Arginine-linked neomycin B dimers: Synthesis, rRNA binding, and resistance enzyme activity

Article abstract of DOI:10.1039/c5md00427f

The nucleotides comprising the ribosomal decoding center are highly conserved, as they are important for maintaining translational fidelity. The bacterial A-site has a small base variation as compared with the human analogue, allowing aminoglycoside (AG) antibiotics to selectively bind within this region of the ribosome and negatively affect microbial protein synthesis. Here, by using a fluorescence displacement screening assay, we demonstrate that neomycin B (NEO) dimers connected by l-arginine-containing linkers of varying length and composition bind with higher affinity to model A-site RNAs compared to NEO, with IC₅₀ values ranging from ~40-70 nM, and that a certain range of linker lengths demonstrates a clear preference for the bacterial A-site RNA over the human analogue. Furthermore, AG-modifying enzymes (AMEs), such as AG O-phosphotransferases, which are responsible for conferring antibiotic resistance in many types of infectious bacteria, demonstrate markedly reduced activity against several of the l-arginine-linked NEO dimers in vitro. The antimicrobial activity of these dimers against several bacterial strains is weaker than that of the parent NEO.

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Arginine-linked neomycin B dimers: synthesis,
rRNA binding, and resistance enzyme activity†‡
Cite this: DOI: 10.1039/c5md00427f
Yi Jin,a Derrick Watkins,b Natalya N. Degtyareva,b Keith D. Green,c
b
c
ab
*
*
Meredith N. Spano, Sylvie Garneau-Tsodikova and Dev P. Arya
The nucleotides comprising the ribosomal decoding center are highly conserved, as they are important for
maintaining translational fidelity. The bacterial A-site has a small base variation as compared with the
human analogue, allowing aminoglycoside (AG) antibiotics to selectively bind within this region of the ribo-
some and negatively affect microbial protein synthesis. Here, by using a fluorescence displacement screen-
ing assay, we demonstrate that neomycin B (NEO) dimers connected by L-arginine-containing linkers of
varying length and composition bind with higher affinity to model A-site RNAs compared to NEO, with
IC50 values ranging from ~40–70 nM, and that a certain range of linker lengths demonstrates a clear prefer-
ence for the bacterial A-site RNA over the human analogue. Furthermore, AG-modifying enzymes (AMEs),
such as AG O-phosphotransferases, which are responsible for conferring antibiotic resistance in many
types of infectious bacteria, demonstrate markedly reduced activity against several of the L-arginine-linked
NEO dimers in vitro. The antimicrobial activity of these dimers against several bacterial strains is weaker
than that of the parent NEO.
Received 23rd September 2015,
Accepted 21st October 2015
DOI: 10.1039/c5md00427f
www.rsc.org/medchemcomm
RNA. The binding of AGs to the aminoacyl (A)-site of the ribo-
some interferes with this recognition process, which results
Introduction
The growing problem of antibiotic resistance presents an
urgent need for the development of new therapeutics to treat
bacterial infections. One class of commonly prescribed broad-
spectrum antibiotics, the aminoglycosides (AGs), combat
Gram-positive and Gram-negative bacteria by selectively bind-
in mistranslation of the mRNA.3–6
Bacterial AG antibiotic resistance is commonly con-
ferred via AG-modifying enzymes (AMEs), namely AG
O-phosphotransferases (APHs), AG N-acetyltransferases
(AACs), and AG O-nucleotidyltransferases (ANTs), which ren-
der their substrate molecules unable to bind at their respec-
tive ribosomal sites of action.7–10 Resistant bacterial strains
carry plasmids containing genes for these enzymes, and any
forthcoming AG antibiotics must be able to bypass this trou-
blesome resistance mechanism.
ing bacterial ribosomes and inhibiting protein synthesis.1,2
A
particularly vulnerable site for antibiotic targeting is the
decoding center of the small ribosomal subunit, which is
responsible for matching the correct aminoacyl-transfer (t)
RNA with the three-nucleotide codon of the messenger (m)
Therefore, when developing novel AGs, it is crucial that (i)
they be highly selective for the bacterial A-site over the
human A-site counterpart, as mutations in human mitochon-
drial ribosomes that result in higher affinity for these ligands
often lead to drug toxicity,11,12 and (ii) they evade the action
of the AMEs responsible for the majority of resistance to
these drugs. In this work, we have combined the two afore-
mentioned molecular design strategies in order to produce
novel compounds that are both poor substrates for AMEs and
display increased affinities for their RNA targets over their
unmodified parent AG. We report the synthesis and screen-
ing of a series of neomycin B (NEO) dimers that are joined by
L-arginine-containing linkers of varying lengths. NEO dimers
with triazole, urea, and thiourea linkages have been shown to
be poor substrates for certain AMEs,13 and both NEO dimers
and L-arginine-conjugated AGs have been reported to increase
a Clemson University, Department of Chemistry, Clemson, SC, 29634, USA.
