Amphiphilic tobramycins with immunomodulatory properties

Article abstract of DOI:10.1002/anie.201500598

Amphiphilic aminoglycosides (AAGs) are an emerging source of antibacterials to combat infections caused by antibiotic-resistant bacteria. Mode-of-action studies indicate that AAGs predominately target bacterial membranes, thereby leading to depolarization

Full text of DOI:10.1002/anie.201500598

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Angewandte
Communications
International Edition: DOI: 10.1002/anie.201500598
German Edition: DOI: 10.1002/ange.201500598
Anti-infective Agents
Amphiphilic Tobramycins with Immunomodulatory Properties**
Goutam Guchhait, Anthony Altieri, Balakishan Gorityala, Xuan Yang, Brandon Findlay,
George G. Zhanel, Neeloffer Mookherjee, and Frank Schweizer*
Abstract: Amphiphilic aminoglycosides (AAGs) are an
emerging source of antibacterials to combat infections caused
by antibiotic-resistant bacteria. Mode-of-action studies indicate
that AAGs predominately target bacterial membranes, thereby
leading to depolarization and increased permeability. To assess
whether AAGs also induce host-directed immunomodulatory
responses, we determined the AAG-dependent induction of
cytokines in macrophages in the absence or presence of
lipopolysaccharide (LPS). Our results show for the first time
that AAGs can boost the innate immune response, specifically
the recruitment of immune cells such as neutrophils required
for the resolution of infections. Moreover, AAGs can selec-
tively control inflammatory responses induced in the presence
of endotoxins to prevent septic shock. In conclusion, our study
demonstrates that AAGs possess multifunctional properties
that combine direct antibacterial activity with host-directed
clearance effects reminiscent of those of host-defense peptides.
actions of AAGs were also reported for amphiphilic neo-
mycin and tobramycin analogues.[12,13]
Encouraged by the multimodal activity of cationic amphi-
philic host-defense peptides (HDPs) in the host-directed
clearance of an infection,[16–18] we developed an interest in
exploring whether AAGs can show HDP-like properties.
AAGs that combine direct antibacterial effects with the
induction of immunomodulatory responses in host immune
cells may display superior efficacy against multiple-drug-
resistant (MDR) bacteria. It is noteworthy that for cationic
amphiphilic HDPs like LL-37, the direct antibacterial activity
is antagonized by physiological concentrations of divalent
cations and polyanions, and other host factors.[16,17] However,
HDP-mediated protection has been observed in several
in vivo infection models, thus suggesting that the broad
range of immunomodulatory activities exhibited by these
peptides is the predominant function of HDPs for the
resolution of microbial infections.[17,19–21] With this in mind,
we set out to explore the potential immunomodulatory
properties of AAGs. We were initially interested in develop-
ing multitargeting AAGs that combine the direct antibacte-
rial effect of AGs with the membrane-targeting effects of
AAGs. We selected tobramycin (1; Scheme 1) as the parent
aminoglycoside since it is indispensable in intravenous or
inhaled therapy to treat P. aeruginosa lung infections in cystic
fibrosis patients.[22] Previous studies have shown that amphi-
philic tobramycin analogues bearing a lipophilic group at C-
6“ or C-5 retain potent antibacterial activity.[12,23] Further-
more, it was shown that C-6”-modified amphiphilic tobramy-
cin targets bacterial membranes as its major mode of
antibacterial action,[12] while C-5-modified tobramycin ana-
logues containing positively charged small hydrophobic
chains retain their capacity to interfere with protein syn-
thesis.[23] Moreover, there is crystallographic evidence that the
C-5 hydroxy group in tobramycin is not involved in direct
contacts to model RNA, thus suggesting that structural
modifications at this position may not interfere with RNA
binding.[24]
T
he world is facing an enormous threat from the emergence
and dissemination of bacteria that are resistant to almost all
currently available antibiotics.[1,2] Two strategies, multiple-
component antibiotic adjuvants[3] and single-component-
based antibacterial polypharmacology[4] are currently under
investigation to combat bacterial resistance. Both strategies
seek to exploit multiple modes of action. Recently, amphi-
philic aminoglycosides (AAGs) have emerged as a source of
antibacterial agents to combat bacterial resistance.[5–14] Mode-
of-action studies have shown that AAGs can show different
modes of action[9,11–13] to AGs, which bind to the 30S
ribosomal subunit, thereby leading to the disruption of
protein synthesis.[15] For instance, it was shown that the
antibacterial effect of a neamine-based AAG against P.
