A Salmochelin S4-Inspired Ciprofloxacin Trojan Horse Conjugate

Article abstract of DOI:10.1021/acsinfecdis.0c00568

A novel ciprofloxacin-siderophore Trojan Horse antimicrobial was prepared by incorporating key design features of salmochelin, a stealth siderophore that evades mammalian siderocalin capture via its glycosylated catechol units. Assessment of the antimicro

Full text of DOI:10.1021/acsinfecdis.0c00568

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Article
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A Salmochelin S-inspired Ciprofloxacin Trojan Horse Conjugate
Thomas Sanderson, Conor Black, James Southwell, Ellis Wilde, Apurva Pandey, Reyme
Herman, Gavin H. Thomas, Eszter Boros, Anne Kathrin Duhme-Klair, and Anne Routledge
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.0c00568 • Publication Date (Web): 11 Aug 2020
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A Salmochelin S4-inspired Ciprofloxacin Trojan Horse Conjugate
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Thomas J. Sanderson[a], Conor M. Black[a], James W. Southwell[a], Ellis J. Wilde[a], Apurva Pandey[b],
Reyme Herman[c], Gavin H. Thomas[c], Eszter Boros*[b], Anne-Kathrin Duhme-Klair*[a], Anne
Routledge*[a].
[a] Department of Chemistry, University of York, Heslington, York, YO10 5DD (UK)
[b] Department of Chemistry, Stony Brook University, 100 Nicolls Road, Stony Brook, New York 11790 (US)
[c] Department of Biology (Area 10), University of York, Wentworth Way, Heslington, York, YO10 5DD (UK).
A novel ciprofloxacin-siderophore Trojan Horse antimicrobial was prepared by incorporating key design features of salmochelin, a
stealth siderophore that evades mammalian siderocalin capture via its glycosylated catechol units. Assessment of the antimicrobial
activity of the conjugate revealed that attachment of the salmochelin mimic resulted in decreased potency, compared to ciprofloxacin,
against two Escherichia coli strains, K12 and Nissle 1917, in both iron-replete and deplete conditions. This observation could be
attributed to a combination of reduced DNA gyrase inhibition, as confirmed by in vitro DNA gyrase assays, and reduced bacterial
uptake. Uptake was monitored using radiolabelling with iron-mimetic 67Ga3+, which revealed limited cellular uptake in E. coli K12.
In contrast, previously reported staphyloferrin-based conjugates displayed measurable uptake in analogous 67Ga3+ labelling studies.
These results suggest that in designing Trojan Horse antimicrobials, the choice of siderophore, and the nature and length of the linker
remains a significant challenge.
KEYWORDS: Siderophores, Antibiotics, Drug Design, Radiolabelling, Bioinorganic Chemistry
The problem of antimicrobial resistance has reached a critical
level and authorities now believe humankind is entering a post-
antibiotic era where minor infections and routine medical pro-
cedures will become major morbidity threats.1-4 New strategies
in the form of new antibiotic targets or modification of current
antibiotics to bypass resistance mechanisms are urgently
needed.4-8 One approach is a Trojan Horse delivery strategy that
targets outer membrane barrier permeability resistance9-13 by
utilizing bacterial iron transport, mediated by siderophores, to
promote active antibiotic uptake; a number of examples can be
found in the literature.14-20 This strategy has met with some suc-
cess, with a number of siderophore-drug conjugates entering
clinical trials.4, 21-23 One example, cefiderocol (Fetroja, Figure
1), was recently approved by the U.S. Food and Drug Admin-
istration (FDA) for treatment of complicated urinary tract infec-
tions (cUTIs).24
including Salmonella spp., and various pathogenic E. coli
strains.25, 28, 29
The salmochelins belong to a class of stealth siderophores,
which maintain their role in bacterial iron acquisition whilst
evading the host innate immune response.30 As a response to an
invading pathogen, the mammalian host secretes immunopro-
tein siderocalin to capture Fe3+-loaded catecholate-based sider-
ophores.31, 32 The glycosyl units in the salmochelins prevent
siderocalin binding, thus allowing the siderophores to avoid
capture. The ability to produce and utilize salmochelins relies
on the iroA gene cluster,30 which encodes the enzymes respon-
sible for the glycosylation of catechol units, the secretion of the
biosynthesized salmochelins, the uptake of their Fe3+ complexes
and finally, the hydrolysis of both the apo- and Fe3+-bound si-
derophores.33 Whilst the salmochelin uptake mechanism,34, 35 is
thought to share similarities with that of enterobactin,18, 36-38 the
enterobactin receptor protein FepA, found in all E. coli, is una-
ble to mediate significant uptake of salmochelin S4, whereas
the outer membrane receptor protein IroN, which is expressed
only in strains that harbor iroA, allows uptake of both salmo-
chelin S4 and enterobactin (see Figure 3).25 This key difference
means that the presence of the iroA gene cluster is considered a
virulence factor.