E-mail: dparya@clemson.edu
b NUBAD, LLC, Greenville, SC, 29605, USA
c University of Kentucky, Department of Pharmaceutical Sciences, 789 South
Limestone Street, Lexington, KY, 40536-0596, USA.
E-mail: sylviegtsodikova@uky.edu; Fax: 859 257 7585; Tel: 859 218 1686
† DPA conceived and designed the dimer compounds and RNA binding experi-
ments. YJ synthesized the dimers. KDG, under SGT supervision, performed MIC
values determination against bacterial strains and experiments against AMEs.
NND assisted with manuscript preparation. NND and DW performed RNA
screening assays. MNS, SGT, and DPA wrote the manuscript. SGT prepared all
figures.
‡ Electronic supplementary information (ESI) available: A figure showing the
preparation of compounds 6–15 (Fig. S1) is presented. Experimental procedures
and images of spectroscopic characterization of all newly synthesized com-
pounds (Fig. S2–S23) and of IC50 plots (Fig. S24 and S25) are also provided. See
DOI: 10.1039/c5md00427f
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ligand affinity for nucleic acids as compared with their
unconjugated counterparts.14–21 Other AG homo- and hetero-
dimers have also been reported to display promise in
enhancing RNA binding, improving antibiotic activity, and/
or resisting the action of AMEs.22–29 Our novel dimers dis-
play higher affinities for both human and bacterial RNA
A-site model constructs than does NEO, as shown by a fluo-
rescence displacement assay, and also show slight binding
preferences among the bacterial and human constructs
used in this study. Significantly, AMEs display very different
activity for these L-arginine-linked NEO dimer substrates
than they do for NEO, and notably, these dimers are very
poor substrates especially for the AG O-phosphotransferases
APHIJ2″)-Ia and APHIJ3′)-Ia. Despite these collective differ-
ences, the dimers' antimicrobial activities are comparable to
those of NEO.
displaces it.34 Fluorescence emission intensity of F-NEO is
decreased when it is bound within the major groove of RNA,
analogously to non-conjugated NEO, as confirmed by docking
experiments. Upon displacement from the RNA target oligo-
mer by a competitive binder, the fluorescence of F-NEO
increases, thereby reporting on ligand binding.
The dissociation constants (Kd) between the F-NEO probe
and A-sites measured by direct titrations were 4 1 nM and
23 3 nM for E. coli and human A-sites, respectively. The
approximate four-to-six-fold difference between affinities of
NEO to E. coli and human A-sites has been previously docu-
mented.35 We assessed the percentage of displacement of
F-NEO by compounds 6–16 by comparison of fluorescence
emission of F-NEO (Fig. 3) in the presence of model A-site
RNAs and again upon addition of a single concentration of
the tested compounds. In order to better distinguish between
NEO-like binders to model A-site RNAs, the ligand concentra-
tion used in competitive binding experiments was approxi-
mately equal to the NEO IC50, where the IC50 concentration is
defined as a concentration at which 50% of F-NEO is
displaced. No increase in emission due to the displacement
of the probe was observed for control molecule 16, indicating
that it did not bind appreciably to either of the A-site RNAs.
All NEO dimers, 6–15, demonstrated stronger binding prefer-
ences for both A-sites as compared with NEO. This result is
consistent with previous findings demonstrating that the
presence of a single positively-charged arginine enhances the
binding ability of NEO dimers to RNAs as compared with
NEO alone.30
We also observed that the linker length affected the bind-
ing affinities of the L-arginine-linked NEO dimers to the A-site
RNAs as shown by IC50 measurements (Table 1 and Fig. S24–
S25‡). Compounds 13 and 14, with 6 and 7 consecutive car-
bons within their linkers, respectively, demonstrated the
strongest affinity for the E. coli A-site of all the compounds
tested. IC50 values were also measured for human A-site.
