Design of membrane targeting tobramycin-based cationic amphiphiles with reduced hemolytic activity

Article abstract of DOI:10.1039/c2md20162c

Tobramycin-based cationic amphiphiles differing in the chemical bond linking their hydrophobic and hydrophilic parts were synthesized and biologically evaluated. Several compounds demonstrated potent antimicrobial activities compared to the parent drug. One analogue exhibited a significant reduction in red blood cells hemolysis, demonstrating that it is possible to maintain the antimicrobial potency of these molecules while reducing their undesired hemolytic effect through chemical modifications. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c2md20162c

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Design of membrane targeting tobramycin-based
cationic amphiphiles with reduced hemolytic activity†‡
Cite this: Med. Chem. Commun., 2013,
4, 120
Ido M. Herzog,a Mark Feldman,a Anat Eldar-Boock,b Ronit Satchi-Fainarob
a
*
and Micha Fridman
Tobramycin-based cationic amphiphiles differing in the chemical bond linking their hydrophobic and
hydrophilic parts were synthesized and biologically evaluated. Several compounds demonstrated potent
antimicrobial activities compared to the parent drug. One analogue exhibited a significant reduction in
red blood cells hemolysis, demonstrating that it is possible to maintain the antimicrobial potency of
these molecules while reducing their undesired hemolytic effect through chemical modifications.
Received 21st June 2012
Accepted 20th July 2012
DOI: 10.1039/c2md20162c
www.rsc.org/medchemcomm
Membranes and cell walls are essential constituents required cell membranes, both Gram-positive and Gram-negative bacte-
for the viability of bacterial cells, and therefore serve as attrac- rial membranes are highly negatively charged due to high
tive targets for the development of antibiotics. Amongst cell- content of anionic lipids such as cardiolipin and phosphati-
wall-targeting antibiotics are several families of peptidoglycan dylglycerol.8,9 Gram-negative bacterial membranes also have the
biosynthesis inhibitors, including b-lactams that irreversibly negatively charged core of lipopolysaccharide (LPS), while
inhibit the activity of the peptidoglycan trans-peptidation negatively charged techoic acids are major constituents of
biosynthetic step,1,2 glyco-peptide antibiotics such as vanco- Gram-positive bacterial cell walls.10,11
mycin that competitively inhibit the trans-peptidation step, and
Hence, both Gram-positive and Gram-negative bacterial
the glyco-lipid antibiotic agent moenomycin A that inhibits the membranes attract positively charged organic compounds
peptidoglycan trans-glycosylation step.3–6 To date, disruption of through ionic interactions. LPS that constitutes the Gram-
the bacterial membrane bi-layer has been poorly exploited as a negative outer membrane leaet is unique to bacteria and
strategy for the development of antibiotics. Bacterial
membrane-disrupting antibiotics offer several advantages over
antimicrobial agents that target intracellular bacterial targets:
rst, membrane disruption is not dependent on the bacterial
cell cycle state and is therefore a promising strategy for the
eradication of dormant bacteria and treatment of persistent
infections.7 Second, antimicrobial agents that act in the extra-
cellular bacterial environment evade intracellular resistance
mechanisms and are expected to maintain prolonged clinical
efficacy. Finally, cell permeability consideration, which is oen
a signicant challenge for drug designers, is not necessary for
the design of membrane-targeting antibiotics. Although pepti-
doglycan exists solely in bacteria, membranes composed of
lipid bi-layers are common to all cells; therefore, avoiding
cytotoxicity to eukaryotic cells through non-selective membrane
disruption is a major challenge. In contrast to most eukaryotic
aSchool of Chemistry, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: mfridman@
post.tau.ca.il; Fax: +972 3-6409293; Tel: +972 3-6408687
bDepartment of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv
University, Tel Aviv 69978, Israel
† This article is part of a MedChemComm ‘New Talents’ issue highlighting the
work of outstanding rising scientists in medicinal chemistry research.
‡ Electronic supplementary information (ESI) available: Synthetic protocols, 1H,
13C NMR assignments and spectra, HRMS, MIC, MBIC, hemolysis assay
Fig. 1 Structures of the Gram-negative bacteria targeting polymyxin B1 and the
procedures are provided. See DOI: 10.1039/c2md20162c
bacterial ribosome targeting aminoglycoside tobramycin.
