis (I), Staphylococcus aureus NorA (J), Staphylococcus epider-
[a] Dr. M. Y. Fosso,+ Dr. K. D. Green, Dr. S. Garneau-Tsodikova
Department of Pharmaceutical Sciences
College of Pharmacy, University of Kentucky
789 S. Limestone, Lexington, KY 40536-0596 (USA)
E-mail: sylviegtsodikova@uky.edu
[b] H. Zhu,+ Dr. K. Fredrick
Department of Microbiology
Center for RNA Biology, Ohio State University
484 W. 12th Avenue, Columbus, OH 43210-1292 (USA)
E-mail: fredrick.5@osu.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201500256.
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Scheme 1. Synthesis of 6’’-thioether TOB variants. a) RSH, Cs2CO3, DMF; b) TFA.
midis (K), Escherichia coli (N), Haemophilus influenzae (H), and
Shigella flexneri (S). Several of these, including compounds
3m–n, 3p, 3x–z, 3aa, 3cc–dd, 3 ff, 3gg, and 3ii were even
capable of inhibiting the growth of various bacterial strains at
an MICꢀ2.4 mgmLÀ1. Furthermore, additional substitution at
the ortho- or para-positions on the aryl ring appeared to im-
prove antibacterial activity. It is worth mentioning that com-
pounds 3m (MIC 2.4 mgmLÀ1), 3p (MIC 1.2 mgmLÀ1), and 3jj
(MIC 9.4 mgmLÀ1) displayed a 16- to 128-fold decrease in MIC
value compare to TOB (MIC>150 mgmLÀ1) against E. coli over-
pendent manner. Several of these (3m–n, 3p, 3x–z, 3aa–dd,
3 ff–hh), all carrying an aryl ring substituent, were more potent
than TOB and retained target specificity.
Two compounds (3 f and 3g) were found to have indistin-
guishably strong antibacterial activity against D7 prrn WT,
A1408G, and G1491U, showing that these compounds act
through a distinct mechanism, independent of h44. These
compounds, substituted with linear alkyl chains (C14 and C16,
respectively) were previously found to have cellulolytic activi-
ty,[13] which might be their primary mechanism of action. Com-
pounds 3d and 3e, with slightly shorter alkyl chains (C10 and
C12, respectively), have lower activity with little to no h44-de-
pendence.
expressing TolC (O), whereas compound 3z (MIC 18.8 mgmLÀ1
was eight times more active than TOB (MIC 150 mgmLÀ1
against Mycobacterium intracellulare (G).
)
)
As these compounds demonstrated comparable or better
potency against E. coli strains, we further investigated their
mechanisms of action. We compared their abilities to inhibit
growth of E. coli D7 prrn containing wild-type (WT), A1408G, or
G1491U ribosomes. Strain D7 prrn lacks all seven chromosomal
rRNA operons and instead contains a single plasmid-borne
rRNA operon.[15] Hence, each of these strains contains a homo-
geneous population of WT or mutant ribosomes. Mutations
A1408G and G1491U target the primary AG binding site of
helix h44 of the 30S subunit, and each mutation confers resist-
ance to a number of AGs.[16]
TOB inhibited E. coli D7 prrn WT (MIC 18.8 mgmLÀ1) and
failed to inhibit either D7 prrn A1408G or G1491U (MIC>
150 mgmLÀ1; Table S2), indicating that TOB inhibits growth by
binding its h44 site, consistent with previous results.[16a,c] Many
of the TOB variants showed a similar activity profile against
these strains, inhibiting growth in an A1408- and G1491-de-
Finally, a number of compounds were found to be virtually
inactive against all the tested strains. These include com-
pounds 3b, 3c, 3h, and 3i (substituted with C6, C8, C18, or C22
linear alkyl chains, respectively), compound 3s (modified with
a 4-tert-butyl thiophenyl group), and compound 3kk (with a 4-
methylcoumarin-7-yl group).