aeruginosa was caused by changes in membrane depolariza-
tion and permeability and not by inhibition of protein
synthesis.[9,11] Strong evidence for membrane-targeting inter-
[*] Dr. G. Guchhait, Dr. B. Gorityala, X. Yang, Dr. B. Findlay,
Prof. F. Schweizer
We report herein our investigations into the antimicrobial
properties of the C-5-substituted amphiphilic tobramycin
analogues 4a–f (Scheme 1), which were prepared from
tobramycin (1) by using phase-transfer catalysis for the
alkylation (Scheme 1). Compounds 4a–f were tested for
antibacterial activity by determining the minimal inhibitory
concentration (MIC) against a panel of bacterial strains,
including tobramycin-resistant clinical isolates (Table S1 in
the Supporting Information). Our results show that the
amphiphilic tobramycin analogues 4d–f, which bear lipophilic
tetradecyl, hexadecyl, and octadecyl ether appendages,
respectively, show good activity against gram-positive bac-
teria (GPBs; MIC = 2–16 mgmLÀ1) and reduced activity
Department of Chemistry, University of Manitoba
Winnipeg, MB, R3T 2N2 (Canada)
E-mail: Frank.Schweizer@umanitoba.ca
Prof. G. G. Zhanel
Department of Medical Microbiology and Medicine
Health Science Centre, Winnipeg, Manitoba, R3T 1R9 (Canada)
A. Altieri, Prof. N. Mookherjee
Department of Internal Medicine and Immunology
University of Manitoba, Winnipeg, MB, R3T 2N2 (Canada)
[**] Funding for this project was provided by CIHR (MOP 119335)
Research Manitoba and NSERC.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201500598.
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LL-37 and its analogues
are typically studied at
concentrations of 2.5 to
10 mm.[26,27] Therefore, we
selected a dose of 10 mm
for further assessment of
the immunomodulatory
activity of these analogues.
Plastic-adherent mac-
rophage-like THP-1 cells
were stimulated with 4a
and 4d–f (10 mm), and the
TC supernatants were
monitored for production
of the pro-inflammatory
cytokines TNF-a and IL-
1b as well as the chemo-
kines Gro-a and IL-8 after
24 h, and production of the
anti-inflammatory cyto-
kine IL-1RA after 48 h of
stimulation. None of the
compounds induced the
production of either TNF-
Scheme 1. Synthesis of amphiphilic tobramycins. Boc=tert-butoxycarbonyl, TBDMS=tert-butyldimethylsilyl,
DMF=N,N-dimethylformamide, TBAB=tetra-n-butylammonium bromide.
(MIC = 16–256 mgmLÀ1
)
against gram-negative bacteria
(GNBs; Table S1). The most active AAG 4 f contains an
octadecyl ether chain and consistently displayed the highest
activity against both GPBs (MIC = 2–4 mgmLÀ1) and GNBs
(MIC = 16–128 mgmLÀ1). Notably, when compared to tobra-
mycin (1), an 8-fold reduction in MIC was observed for 4 f
against resistant GNB strains including a tobramycin-resist-
ant E. coli strain and a tobramycin-resistant P. aeruginosa
strain, while a 4-fold or higher reduction was observed against
tobramycin-resistant S. maltophilia. By contrast, the poorly
amphiphilic tobramycin analogue 4b, which contains
a weakly lipophilic hexyl ether chain, showed poor antibac-
terial activity (MIC ꢀ 128 mgmLÀ1) against most bacterial
strains tested (Table S1). Overall, the activity of the AAGs
4d–f is comparable to the antibacterial activity often observed
for antimicrobial peptides and HDPs like LL-37.