S
O
H2N
NH
O
N
S
O
Cl
N
N
O
N
OH
OH
O
N
O
H
O
OH
Cefiderocol
Figure 1. Structure of FDA-approved Trojan Horse antibiotic
cefiderocol (antibiotic unit highlighted in red).
Salmochelin siderophores, identified in 2003,25 are a family of
siderophores consisting of glycosylated variants of the high-af-
finity tris-catecholate siderophore enterobactin produced by
many of the Enterobacteriaceae.26 The family derives from the
di-glycosylated hexadentate salmochelin S4 (Figure 2) and in-
cludes a series of di- and mono-glycosylated hydrolysis prod-
ucts.27 Salmochelins are biosynthesized by a range of bacteria,
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O
OH
OH
O
OH
OH
N
N
N
H
3
N
O
O
NH
O
HO
OH
HN
O
O
R
HN
NH
O
O
O
O
O
HN
O
O
O
O
O
O
OH
O
OH
O
O
O
NH
O
HN
O
HO
OH
H
O
HO
O
HO
OH
O
N
N
NH
S
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H
HN
O
HO
OH
OH
OH
COOH
O
OH
OH
OH
O
HO
OH
OH
HO
OH
Linear
Enterobactin
Dimer
OH
Enterobactin
Figure 4. Salmochelin- lactam conjugate designed and synthe-
sised by Nolan et al. (antibiotic component in red, chemical linker
in blue, and siderophore in black).
OH
OH
OH
O
As part of our wider investigation into fluoroquinolone sidero-
phore conjugates,40-42 we herein report the design and synthesis
of a first generation ciprofloxacin-salmochelin S4 inspired Tro-
jan Horse antimicrobial 1 (Figure 5), designed to evade the
mammalian siderocalin immune response and to selectively tar-
get bacteria expressing salmochelin transport machinery.
HO
HO
OH
O
HO
HO
OH
HN
O
O
O
O
OH
O
NH
O
O
O
OH
OH
O
HN
HO
OH
O
OH
OH
HO
OH
HO
OH
O
HO
HO
OH
Salmochelin S4
HN
Figure 2. Chemical structures of siderophores: Enterobactin, Lin-
ear Enterobactin Dimer and Salmochelin S4, with characteristic
glycosylated catechol units of Salmochelin S4 highlighted.
O
O
OH
N
NH
F O
N
N
HO
OH
O
O
HO
1
Figure 5. Salmochelin S4-inspired Trojan Horse antimicrobial.
Results and Discussion
Design and Synthesis
In our approach to the design of a first generation salmochelin
S4-inspired ciprofloxacin conjugate, the salmochelin S4 struc-
ture was simplified to a synthetically accessible structure, com-
prising an aliphatic link based on L-lysine between the catechol
units, and a reduction of siderophore denticity from hexadentate
to tetradentate. L-lysine was utilised to mimic the chirality and
exact length of the backbone between the catechol moieties in
salmochelin in a hydrolytically-stable manner; the ester back-
bone in salmochelin is prone to hydrolysis under physiological
conditions. The tetradentate siderophore mimic allows for im-
proved synthetic tractability than if the full tri-serine scaffold of
salmochelin S4 was used. The carboxylic acid group of the ly-
sine moiety provides an attachment point for the antimicrobial.
It was shown previously that tetradentate, diamine-linked
bis(catecholates) can function as siderophore components in
Trojan Horse antimicrobials, which were shown to penetrate at
least the outer membrane of Gram-negative bacteria, including
E. coli.43, 44
Figure 3. Simplified schematic representation of the outer mem-
brane receptor proteins involved in Fe3+-siderophore uptake in all
E. coli (left) and pathogenic E. coli capable of producing salmo-
chelin (right).
The production of siderocalin by the host may limit the appli-
cation of catecholate siderophores in Trojan Horse antimicrobi-
als, unless the siderophore component is structurally modified
to evade the immune response, whilst maintaining high affinity
for Fe3+. The use of salmochelin-based siderophore components
offers the opportunity of specifically targeting bacteria that ex-
press the salmochelin transport machinery, whilst minimizing
disruptions to the host microbiome. This approach has been suc-
cessfully demonstrated by Nolan et al. with the chemoen-
zymatic synthesis of glucosylated enterobactin--lactam conju-
gates (Figure 4).39
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We anticipated compound 1 to possess similar iron-binding
benzoyl ester 8 via a transient t-butyloxycarbonyl nitrogen pro-
tection/deprotection strategy. It was then coupled to NN-
diboc-L-lysine via EDC-mediated amide formation to give 9.