Some AGs are potent inhibitors of EF-G-dependent translo-
cation.[3,7,17] This is believed to be due to the stabilization of A-
site tRNA, which occurs when these antibiotics bind their pri-
mary site in h44. We suspected that TOB would also inhibit
translocation, allowing effects of the 6’’-substituents to be
compared in vitro. To test this, we purified WT and A1408G
ribosomes from the corresponding D7 prrn strains and used
toeprinting to measure the extent of translocation in the pres-
ence of various concentrations of TOB. The resulting data were
fitted to the Hill equation. TOB strongly inhibited translocation
of WT ribosomes (IC50 16 mm; Figure 1, Table 1). Interestingly,
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*
Figure 1. Effects of TOB variants on ribosomal translocation. The extent of EF-G-dependent translocation was measured in control (WT; ) and mutant
&
(A1408G; ) E. coli ribosomes with various concentrations of TOB variants.
the Hill coefficient derived from the curve fitting was consis-
Table 1. Translocation inhibition activities of TOB variants in wild-type
(WT) or mutant A1408G E. coli ribosomes.
tently less than 1 (0.5–0.7), raising the possibility that TOB
binds its primary (h44) and secondary (H69)[5a] sites in a nega-
tive cooperative manner. With A1408G ribosomes, TOB had
substantially reduced potency (IC50 700 mm), and the data
could be well fit with a Hill coefficient of either 1 or >1, de-
pending on the particular experiment. These data show that
inhibition of translocation by TOB normally depends on the
primary h44 site, and mutation of that site qualitatively
changes the concentration dependence of inhibition.
IC50 [mm]
Control (WT)
IC50 [mm]
Control (WT)
AG
A1408G
AG
A1408G
TOB
3e
3k
3m
3o
3u
16
>1000
330
700
n.d.
660
3d
3h
3l
3n
3p
3v
>1000
>1000
230
n.d.
n.d.
>1000
250
>1000
990
44
47
100
>1000
430
5
36
100
620
650
210
370
240
180
130
410
<1; 360[a]
72
3x
3z
<1; 210[a]
<1; 280[a]
19
310
350
140
230
320
270
100
Next, the effects of several TOB variants on translocation
were analyzed (Figure 1, Table 1). Many behaved similarly to
the parent compound, although differences correlating with
the structure of the substituent were observed. Four com-
pounds inhibited translocation more strongly than TOB in both
WT and A1408G ribosomes. Three of these (3n, 3aa, 3dd)
have ortho-substituted thioaryl groups, whereas the fourth car-
ries a naphthyl moiety (3jj). For the former compounds, IC50
values similarly decreased for WT and mutant ribosomes. Com-
pound 3jj, on the other hand, exhibited some loss of specifici-
3w
3y
3aa
3cc
3ee
3gg
3ii
7.7
3bb
3dd
3 ff
3hh
3jj
<5; 290[a]
33
1.2
<1; 250[a]
66
39
350
11
[a] Curves in these cases exhibited complex concentration dependence.
Data were fitted to the sum of two Hill functions, and the reported
values correspond to the two deduced inflection points (IC50 values).
n.d.=not determined.
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ty, as the ratio of IC50 values (WT vs. A1408G) dropped from 44
to 9. Compounds 3u and 3x (with o-methoxythiophenyl or
o-fluorothiophenyl groups) were notably poorer inhibitors of
translocation.
constraints of the h44 site. Further lengthening of the alkyl
chain to C10 through C16 (3d–g) restored antibacterial activity,
although this activity was h44-independent. Indeed, these
compounds do not appear to target the ribosome, as 3 f failed
to inhibit in vitro translation,[13] and 3e failed to inhibit trans-
location (Figure 1). Rather, these compounds target the cell
membrane and probably act through bacteriolysis.[13] Com-
pounds with even longer chains (3h, C18; 3i, C22) lost potency,
likely due to diminished bacteriolytic activity.
Compounds with meta-substituted thioaryl groups (3o, 3v,
3y, 3bb, 3ee) were generally less potent translocation inhibi-
tors than their ortho-substituted counterparts and had reduced
specificity for WT ribosomes (Figure 1, Table 1). As with the
ortho-substituted set, the methoxythiophenyl- and fluorothio-
phenyl-modified compounds (3v and y) were the weakest in-
hibitors of the group.
As quite a number of our TOB variants demonstrated good
antibacterial activity, we evaluated their susceptibility to AMEs,
which greatly contribute to bacterial resistance to AGs. We
measured the relative activities of four AMEs with our TOB
variants and compared them to that of TOB itself (Figure 2).