Next, we explored the immunomodulatory properties of
the most potent amphiphilic tobramycin ether analogues 4d–
f, while the nonamphiphilic tobramycin methyl ether 4a
served as a negative control. There is little data on the
immunomodulatory effects of tobramycin, although it has
been suggested that a tobramycin–copper complex may
display anti-inflammatory properties.[25] We monitored the
cytotoxic effects of amphiphilic tobramycin analogues in
human monocytic THP-1 cells (ATCC TIB-202). The release
of lactate dehydrogenase was monitored in the tissue culture
(TC) supernatants after 24 h stimulation to assess cytotoxicity.
The amphiphilic tobramycin ether analogues 4d–f showed
negligible or less than 10% cytotoxicity at 5–10 mm, with
a dose-dependent cytotoxicity response between 20 and
80 mm. By contrast, the nonamphiphilic control analogue 4a
showed 20% cytotoxicity at all concentrations tested (Fig-
ure S1 in the Supporting Information). It should be noted that
the immunomodulatory properties of the HDP cathelicidin
Figure 1. Plastic-adherent human macrophage-like THP-1 cells were
stimulated with the tobramycin analogues (10 mm). TC supernatants
were monitored after 24 h by ELISA for the production of the cytokines
TNF-a and IL-1b, as well as the chemokines IL-8 and Gro-a. The
values shown are an average of at least three independent exper-
imentsÆstandard error (* p<0.05).
a, IL-1b, or Gro-a (Figure 1). Likewise no production of the
IL-1-antagonist IL-1RA was observed (data not shown). By
contrast, AAGs 4d–f, but not 4a, significantly induced the
production of the chemokine IL-8 in macrophages (Figure 1).
Previous studies have shown that HDPs such as LL-37 and
indolicidin show similar activity: they do not induce the
production of TNF-a but are potent inducers of the neutro-
phil chemokine IL-8.[27,28] However, LL-37 can also induce the
production of other chemokines such as MCP-1 and Gro-a,
which act as chemoattractants for other leukocytes such as
macrophages.[27,29] The fact that 4d–f selectively induced IL-8
but not Gro-a suggests that these analogues may be
selectively chemoattractant to neutrophils. Since the chemo-
kine IL-8 is a potent neutrophil chemotactic factor required
for the resolution of infections,[30,31] our results suggest that
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the three AAGs 4d–f may, in addition to their antibacterial
activity, be able to mediate the recruitment of immune cells, in
particular neutrophils, to the site of infection.
Previous studies have demonstrated that amphiphilic
cationic HDPs, such as LL-37, can neutralize bacterial
products such as lipopolysaccharide (LPS) and switch the
signaling of Toll-like receptors to the NF-kB pathway induced
by bacterial ligands to control bacterial infections and
pathogen-induced inflammation.[27,32] Since the primary
target cell type involved in the immunomodulatory activity
of HDPs and their analogues has been shown to be macro-
phages,[33] we monitored LPS-induced cytokine production in
the presence and absence of 4d–f and 4a in THP-1 macro-
phages after 24 h stimulation as previously described.[26,27] The
analogues 4d–f abrogated the production of LPS-induced
pro-inflammatory TNF-a when the compounds were added
either at the same time as LPS stimulation (Figure 2A) or
30 min prior to LPS stimulation (Figure 2B). Furthermore,
4d and 4e significantly suppressed LPS-induced IL-1b
production when added either at the same time or 30 min
prior to LPS stimulation (Figure 2). The analogue 4 f signifi-
cantly suppressed LPS-induced IL-1b production when added
simultaneously with LPS stimulation (Figure 2A) but not
when added 30 min prior to stimulation (Figure 2B). The
analogues 4d–f also significantly suppressed the LPS-induced
production of chemokines IL-8 and Gro-a by between 50 and
70% when added either simultaneously or 30 min prior to
LPS stimulation, but they did not completely neutralize
chemokine production (Figure 3). The nonamphiphilic ana-
logue 4a did not suppress the LPS-induced production of
Figure 3. Plastic-adherent human macrophage-like THP-1 cells were
stimulated with 10 ngmLÀ1 of bacterial LPS (gray=without LPS,
black=with LPS) in the presence and absence of the tobramycin
analogues (10 mm). The tobramycin analogues were added either at
the same time as LPS stimulation (A) or 30 min prior to LPS
stimulation (B). TC supernatants were monitored after 24 h by ELISA
for the production of the chemokines IL-8 and Gro-a. The values
shown are an average of at least three independent experimentsÆ
standard error.