properties to the tetradentate linear enterobactin dimer45 (Figure
2) and a previously described salmochelin S1 mimic,46 as both
feature similar 2,3-dihydroxybenzamide iron-chelating moie-
ties attached to a 5-atomic backbone. We have previously in-
vestigated the Fe3+ coordination chemistry of these two tetra-
dentate bis(catecholates) by using both UV-vis and CD spec-
troscopy and observed rapidly equilibrating mixtures of 1:1 and
2:3 complexes, both monomers and dimers. In compound 1,
however, the deprotonated carboxylic acid and adjacent car-
bonyl donor of the ciprofloxacin moiety could act as an addi-
tional third iron chelating unit and hence affect the iron-binding
properties.47, 48
Deprotection of the lysine-associated protecting groups yielded
10. Lysine functionalized ciprofloxacin was coupled with glu-
cosylated catechol 7 to give benzyl-protected salmochelin-in-
spired conjugate 11. Global debenzylation with Pearlman’s cat-
alyst52 furnished salmochelin-inspired ciprofloxacin conjugate
1 in a moderate yield. Experimental details are provided in the
SI.
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O
OMe
OBn
OBn
O
OMe
OH
OMe
O
OMe
OBn
OAc
O
The C5-glycosyl-2,3-dihydroxybenzoyl units of salmochelin
S4 (highlighted in Figure 2) were amalgamated into the design
but connected via NNof L-lysine, with the C-terminus
providing an appropriate handle for attachment of the parent an-
tibiotic, ciprofloxacin. Suitably functionalized catechol units
were synthesized using previously described methodologies.
Commercially available methyl 3-methoxysalicylate 2 was io-
dinated with iodine monochloride,49 then demethylated. This
was followed by benzylation of the free phenolic hydroxyl
groups to give 3. Acetyl-protected -glucose was installed via
nickel-promoted Negishi coupling using an adapted literature
procedure50,51 to give aryl-C-glucoside 4. The glucosyl acetyl
protecting groups of 4 were substituted with benzyl ethers, re-
sulting in the formation of two compounds 5 and 6, which were
combined, then hydrolysed to give the free carboxylic acid 7
(Scheme 1).50
i,ii,iii
iv
I
OBn
AcO
OAc
O
2
OAc
3
4
O
OR
OBn
OBn
OH
OBn
OBn
OBn
OBn
v
vi
O
O
BnO
OBn
BnO
OBn
OBn
OBn
5 R=OMe
6 R=OBn
7
Scheme 1. i) AgNO3, ICl, pyridine, CHCl3, 68%; ii) 1. BBr3,
CH2Cl2; 2. H2SO4, CH3OH, 91%; iii) Benzyl bromide, NaI, DMF,
72%; iv) 1-bromo--D-glucose tetraacetate, Zn, LiCl, Ni(COD)2,
tBuTerpy, DMF, 63%; v) 1. Na2CO3, MeOH; 2. Benzyl bromide,
NaH, Bu4NI, DMF, 5 + 6 74%; vi) NaOH, THF:MeOH (3:1), 87%.
In order to furnish a suitably functionalized antimicrobial to al
low the salmochelin analogue to be constructed, L-lysine-ap-
pended ciprofloxacin 10 was synthesized as shown in Scheme
2. Commercially available ciprofloxacin was converted into its
O
O
O
N
O
O
O
F
F
F
OBn
OBn
OH
i
ii
N
N
BocHN
BocHN
N
N
N
H2N
TFA
N
HN
4
8
9
O
OBn
OBn
iii
O
O
N
O
BnO
BnO
OBn
O
F
OBn
TFA
NH3
OBn
BnO
OBn
N
v
TFA
H3N
iv
HN
1
N
BnO
4
O
O
OBn
O
N
NH
10
N
N
F
O
BnO
OBn
O
O
11
BnO
Scheme 2. i) 1. Boc2O, NaOH, 1,4-dioxane, H2O; 2. Benzyl bromide, Cs2CO3, DMF; 3. TFA, CH2Cl2, 51%; ii) N N-Boc-lysine, EDC.HCl,
HOBt.H2O, DIPEA, DMF, 55%; iii) TFA, CH2Cl2, 100%; iv) 7, EDC.HCl, HOBt.H2O, DIPEA, DMF, 42%; v) Pd(OH)2/C, H2, MeOH:
EtOAc (1:1), 45%.