Measured IC50 values of these compounds for the human
A-site were similar to those measured for the E. coli A-site
(Table 1). The affinities of compounds 7, 11, 12, and 13 were
higher for the E. coli A-site than for the human homologue,
indicating that the consecutive carbons chains within the
arginine linkers have an optimum length of 4–6 carbons, and
that the presence of this carbon chain assists in distinction
between two A-sites. Since the reported IC50 values were
determined by F-NEO displacement and are therefore relative
to F-NEO affinity for each RNA, and since F-NEO has a ~5.8-
fold larger affinity for the E. coli versus the human A-site
RNA, we can report that all of the L-arginine-linked NEO
dimers have a higher affinity for the E. coli A-site than does
NEO, and most likely preferences for one A-site for another
are actually larger than the reported IC50 values imply. There-
fore, we have calculated selectivity factor that indicates the
preferential affinity ratio of each compound for the E. coli
A-site as compared with the human A-site. The affinity of
NEO for the E. coli A-site is five times stronger than for the
human homologue.
Results and discussion
Synthesis of L-arginine NEO dimers
We successfully achieved the synthesis of nine novel triazole-
linked L-arginine-NEO dimers (compounds 7–15) by using a
high-yielding robust synthetic “click chemistry” approach
followed by standard 9-fluorenylmethoxycarbonyl (Fmoc)-
based solid-phase peptide synthesis (Fig. 1). Our initial focus
was to synthesize the terminal dialkyne linker 4 in order to
achieve triazole-linked NEO dimers via click chemistry. To
this end, we synthesized compound 4 in two steps from ethyl
3,5-dibromobenzoate (2) via Sonogashira–Hagihara cross-
coupling followed by removal of the 2-hydroxyisopropyl group
of compound 3 to generate free terminal alkynes in molecule
4 (Fig. 1A). We synthesized the Boc-protected NEO azide from
commercially available NEO in three steps30 and further
coupled it with the dialkyne compound 4 to successfully pro-
duce the triazole-linked NEO dimer 5 via click chemistry
(Fig. 1A). The presence of a free carboxyl functional group in
5 proved to be extremely beneficial for further modifications
using solid-phase peptide chemistry. To this end, we rapidly
generated a library of L-arginine-NEO dimers 7–15 with vary-
ing chain lengths by using Fmoc-based solid-phase peptide
chemistry (Fig. 1B and S1B‡). Compound 5 was further
deprotected to obtain NEO dimer 6 with a free carboxyl group
(Fig. S1A‡), which was used as a control molecule for the
binding study. Detailed experimental procedures and charac-
terization data (NMR spectra and HRMS/MALDI-TOF MS; Fig.
S2–S23‡) for compounds 6–15 are presented in the ESI.‡
Screening of compounds 6–16 for binding to model A-sites
In order to establish relative affinity of the L-arginine-linked
NEO dimers for human and bacterial ribosomal A-sites, we
assessed dimers 6–15, NEO, and a control molecule 16
(Fig. 1C), along with a 27-base model A-site RNA oligomers
representing E. coli and human rRNA sequences by using a
fluorescence displacement assay (Fig. 2).31–33 In this ligand
binding assay, F-NEO, a conjugate of NEO and fluorescein,
serves as a fluorescent reporter of binding when a ligand
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Fig. 1 Synthetic schemes for the preparation of A. compound 5, and B. arginine-linked NEO dimers 6–15. C. Structure of compound 16 used as a
control in binding studies to model A-sites.
Antibacterial activity
weight of our compounds (ranging from 2818 to 3284 g
Having studied the binding of our compounds to model A-
sites, we next investigated our L-arginine-linked NEO dimers
6–15 for their inhibition of antimicrobial growth against
three bacterial strains: Bacillus cereus ATCC 11778, Staphylo-
coccus epidermidis ATCC 12228, and Staphylococcus aureus
ATCC 25923. The minimum inhibitory concentration (MIC)
values for dimers 6–15 and NEO, which served as a control,
are summarized in Table 2. Due to the large molecular
mol−1) we provide the MIC values in μM, which we feel is
best suited for larger molecules. However, as it is standard
practice to provide MIC values in μg mL−1 in microbiology,
we also included these values into parentheses in Table 2. A
range is reported for the concentrations at which a growth
inflection point was not observed. While most of the NEO
dimers showed little activity against S. epidermidis and S.