Many of the compounds with para-substituted aryl rings
(3w, 3z, 3cc, 3 ff) gave complex inhibition curves that failed to
fit the standard Hill equation (Figure 1). In these cases, the
lowest concentration of drug tested (10 mm) resulted in a sub-
stantial degree of inhibition, and further inhibition occurred
only gradually over the higher range of drug concentrations.
Although the basis of this complexity remains unclear, we
speculate that it might arise from two distinct populations of
ribosomal complexes, one being considerably more sensitive
to AG inhibition than the other. Accordingly, the data were
fitted to the sum of two Hill functions, yielding inflection
points (i.e., IC50 values) for the two putative distinct popula-
tions (Table 1). In the context of the cell, potent inhibition of
even a small subset of translating ribosomes would have dele-
terious consequences, consistent with the enhanced biological
activity of these variants (Table S2). Compound 3x, which car-
ries an ortho-fluorothiophenyl substituent, showed a similarly
complex inhibition curve (Figure 1). Finally, compounds 3d,
3e, and 3h, with linear alkyl chains, failed to inhibit transloca-
tion in vitro (Figure 1, Table 1).
Compounds 3n, 3aa, and 3dd, with ortho-substituted aryl
groups, thus appear to be more potent inhibitors of both bac-
terial growth and ribosomal translocation than TOB in E. coli
strains. In these cases, inhibition of ribosomes in vivo and in
vitro remains strictly dependent on A1408, showing that
higher potency comes without loss of target specificity. These
TOB variants thus bind the primary h44 site with increased af-
finity. The way in which these 6’’-aryl substituents promote
TOB binding remains to be determined. Co-crystal structures
of ribosomes bound to related AGs provide some clues to the
basis of enhanced binding.[5a] Rings I and II of these TOB var-
iants make similar sets of contacts to nucleotides 1407–1409
and 1491–1495 of helix h44. Ring III of gentamicin contacts nu-
cleotides 1405–1407, forming hydrogen bonds with G1405 and
C1407. Presumably, the analogous ring III of TOB occupies
a similar position. The 6’’-aryl groups of the TOB variants might
form a favorable stacking interaction with a nearby rRNA nu-
cleotide, such as C1404, and thereby stabilize binding.
Figure 2. Bar graph showing the relative initial rates of reactions of the
listed AMEs with variants 3a–jj. Rates are normalized to TOB. *indicates that
3 f with AAC(6’)-Ib’ had activity >200% and is not shown here.
For the linear alkyl substituted compounds, potency and
mechanism of action changed as a function of chain length.
Compound 3a, with the shortest chain (C4) of the series, had
biological activity indistinguishable from TOB, inhibiting cell
growth in an A1408- and G1491-dependent manner. When the
length of the chain was increased to C6 (3b), antibacterial
activity was completely lost (Table S2), presumably due to
a steric clash with the ribosome, shedding light on the spatial
Although an increase in catalytic activity of AAC(3)-IV[1a] and
ANT(4’)-Ia[18] was noticeable in the majority of TOB variants
with aliphatic substituents (3a–c, 3 f, 3k, 3l), in general, TOB
variants bearing an aromatic ring appeared to be poorer sub-
strates of these AMEs. This implies that TOB variants with ali-
phatic substituents were more susceptible to modifications by
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ring. In light of the information gathered from the crystal struc-
tures of various AMEs with our parent drug TOB (e.g., AAC(2’)-
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4JD6),[21] along with that of AMEs with other AGs (e.g., AAC(2’)-
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several water molecules and amino acid residues with acidic
side chains. Replacement of the hydroxy group at position 6’’
with a hydrophobic moiety might thus weaken the interactions
of the resulting AGs in the binding pocket, reducing their sus-
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group, exhibited the most potent inhibitory activity against
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Acknowledgements
This work was supported by startup funds from the University of
Kentucky (S.G.-T.), and by NIH grants AI090048 (S.G.-T.) and
GM072528 (K.F.). We thank Aishwarya Devaraj for help with toe-
printing experiments.
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Keywords: aminoglycoside · antibacterial agents · bacterial
resistance · drug-modifying enzymes · translocation
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Manuscript received: May 19, 2015
Accepted article published: May 28, 2015
Final article published: June 17, 2015
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