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Fe3+ Complex Formation
to a much lesser degree than uptake via porins.59 However, it is
unlikely 1, with an projected polar surface area of 401 Å,60
would display a similar capacity for passive diffusion as ciprof-
loxacin, with a corresponding approximated polar surface area
of 82 Å, in its zwitterionic form; a high polar surface area cor-
relates strongly with decreased membrane permeability.61
The Fe3+ complex of 1 (1:1 ratio) was prepared by combining
equimolar amounts of 1, dissolved in DMSO, and FeCl3 hexa-
hydrate, dissolved in water. The solvents were removed in
vacuo and the resulting residue was taken up in a volatile buffer
(ammonium acetate) to enable the species formed at pH 7.4 to
be examined by native ion mass spectrometry (experimental de-
tails are provided in the SI). Complexation was confirmed by
UV/vis spectroscopy (Figure S1). The observed λmax of the lig-
and-to-metal charge transfer band at 544 nm is consistent with
the prevalence of a chromophore in which two catecholate units
are coordinated to Fe3+, i.e. an equimolar Fe3+-to-1 ratio.46
It was hoped that the presence of the salmochelin transport ma-
chinery in Nissle 1917 would support active uptake of the Fe3+
complex of 1 and hence increase its antibacterial activity,
thereby allowing the selective targeting of this strain.
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The antibacterial activity assays were carried out in MOPS Ac-
etate minimal media,62 either in the presence of 100 μM Fe3+
(iron replete conditions) or with no added Fe3+ (iron deplete
conditions, <18 pM Fe). Details of the composition and iron
content of the media are provided in the SI.
Interestingly, the negative ion ESI mass spectrum showed evi-
dence for the formation of both 1:1 and 2:2 species, the latter
consisting of two Fe3+ cations and two 15- ligands (Scheme S1).
The most abundant ions were observed at m/z 553.1336,
583.1437, 737.8462 and 1107.2695. Based on their isotope pat-
terns and charges, these peaks could be assigned to the acetate
adduct of the monomeric species [Fe3+L5-+acetate-+H+]2-, and
the dimeric species [(Fe3+L5-)2]4-, [(Fe3+L5-)2+H+]3- and [(Fe3+L5-
)2+2H+]2- (Figure S2). The equilibrium shifts slowly from the
monomeric towards dimeric species over the course of several
days (Figures S2a-c).
As expected, in E. coli K12 (BW25113), which lacks the IroN
transporter, 1 demonstrated a much lower antibacterial activity
than ciprofloxacin under both iron replete and iron deplete con-
ditions. Disappointingly, when the activity was tested against
Nissle 1917, capable of expressing IroN, a similar lack of activ-
ity of 1 was observed, suggesting that active uptake of 1 is not
taking place (Table 1). This observation led to a further investi-
gation of factors limiting uptake/activity (vide infra).
Table 1. MIC values of conjugate 1 and ciprofloxacin (cipro)
determined in acetate/MOPS minimal media under iron replete
(100 μM Fe3+) and deplete (<18 pM Fe) conditions.
The composition of these ions suggests that the acetate in the
1:1 species and the carboxylate and keto O-donors of ciproflox-
acin in the 2:2 species may be recruited to complete the six-fold
coordination sphere of the iron center. Due to the rigid nature
of ciprofloxacin, its O-donor atoms can only coordinate to an
adjacent iron centre, and this may trigger dimer formation. In
addition, H-bonding interactions with to the glucose unit on the
siderophore may support dimer formation.
E. coli K12 (BW25113)
Fe replete Fe deplete
E. coli Nissle 1917
Fe replete Fe de-
plete
1
>100 μM 75 μM
>100 μM 100 μM
cipro
1 μM
1 μM
0.1 μM
8 μM
In addition, a solution containing Fe3+ and conjugate 1 in a 2:3
ratio was prepared in an analogous way. The λmax of the ligand-
to-metal charge transfer band in the UV/vis spectrum did not
shift significantly (Figure S1) and the most abundant ions ob-
served in the negative ion ESI mass spectrum were those as-
signed to 1:1 and 2:2 species, as above, plus free ligand (conju-
gate 1). The formation of a 2:3 complex (Scheme S1) was not
apparent (Figure S3). This suggests that the coordination of a
third ligand is not favoured in this case, potentially due to steric
hindrance or competitive binding of the acetate or keto and/or
carboxylate O-donors of the ciprofloxacin unit.
Antibacterial Activity vs E. coli K12 (BW25113)
Conjugate 1 was added to the growth media at varying concen-
trations (0 – 100 μM, Figure 6). At higher concentrations of 1
in iron-replete conditions immediate Fe3+-chelation was evident
by the emergence of a characteristic purple color due to Fe3+-
siderophore complex formation.45 Due to the significant absorb-
ance of these complexes at 600 nm, the optical density of the
cell cultures is displayed at 800 nm (OD800). As expected, at
plateaued bacterial growth (after 48 h) and in the absence of
antimicrobial agents, the bacterial cell density obtained in Fe3+
replete media was more than twice as high than that obtained
under Fe3+ limitation (Figure 6, Figure S6).