aureus, all dimers had a profound inhibitory effect on the
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Table 2 MIC values in μM (and in μg mL−1 into parentheses) of com-
pounds 6–15 and NEO
B. cereus
S. epidermidis
S. aureus
Compound
ATCC 11778
ATCC 12228
ATCC 25923
NEO
6
7
8
9
≤0.1 (≤0.1)
3–6 (9–18)
3–6. (10–19)
1.6 (5)
0.2 (0.2)
13 (35)
>50 (>154)
>50 (>157)
50 (158)
0.2 (0.2)
13 (35)
>50 (>154)
50 (157)
50 (158)
0.8 (2.5)
10
11
12
13
14
15
1.6–3 (5–10)
3–13 (10–40)
1.6–3 (5–10)
3 (10)
3–6 (10–19)
3–6 (10–20)
>50 (>159)
>50 (>159)
>50 (>160)
>50 (>161)
25 (81)
>50 (>159)
>50 (>159)
>50 (>160)
>50 (>161)
>50 (>161)
>50 (>164)
Fig. 2 rRNA A-site constructs. Nucleotides implicated in AG binding
are shown in a turquoise box in the E. coli and human A-site struc-
tures. Nucleotides that match part of the crystal structures used for
modelling in Fig. 5 are shown in a pink box in the E. coli and human
A-site structures. Nucleotides are colored as follow: A = green, G =
red, C = yellow, and U = blue.
50 (164)
dimers showed promising activity (MIC values ranging from
0.8–13 μM) against B. cereus, with dimers 8 and 9 being the
best with MIC values of 1.6 μM and 0.8 μM, respectively. As
observed previously, the positive charge of the L-arginine
group in the linker is likely to assist with bacterial membrane
penetration and binding to the negatively charged RNA.
Aminoglycoside-modifying enzyme (AME) activity assays
Since the alteration of AG functional groups by AMEs is the
most common mechanism of bacterial AG resistance, we
finally investigated our NEO dimers 6–15 as potential sub-
strates of N-acetyltransferase (AAC) and O-phosphotransferase
(APH) resistance enzymes (Fig. 4). For the AACs, we selected
AACIJ2′)-Ic from Mycobacterium tuberculosis,36–38 AAC(3)-IV
from Escherichia coli,39,40 and AACIJ6′)-Ie from the bifunc-
tional AACIJ6′)-Ie/APHIJ2″)-Ia from S. aureus,39,41–43 which are
known to acetylate NEO at the 2′-, 3, and 6′-position, respec-
tively. We also elected to test against Eis, a unique AAC
found in various bacterial strains, which is known to multi-
acetylate AGs at a variety of positions.38,44–48 For the APHs,
Fig. 3 Bar graph showing the percentage of displacement of F-NEO
by compounds 6–16. NEO is shown as a control.
growth of the spore-forming B. cereus, demonstrating the
selectivity of our compounds towards certain bacterial
strains. Interestingly, compound 6, which does not contain
arginine, showed better inhibition of S. aureus and S. epi-
dermidis than the L-arginine-containing NEO dimers 7–15. All
Table 1 IC50 values (nM) and selectivity factors for E. coli over human
A-site of compounds 6–15 and NEO
Compound
E. coli A-site
Human A-site
76 13
Selectivity factor
NEO
6
7
8
9
10
11
12
13
14
15
87
51
55
62
70
56
52
54
44
48
60
6
6
1
2
3
8
2
4
2
2
3
5.0
5.6
7.4
5.3
4.7
5.3
7.4
7.2
7.3
5.2
5.9
50
71
58
58
52
70
68
56
43
62
5
1
2
1
3
8
6
1
1
3
Fig. 4 Bar graph showing the relative initial rates of the listed AMEs
with NEO and L-arginine-linked NEO dimers 6–15. All rates were
normalized to the parent NEO.