120 | Med. Chem. Commun., 2013, 4, 120–124
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serves as a target for the antimicrobial agent polymyxin B1 analogue had the least hemolytic activity. We hypothesized that
(Fig. 1). Polymyxin B1 composed of a cyclic cationic decapeptide the aliphatic alkyl chains and the AG scaffold are required for
with an N-terminal hydrophobic residue is a potent and clini- optimal antimicrobial activity but that altering the link between
cally used antibiotic that binds to the negatively charged LPS these two segments should not have a dramatic effect on the
core and disrupts the outer membrane of Gram-negative antimicrobial performance, yet may affect the specicity of
bacteria.12,13 The potency and broad-spectrum activity of poly- these compounds towards different membranes. To test this
myxin B1 against Gram-negative bacteria demonstrate the hypothesis, we chose to evaluate several types of chemical
potential that lies in the development of membrane-targeting bonds between the aliphatic chain and tobramycin (1). We
antibiotics. In recent years, several studies have demonstrated compared the thioether-linked analogues (Scheme 1A, 2a–c) to
the potential of positively charged aminoglycosides (AGs) as sulfone-linked analogues (3a–c), triazole ring-linked analogues
scaffolds for the development of membrane-targeting cationic (Scheme 1B, 4a–c), and amide bond-linked analogues (Scheme
amphiphilic antimicrobial agents by the attachment of hydro- 1C, 5a–c). The thioethers 2a–c were prepared from the penta-
phobic residues to one or more positions on the AG.14–17
NH-Boc-600-O-trisyl tobramycin (Scheme 1A, 1a) as previously
We have been particularly interested in tobramycin (1) based reported.18,19 Oxidation of the protected thioether analogues
cationic amphiphiles since similar to polymyxin B1, this AG also (1b–d) using mCPBA followed by the removal of the NH-Boc
contains ve primary amines which are positively charged protecting groups in neat TFA yielded the sulfone analogues
under physiological conditions (Fig. 1). We recently demon- (3a–c). The 600-O-trisyl group of 1a was replaced by an azide to
strated that the attachment of aliphatic chains to the 600-posi- yield compound 1e,20 which served as a precursor for the
tion of tobramycin resulted in potent antimicrobial agents and preparation of the triazole analogues (Scheme 1B, 4a–c).
provided evidence for their membrane-disruption activity.18
Microwave-heated click reaction using 1e and terminal alkynyl
The most potent and broad-spectrum antimicrobial activity aliphatic chains, followed by the removal of the NH-Boc groups,
was observed for thioether analogues containing C12-, C14-, and yielded the desired triazole analogues 4a–c. Reduction of the 600-
C
16-linear alkyl chains (Scheme 1A, 2a–c). The aliphatic chain azido group of 1e under the Staudinger reaction conditions
length affected not only the antimicrobial activity but also the resulted in superior yields (80%) of the 600-amino tobramycin
level of undesired red blood cell (RBC) hemolysis; the C12 analogue 1f (Scheme 1C) compared to the reduction of the azide
Scheme 1 Synthesis of amphiphilic tobramycin analogues: Reagents and conditions: (a) R0SH, Cs2CO3, DMF, 25–60 ꢀC, 63–92%; (b) neat TFA, rt; (c) mCPBA (3 equiv.),
CHCl3, rt; (d) NaN3, DMF, 60 ꢀC, 12 h, 91%; (e) R0CCH, CuSO4$5H2O (0.1 equiv.), sodium ascorbate (0.2 equiv.), DMF, microwave irradiation, 87–94%; (f) PMe3 (1 M in
THF, 1.1 equiv.), 0.01 M aqueous NaOH/THF: 1/20, rt, 80%; and (g) R0COOH, HBTU, DIEA, DMF, 71–86%.
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under catalytic hydrogenation conditions (H2, Pd/C, MeOH). highly tobramycin resistant: the MIC of tobramycin against S.
Compound 1f served as the precursor for the preparation of the pyogenes M12 (strain A) was 64 mg mLꢁ1; the thioether 2b, triazole
amide analogues (Scheme 1C); 1f was coupled to linear aliphatic 4b, and the amide analogue 5b were 16 to 32 times more potent
carboxylic acids using HBTU (71–86% yield), and the NH-Boc against this strain (2 mg mLꢁ1 for 2b, and 4 mg mLꢁ1 for 4b and
groups were removed to yield the amide-linked analogues 5b). A signicant improvement in antimicrobial activity of the
(Scheme 1C, 5a–c).
semi-synthetic analogues compared to that of tobramycin was
The minimum inhibitory concentrations (MICs) of the semi- also observed in the case of S. mutans UA159 and S. epidermidis
synthetic tobramycin amphiphiles were determined for 11 Gram- ATCC35984 (strains C and G, respectively). Although most of the
positive and Gram-negative strains (Table 1). Amongst the Gram- synthetic analogues were not active against the tested P. aerugi-
positive bacteria were pathogenic strains such as Streptococcus nosa (strain I), the C12 chain triazole analogue 4a and amide
pyogenes M12 (strain A), a hospital isolate of methicillin-resistant analogue 5a demonstrated improved antimicrobial activity
Staphylococcus aureus (MRSA; strain B), and vancomycin-resistant against this strain relative to tobramycin (MICs ¼ 64 and 32 mg
Enterococcus (VRE; strain D) with high levels of resistance to mLꢁ1, respectively, and MIC > 128 mg mLꢁ1 for tobramycin).