Antibacterial Activity Testing
The antibacterial activity of both 1 and the parent drug ciprof-
loxacin was tested against two bacterial strains: 1. E. coli K12
(BW25113), a common laboratory strain that does not express
IroN, the outer membrane receptor protein required for active
salmochelin uptake,25 and 2. E. coli Nissle 1917, a probiotic
strain that expresses IroN and is able to utilize salmochelin.29 In
addition to serving as a negative control, the K12 strain was in-
vestigated to confirm that non-pathogenic bacterial strains that
are unable to utilize salmochelin remain unaffected. The size of
1 renders it unlikely to be taken up passively via porins, OmpF
or OmpC.53-57 OmpF is considered the main uptake pathway for
ciprofloxacin, but has a molecular weight limit of around 600
Da.58 In the absence of active transport or porin-mediated up-
take, passive diffusion across the outer membrane is a possibil-
ity; this has been suggested to occur for ciprofloxacin, although
a)
b)
Figure 6. E. coli K12 (BW25113) growth in minimal media
(MOPS Acetate) in the presence of 1 at t = 48 h in a) iron replete
(striped bars) and b) iron deplete conditions (filled bars).
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In iron replete media, ciprofloxacin, the parent antimicrobial of
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1, was active at low concentrations between 0.1-1 M (Figure
7a), whilst 1 only showed growth suppression once the concen-
tration started to approach that of Fe3+ in the growth medium
(>75 M). In iron deplete conditions, a clear growth inhibitory
effect was already observed at a 25 M concentration of 1.
Whilst the latter might be an indication of salmochelin-medi-
ated uptake of 1, the attenuation of bacterial growth may also
be attributed to the competition of 1 with native siderophores
for the more limited Fe3+ resource, starving the cells of the vital
nutrient. Again, the antibacterial activity of 1 was much lower
than that of the parent antibiotic ciprofloxacin (~75x, Figure
7b). Hence, to further explore the relationship between sidero-
phore-mediated Fe3+ uptake and the antibacterial activity of 1,
a radiolabelling study with the Fe3+-mimetic 67Ga3+ was under-
taken.
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Figure 8. Time-dependent, radiochemical bacterial uptake studies
in E. coli K12 (MG1655) of 67Ga-1 in iron replete (striped bars) and
iron deplete media (solid bars). Error bars calculated as standard
deviation of n = 5.
a)
b)
67Ga-1 was sufficiently inert for subsequent uptake experi-
ments, we monitored complex inertness in LB broth over the
course of 2 hours, during which no significant de-chelation was
observed. Next, we assessed the time dependent bacterial up-
take of 67Ga-1 in E. coli K12 (MG1655) in iron replete LB over
2 hours. The percentage of internalized 67Ga-1 was <10% of the
total 67Ga and comparable to the uptake of a 67Ga-citrate control.
The results in iron deplete LB media were similar, with <10%
internalization, comparable to that seen with 67Ga-citrate (Fig-
ure 8).
Figure 7. E. coli K12 (BW25113) growth in minimal media
(MOPS Acetate) in the presence of ciprofloxacin at t = 48 h in a)
iron replete (striped bars) and b) iron deplete conditions (solid
bars).
To confirm if this measured uptake mirrored low transport into
bacteria, with the resultant absence of activity, the study was
expanded to assess three compounds previously reported by
us, staphyloferrin A-inspired conjugates 12, 13 and 14 (Figure
9).40 These conjugates retain and mirror the original sidero-
phore structure more closely, and provide a 6-coordinate lig-
and environment for corresponding Fe3+ and Ga3+ complexes.
These compounds showed activity against E. coli NCTC10418
and some inhibition of DNA gyrase in vitro, albeit at a lower
level than the parent ciprofloxacin.41 All three compounds
have an estimated polar surface area of 329 Å when fully pro-
tonated. In analogy to 67Ga-1, 67Ga-12, 67Ga-13 and 67Ga-14
were synthesized under identical conditions.