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we selected APHIJ2″)-Ia and APHIJ3′)-Ia,13,49 which commonly
phosphorylate NEO. The activity of the enzymes for each
compound was normalized to that of NEO. It is important
to note that we did not test our NEO dimers 6–15 against
ANTs due to the facts that (i) most ANTs modify streptomy-
cin or spectinomycin,7 and (ii) the bacterial strains against
which these compounds were tested in this study are not
known to contain ANTs that modify NEO.13
Interestingly, the AAC(3)-IV enzyme that acetylates the
3-amino group of NEO was found to be similarly or more
active against certain dimers (6 and 8–12) than NEO. Its activ-
ity was found to be inversely proportional to linker length,
and gradually decreased with increasing linker length for
L-arginine-containing linkers. Compound 7 (n = 0), was modi-
fied by AAC(3)-IV at approximately 30% of the rate when com-
pared to the NEO modification.
All other AMEs tested demonstrated much lower enzy-
matic activity towards compounds 6–15, which were found
to be poorer substrates for these enzymes than NEO. The
most likely explanation for these positive results is that
steric hindrance from the bulky second NEO and/or the
Fig.
5
Docked structures of the L-arginine-linked NEO dimer 11
(depicted as orange stick) with the E. coli (right) and human RNA (left)
A-sites. The nucleoside bases are colored as follow: A = green, G =
red, C = pale yellow, and U = blue. The pink box represent the residues
that are identical to those presented in Fig. 2.
increase in molecular dynamics afforded by the flexible Conclusions
linker prevents proper and/or stable binding of the NEO
In summary, we have designed a series of NEO dimers with L-
group at the active site of the enzyme. In support of this
argument, compound 15 with longest L-arginine tail is the
least susceptible substrate for the AMEs tested.
arginine-containing linkers of varying lengths to target the
ribosomal A-site. These dimers displayed higher affinity for
both human and bacterial RNA A-site model constructs than
the parent NEO does, as shown by a fluorescence displace-
ment assay. These compounds also showed slight binding
preferences among the bacterial and human A-site model
constructs, with a carbon linker length of 4–6 being optimal
for E. coli A-site preference over the human sequence. The
dimers displayed antimicrobial effects that were weaker than
their parent AG, NEO. It is interesting to note that dimer 6
also displayed significant antibacterial activity. Significantly,
AMEs showed very different activity for the arginine-linked
NEO dimer substrates than they do for NEO. As compared
with NEO, our dimers are very poor substrates for several
AMEs, especially for the phosphotransferases APHIJ2″)-Ia and
APHIJ3′)-Ia, as well as for the AACIJ6′)-Ie/APH-(2″)-Ia. Our
L-arginine-linked NEO dimers therefore present a potential
avenue for the development of therapeutics to combat AG
antibiotic-resistant infections.
Overall, the O-phosphotransferases APHIJ2″)-Ia and APHIJ3′)-
Ia, as well as AACIJ6′)-Ie/APH-(2″)-Ia showed greatly reduced
activity against the dimers. Since phosphorylation of AG func-
tional groups by APHIJ3′)-Ia is one of the most commonly
encountered AG antibiotic resistance mechanisms,10 these
results indicate that our L-arginine-linked dimers are a prom-
ising avenue for future development as antimicrobial
therapeutics.
A computer generated model of dimer 11 bound to the two
RNA A-sites
Docking experiments using Auto Dock were performed
with dimer 11, as it showed a significant difference in
binding between the two A-sites. These docking experi-
ments were performed to offer a potential explanation of
our experimental findings. Indeed, different binding modes
were observed when we docked 11 with E. coli as well as
human RNA A-sites. In the 11-E. coli RNA A-site complex,
NEO rings interact in the same region of the RNA (Fig. 5,
please see pink boxed region), as established for AG (NEO)
binding with E. coli RNA A-site (Fig. 2). However, in the
binding of 11 with human RNA A-site, NEO rings interact
with RNA bases away from adenines A1490–A1493 (Fig. 5).
The difference in binding sites may also cause the
observed difference in binding site of the L-arginine
containing side chain of 11 (Fig. 5). Together, these two
factors likely contribute to the seven-fold difference in
binding of 11 with E. coli RNA A-site as compared to
human RNA A-site.
Acknowledgements
This work was supported by the National Institutes of Health
(NIH) grants 2R42GM097917 (D. P. A.) and AI090048 (S. G.-T.)
and by startup funds from the College of Pharmacy at the
University of Kentucky (S. G.-T.). We thank Dr. Sayantan
Bhaduri for assistance with the description of the synthetic
scheme. We thank Dr. Souvik Sur for modelling.
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