tobramycin (MIC ¼ 64 mg mLꢁ1 for strain A and >128 mg mLꢁ1 for
The antibacterial activity of tobramycin and six out of the 12
strains B and D). Amongst the Gram-negative isolates were the synthetic analogues was better against O-antigen positive
pathogenic and highly tobramycin resistant (MIC > 128 mg mLꢁ1
)
S. sonnei (strain J) than against the corresponding O-antigen
Pseudomonas aeruginosa (ATCC33347; strain I) and Shigella sonnei, negative (strain K). This difference may be explained by the
which is responsible for the severe foodborne disease shigellosis. higher overall negative charge of the membrane of the O-
Two types of S. sonnei were tested: O-antigen positive (strain J), antigen positive S. sonnei, which contains the negatively
and O-antigen negative (strain K).21 In general, analogues with a charged 2-acetamido-2-deoxy-L-altruronic acid.22
C14 linear aliphatic chain (2b, 3b, 4b, and 5b) exhibited the most
It was previously demonstrated that low micromolar
potent antimicrobial activity, which was in most cases one to two concentrations of saturated fatty acids inhibit the formation
double dilutions more potent than the activity of the corre- of biolms formed by S. aureus and Listeria monocytogenes
sponding C12 and C16 linear aliphatic chain analogues. The strains.23 The most potent biolm growth inhibitors were C12
–
chemical links between the AG and the aliphatic chain did not
C14 aliphatic chain carboxylic acids. We therefore determined
have signicant effects on MIC values against the tested strains the minimal biolm inhibition concentration (MBIC) values
with the exception of the sulfone linked analogues 3a–c. These for each of the C12 and C14 chain tobramycin analogues
analogues were less potent than the corresponding un-oxidized (Table 2). MBIC tests were performed using S. mutans UA159
thioether analogues 2a–c. Some of the amphiphilic tobramycin and S. epidermidis ATCC35984 grown under biolm-forming
analogues demonstrated high potency against strains that were conditions. Compared to tobramycin (MBIC range of 64–128
mg mLꢁ1), the tested analogues demonstrated improved bio-
lm growth inhibition properties (MBIC range of 4–32 mg
Table 1 MIC values (mg mLꢁ1) of tobramycin and its amphiphilic analogues
mLꢁ1) against the tested strains (Table 2). However, the MBIC
MICs (mg mLꢁ1) for tested bacterial strainsb
values of the tested compounds were identical or no more
than one double dilution lower than their MIC values against
strains C and G. We therefore conclude that these compounds
have no specic biolm growth inhibition properties against
the tested strains, and that their MBIC values result from their
antibacterial activity.
AGa
A
B
C
D
E
F
G
H
I
J
K
1
64 >128 128 >128 >128 16 128 <1 >128
16
32
16
32
32
64
32
64
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
8
2
4
64
32
64
8
2
4
64 128 16
16 64
64 128 16
8
4
8
8
4
4
128
128
8
>128
32 >128 16
64 >128 64 64 128 >128 >128 >128
4
8
8
4
16
16 128
4
8
64
32
128
64
4
8
4
4
8
4
4
4
32
32
64 16 16 16 128
64 16 16 >128
32
64
8
32 128
Table 2 Biofilm growth inhibition. MBIC values (mg mLꢁ1) of the amphiphilic
tobramycin analogues and tobramycin (1)
32 128 16
32 128
16 128 16
64 128 16
32
32 128
8
8
8
8
4
4
16
4
4
8
4
64
128
64
32
64
32
64
8
32
>128 128
AGa
32 128 128
32
32
64
8
8
128
128
16
32
32
32
Strainb
1
2a
2b
3a
3b
4a
4b
5a
5b
4
a
b
AG ¼ aminoglycoside. MIC values were determined against Gram-
positive bacterial strains: A, S. pyogenes serotype M12 (strain
MGAS9429); B, MRSA; C, S. mutans UA159; D, VRE; E, E. faecalis
ATCC29212; F, S. aureus ATCC9144; G, S. epidermidis ATCC35984; H,
C
G
64
128
4
16
4
8
16
32
4
8
4
16
4
16
8
8
4
8
a
b
AG
¼
aminoglycoside.