67Ga-radiolabeling and Bacterial Uptake
Hexadentate siderophores exhibit exceptionally high affinity
for both Fe3+ and the non-redox active, Fe3+-mimetic Ga3+ (KD
> 10-30 M).63 Functionalized Ga3+ and Fe3+ siderophore com-
plexes are both efficiently recognized by bacterial siderophore
membrane transporters, indicating that bacteria cannot distin-
guish the trivalent ions at the time of siderophore-mediated en-
try. Ga3+ represents an ideal surrogate to study the behaviour of
Fe3+ complex species.64,65 Commercial availability of two radi-
oactive imaging isotopes of Ga3+, 68Ga (t1/2 = 1.1 h), a positron
O
emission tomography (PET) imaging isotope, and 67Ga3+ (t1/2
=
F
COOH
3.3 d), a longer lived isotope utilized for single photon emission
computed tomography (SPECT) provides opportunities to study
the stability and pharmacokinetics of Ga-siderophore com-
plexes in vitro and in vivo. Indeed, this approach has already
been explored for the imaging of fungal infections in rats, tak-
ing advantage of the siderophore-mediated uptake of 68Ga in
fungi such as A. fumigatus,66 as well as the assessment of a
novel desferrichrome-based conjugate that exhibited enhanced
potency in Gram-positive and Gram-negative strains.19
Here, we elected to use 67Ga, as the longer half-life of this iso-
tope permits extensive long-term stability and internalization
studies. It has been shown that 67Ga-labelled deferoxamine
(DFO) retains active uptake via bacterial Fe3+-transport in S. au-
reus with DFO acting as a xenosiderophore.67,68 In order to syn-
thesize 67Ga-1, we first transformed 67Ga-citrate to GaCl3 and
monitored the radiolabelling of 1 by radio-HPLC.19 Complexa-
tion proceeds quickly with quantitative yield, producing an ap-
parent molar activity of 90 nmol/MBq. In order to probe if the
N
N
O
N
H
O
N
*
NH
12 n=1 (R)-orn
13 n=1 (S)-orn
14 n=2 (S)-lys
n
HOOC
O
OH
COOH
OH
COOH
HOOC
Figure 9. Structures of staphyloferrin A-ciprofloxacin conjugates
12-14.
The radiolabeled complexes were less stable than 67Ga-1 in both
iron replete and iron deplete LB broth, exhibiting percentage of
intact radiochemical complexes ranging from 67Ga-14 (70%),
67Ga-12 (60%), to 67Ga-13 (50%). No further trans-chelation
was observed after 9 h, indicating that a relative equilibrium
state is reached. In iron deplete LB broth, complex stability was
marginally lower but again maintained over 9 h.
To assess relative complex inertness in comparison with the 6-
coordinate chelator ethylene-diamine-tetraacetate (EDTA), the
radiochemical gallium complexes were challenged with a 10-
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fold excess with respect to conjugate concentration. Complex
However, the antimicrobial activity of 1 was found to be similar
inertness was monitored using radio-HPLC. After 2 hours, the
to that observed with the K12 strain. In iron replete media, clear
growth inhibition was only seen at 100 μM concentrations and
above, when the concentration of 1 reached the 100 μM con-
centration of Fe3+ (Figure 11a). This suggests that under these
conditions, the sequestration of Fe3+ by the siderophore unit of
1 becomes so significant that it deprives the growth medium of
this vital nutrient. The fact that ciprofloxacin already suppresses
growth at ~1000x lower concentrations (0.1 μM, Figure 12a), is
consistent with a lack of bacterial uptake of 1.
relative inertness can be ranked as follows: 67Ga-14 > 67Ga-12
>
67Ga-13 > 67Ga-1 (Figure S11). This result indicates that in
the presence of a six-coordinate, competing chelator, the trans-
chelation from a 4-coordinate donor such as 1 occurs rapidly;
the 6-coordinate, staphyloferrin-based conjugates provide a
more inert coordination environment, with the lysine-derived
structure, 67Ga-14, showing the best stability in this challenge
assay.
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Uptake experiments in E. coli K12 (MG1655) in both iron re-
plete and deplete LB broth were assessed in comparison with
67Ga-1. Although still low (up to 14.7% after 0.33 h for 67Ga-
13, Figure 10), uptake of all three conjugates was higher than
observed with 67Ga-citrate control, and marginally higher than
67Ga-1, suggesting a very modest degree of translocation
through bacterial cell membranes, corroborating antibacterial
activity results obtained in previous work.40-42
In iron deplete media, a slight growth inhibitory effect is already
evident at a 1 μM concentration of 1 (Figure 11b). This obser-
vation is consistent with both a fiercer competition between the
siderophore unit of 1 and native siderophores for the small
amount of Fe3+ available in the deplete medium, and the sider-
ophore-mediated uptake of 1. However, if active uptake is oc-
curring, the lower activity of 1 compared to that of ciprofloxa-
cin (Figure 12b), suggests that it is insufficient to compensate
for the decrease in uptake of the antimicrobial via porins and
passive diffusion caused by attachment of the salmochelin unit
in 1, even though the expression of high-affinity siderophore
transport proteins, such as IroN, should be upregulated under
Fe3+ limited conditions.69 The observation that the activity of 1
vs. E. coli Nissle 1917 is significantly lower than that of its par-
ent drug ciprofloxacin led us to consider poor binding to the
drug target DNA gyrase as an additional possible reason for the
observed reduction in potency.
b)
a)
Figure 10. Time-dependent, radiochemical bacterial uptake studies
in E. coli K12 (MG1655) of 67Ga-staphyloferrin conjugates 12-14
in iron replete (striped bars) and iron deplete media (solid bars).