either pro-inflammatory cytokines or chemokines under any
conditions (Figure 3 and Figure 4). Next, to determine
whether the ability of the analogues to suppress LPS-induced
cytokine production was due to binding to LPS, we tested the
activity of the analogues in LPS-primed macrophages. The
cells were stimulated for 30 min with LPS, followed by
removal of the TC media and washing of the cells to ensure
the removal of residual LPS in the TC media. Subsequently,
the cells were stimulated with the tobramycin analogues
30 min after LPS stimulation. The analogues 4d and 4e
significantly suppressed LPS-induced TNF-a production,
even when added 30 min after LPS stimulation (Figure 4A),
which suggests that the effect of 4d and 4e in controlling LPS-
induced TNF-a may be due to the alteration of intracellular
signaling mechanisms rather than direct LPS binding. How-
ever, none of the compounds significantly altered the LPS-
induced production of either IL-1b, IL-8, or Gro-a when
added 30 min after LPS stimulation (Figure 4). This is
consistent with previous studies demonstrating that HDPs
such as cathelicidins LL-37 and BMAP-27 inhibit TNF-
a production,[34] whereas defensin HNP-1 promotes IL-1b
production[35] in LPS-primed macrophages. Previous studies
have also demonstrated that HDPs, for example, LL-37 and
BMAP-27, induce the expression of several chemokines and
do not neutralize LPS-induced chemokines.[27,29,36] Taken
together, these results suggest that that the selective modu-
lation of endotoxin-induced inflammatory responses by
certain HDPs, as with 4d and 4e, maybe in part mediated
through alteration of the intracellular signaling downstream
of pattern-recognition receptors in macrophages.
Figure 2. Plastic-adherent human macrophage-like THP-1 cells were
stimulated with 10 ngmLÀ1 of bacterial LPS (gray=without LPS,
black=with LPS), in the presence and absence of the tobramycin
analogues (10 mm). The tobramycin analogues were added either at
the same time as LPS stimulation (A) or 30 min prior to LPS
stimulation (B). TC supernatants were monitored after 24 h by ELISA
for the production of the pro-inflammatory cytokines TNF-a and IL-1b.
The values shown are an average of at least three independent
experimentsÆstandard error.
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Figure 4. Plastic-adherent human macrophage-like THP-1 cells were
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In summary, our results demonstrate for the first time that
AAGs, besides their direct antibacterial activity, can also
induce immunomodulatory responses at concentrations that
are nontoxic to host cells. The multimodal activity of AAGs,
whereby direct antibacterial activity is combined with an
immunomodulatory response, is encouraging since immuno-
modulatory compounds are becoming increasingly important
in anti-infective therapy. Although AAGs were originally
designed to overcome bacterial resistance by targeting both
ribosmal RNA and the bacterial membrane, our study shows
that AAGs can also influence host immune responses. We
have shown that AAGs can boost the innate immune
response, specifically the recruitment of immune cells such
as neutrophils required for resolution of infections. Further-
more, AAGs can selectively control inflammatory responses
induced in the presence of endotoxin to prevent septic shock.
As with certain amphiphilic HDPs, for example, LL-37,
AAGs exhibit modest direct antibacterial activity and immu-
nomodulatory properties for the control of both infection and
pathogen-induced hyperinflammation. AAGs thus represent
a promising avenue for the development of multifunctional
molecules for the prevention or treatment of bacterial
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Received: January 21, 2015
Published online: April 1, 2015
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