S. mutans UA159, C; S. epidermidis
S. epidermis ATCC12228 and Gram-negative bacterial strains: I, P. ATCC35984, G. All strains were tested by using the double-dilution
aeruginosa ATCC33347; J, S. sonnei clinical isolate 6831 (O-antigen method (starting from 128 mg mLꢁ1). S. mutans biolm was grown in
positive); and K, S. sonnei clinical isolate 6831 (O-antigen negative). All BHI + sucrose 2%, at nal dilution 1 : 100. S. epidermidis biolm was
strains were tested by using the double-dilution method (from a grown in TSB + glucose 1%, at nal dilution 1 : 100. Biolms were
starting concentration of 128 mg mLꢁ1). All experiments were stained using crystal violet. All experiments were performed in
performed in triplicate, and results were obtained from two different triplicate and results were obtained from two different sets of
sets of experiments.
experiments.
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Fig. 2 Laboratory rat RBC hemolysis assay. Amphiphilic tobramycin analogues were incubated with RBCs isolated from a laboratory rat at concentrations of (A) 32 mg
mLꢁ1, (B) 64 mg mLꢁ1, and (C) 128 mg mLꢁ1 for 1 hour at 37 ꢀC. All experiments were performed in triplicate, and results are the average from two different sets of
experiments using blood samples from two laboratory rats.
Finally, the hemolytic activity was determined using a at the same concentration (0.0 ꢃ 0.4%). At 128 mg mLꢁ1, the
hemolysis assay using laboratory rat RBCs (Fig. 2A–C).18 The triazole analogue 4a caused extensive hemolysis (89.1 ꢃ 1.6%),
MIC and MBIC values were signicantly lower than the the thioether 2a caused 71.6 ꢃ 8.3% hemolysis, yet the amide
concentrations required for 100% hemolysis for some of the analogue 5a caused signicantly less hemolysis (10.2 ꢃ 0.8%).
analogues (Fig. 2). In most cases, the MIC range of analogues The lowest hemolytic activity at all of the tested concentrations
with the C14 aliphatic chain was 2–32 mg mLꢁ1; these analogues was observed for the C12 sulfone analogue 3a, however, this
caused signicant hemolysis (ꢂ23 to 43%) at 32 mg mLꢁ1. All of compound had poor antimicrobial activity against the tested
the C14 aliphatic chain analogues caused extensive hemolysis strains. In contrast, while the C12 amide analogue 5a was potent
(74.4 ꢃ 5.5% to 100%) at a concentration of 64 mg mLꢁ1. The C16 against several of the tested bacterial strains, and was the most
aliphatic chain analogues also caused high levels of hemolysis potent analogue against the tested P. aeruginosa (strain I), it
at 64 mg mLꢁ1 (37.9 ꢃ 5.1% to 81.8 ꢃ 2.3%).
caused the lowest levels of hemolysis at a concentration which
No direct correlation between the antibacterial potency and was 16–32 times higher than the MIC values of this compound
the hemolytic activity was detected for the thioether, triazole, or against several of the tested strains.
amide analogues. As initially hypothesized, the hemolytic
activity of the different tobramycin analogues was affected by
the type of bond between the aliphatic chain and the AG scaf-
Conclusions
fold. The most dramatic effect was observed for the C12 aliphatic In conclusion, 12 600-aliphatic chain tobramycin analogues
chain analogues. At 64 mg mLꢁ1, the triazole C12 aliphatic chain differing in the chemical linkage between the AG and the
analogue 4a demonstrated the highest hemolytic effect (26.3 ꢃ hydrophobic chain (thioether, sulfone, triazole, and amide
2.7%) of the C12 aliphatic chain tobramycin analogues. The C12 bonds) and in the length of their hydrophobic linear aliphatic
aliphatic chain amide analogue 5a caused almost no hemolysis chain (C12, C14, and C16 chains) were synthesized and
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evaluated for their antimicrobial activity against 11 bacterial
strains. Of the three chain lengths tested, the C14 aliphatic
chain analogues were the most potent antimicrobial agents,
and were in most cases one or two double dilutions more
potent than the corresponding C12 and C16 chain analogues. In
some cases, the antimicrobial activity was at least 32-fold more
potent than that of the parent AG tobramycin (1). Finally, RBC
hemolysis tests revealed that there was no linear correlation
between the antimicrobial potency and the hemolytic activity
of the amphiphilic tobramycin analogues. Both the aliphatic
chain length and the type of chemical linkage between the
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Acknowledgements
This work was supported by the FP7-PEOPLE-2009-RG Marie
Curie Action: Reintegration Grants (grant 246673). We thank
Profs Itzhak Ofek, Dani Cohen (Tel Aviv University) and Doron
Steinberg (The Hebrew University of Jerusalem) for the gi of
bacterial strains.
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