Error bars calculated as standard deviation of n = 5.
Antibacterial Activity vs E. coli Nissle 1917
The antibacterial activity of 1 and ciprofloxacin were also as-
sessed against a strain of E. coli capable of producing and trans-
porting salmochelin, E. coli Nissle 1917. The probiotic Nissle
1917 shares many characteristics with uropathogenic E. coli
strains, including the ability to produce several siderophores,
specifically salmochelin, enterobactin, yersiniabactin and aero-
bactin, to be able to adapt to environmental challenges.29 It was
hoped that the presence of the salmochelin transport machinery,
including the outer membrane transporter IroN,25 would in-
crease the bacterial uptake and hence antibacterial activity of 1
in Nissle 1917.
Figure 12. E. coli Nissle 1917 growth in minimal media (MOPS
Acetate) in the presence of ciprofloxacin at t = 48 h in a) iron re-
plete (striped bars) and b) iron deplete conditions (filled bars).
Gyrase Inhibition Assay
A key factor in the antimicrobial activity of ciprofloxacin con-
jugates is their continued ability to inhibit DNA gyrase, the cy-
toplasmic drug target of ciprofloxacin. In order to investigate
the impact of salmochelin-inspired siderophore conjugation on
the ability of ciprofloxacin to inhibit gyrase, 1 was evaluated in
an in vitro assay using a commercial DNA gyrase supercoiling
assay. Initially, 1 was studied over a range of concentrations
(0.5-20 μM) and no inhibition of gyrase activity was observed,
as indicated by no reduction in the presence of supercoiled
DNA plasmids on agarose gels (Figure 13). Inhibition of the
intracellular drug target DNA gyrase by 1 was detectable at con-
centrations of 30 μM and above, with complete inhibition at 75
μM (Figure 14) indicating a decrease in gyrase inhibitory activ-
ity in comparison with the parent drug (10 μM). However, this
moderate decrease in gyrase inhibitory activity does not explain
the drastic drop in the antibacterial potency of 1.
a)
b)
Figure 11. E. coli Nissle 1917 growth in minimal media (MOPS
Acetate) in the presence of 1 at t = 48 h in a) iron replete (striped
bars) and b) iron deplete conditions (solid bars).
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These results suggest that one major obstacle in the successful
application of salmochelin-based Trojan Horse antibiotics with
an intracellular drug target is the delivery of the conjugate into
the bacterial cell via Fe3+-siderophore transporters.
The observed formation of dimeric 2:2 species by native mass
spectrometry provides a potential explanation for the observed
lack of cellular uptake of conjugate 1. If the tendency to form
dimers also applies to biological media, it is unlikely that these
dimers would be recognized by the outer membrane receptor
IroN and fit through the iron-salmochelin transporter.
7
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10
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12
13
14
15
Hence, our first generation conjugate, 1, the tetradentate mimic
of the hexadentate siderophore salmochelin S4, appears poorly
suited to the targeting of the salmochelin-mediated uptake path-
way. A similar relationship was observed for enterobactin-
ciprofloxacin Trojan Horse conjugates by Nolan et al.,18 who
observed that an intact hexadentate enterobactin unit was re-
quired for good antibacterial activity, whereas tetradentate or
bidentate equivalents displayed poor activity, possibly due to
reduced recognition by the corresponding outer membrane re-
ceptor.18 It is also conceivable that the lack of a flexible linker
between the siderophore component and ciprofloxacin renders
1 too rigid and bulky to be able to pass through the IroN trans-
porter, or the closely linked ciprofloxacin directly impedes si-
derophore binding.
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Figure 13. DNA gyrase assay of A) ciprofloxacin and B) conjugate
1. 0 µM = DMSO control. + = positive control, with DNA gyrase
present without the antimicrobial. - = negative control, no DNA
gyrase or antimicrobial.
The poor cellular uptake shown in the case of 1, along with its
increase in activity in iron deplete media, may point to extracel-
lular Fe3+ sequestration as an additional mechanism of action,
alongside DNA gyrase inhibition.
Figure 14. DNA gyrase assay of conjugate 1 at concentrations
ranging from 100-20 µM.
Whilst the lack of significant uptake and activity of 1 is disap-
pointing, it offers some direction to a future second generation
design. The size and polar surface area of the conjugate are suf-
ficient to prevent passive uptake via porin channels or passive
diffusion through the cell membrane, suggesting that optimiza-
tion of the siderophore component and linker to boost uptake in
salmochelin-utilizing strains could be sufficient to transform
similar conjugates into narrow-spectrum antimicrobials. In ad-
dition, while the application of a biolabile linker designed to
cleave the ciprofloxacin from the siderophore inside the cell
could retrieve the DNA inhibitory activity, a biolabile link will
not increase antibiotic efficacy unless the conjugate is deliv-
ered, at an appropriate level, into the bacterial cytoplasm. Fu-
ture studies will be focused on optimizing active conjugate
transport into bacterial cells using a combination of targeted
chemical synthesis and radiolabelled 67Ga complex uptake stud-
ies.
This, plus the lack of higher activity vs. the Nissle 1917 strain,
suggests that poor active transport of 1 is a major factor in the
observed lack of antimicrobial activity against both strains of E.
coli.
Summary and Conclusions
A ciprofloxacin siderophore conjugate 1 has been synthesized
by linking glycosylated catechol units 7, found in salmochelin
S4, to L-lysine modified ciprofloxacin 10. The resulting sider-
ophore-ciprofloxacin conjugate was designed to evade the
mammalian immune response, and to selectively target patho-
genic bacteria that express the salmochelin receptor IroN. Initial
screening of 1 against a common laboratory strain of E. coli
lacking the IroN transporter demonstrated much reduced anti-
bacterial activity compared to the parent antibiotic ciprofloxa-
cin, with concentrations ca. 250-1000x higher than required to
obtain similar activity in identical conditions. Increased anti-
bacterial activity was observed in iron deplete media. Probing
cellular uptake via 67Ga labelling suggested the absence of sig-
nificant bacterial cell uptake of 67Ga-1, consistent with the ab-
sence of IroN.
ASSOCIATED CONTENT
Supporting Information. Full experimental details and screening
methodology. This material is available free of charge via the In-
ternet at http://pubs.acs.org.
When the activity was examined vs. Nissle 1917, a strain capa-
ble of expressing IroN, a similar concentration dependence was
observed, with 1 again requiring concentrations 250-1000x
higher than ciprofloxacin to obtain similar growth inhibition,
suggesting the presence of IroN was not leading to significantly
greater uptake. Again, higher activity was observed in iron de-
plete media.
AUTHOR INFORMATION
Corresponding Authors
*Mailing Addresses:
Department of Chemistry, University of York, Heslington, York
YO105DD, United Kingdom. Tel: +44 (0) 1904 322501. Fax: +44
(0) 1904 322516. E-mail: anne.routledge@york.ac.uk (A.R.),
anne.duhme-klair@york.ac.uk (A.-K.D.-K.).
Department of Chemistry, Stony Brook University, 100 Nicolls
Road, Stony Brook, New York 11790, USA. Tel: (631) 632-
8572. E-mail: eszter.boros@stonybrook.edu (E.B.).
Whilst the DNA gyrase inhibitory activity of 1 was significantly
lower than that of the parent drug, the relatively moderate de-
crease in gyrase inhibitory activity does not explain the drastic
drop in the antibacterial potency of 1.
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conjugates as theranostics for imaging and treatment of bacterial in-
fections. Angew. Chem. Int. Ed. 56, 28, 8272-8276.
(16) Ghosh, M., Miller, P. A., Möllmann, U., Claypool, W. D.,
Author Contributions
Schroeder, V. A., Wolter, W. R., Suckow, M., Yu, H., Li, S., Huang,
W., Zajicek J., and Miller, M. J. (2017) Targeted antibiotic delivery:
selective siderophore conjugation with daptomycin confers potent ac-
tivity against multidrug resistant Acinetobacter baumannii both in
vitro and in vivo. J. Med. Chem. 60, 4577-4583.
(17) Liu, R., Miller, P. A., Vakulenko, S. B., Stewart, N. K.,
Boggess, W. C., and Miller, M. J. (2018) A synthetic dual drug
sideromycin induces gram-negative bacteria to commit suicide with a
gram-positive antibiotic. J. Med. Chem. 61, 3845-3854.
(18) Neumann, W., Sassone-Corsi, M., Raffatellu M., and Nolan,
E. M. (2018) Esterase-catalyzed siderophore hydrolysis activates an
enterobactin–ciprofloxacin conjugate and confers targeted antibacte-
rial activity. J. Am. Chem. Soc. 140, 15, 5193-5201.
(19) Pandey, A., Savino, C., Ahn, S. H., Yang, Z., Van Lanen, S.
G., and Boros, E. (2019) Theranostic gallium siderophore ciprofloxa-
cin conjugate with broad spectrum antibiotic potency. J. Med. Chem.
62, 21, 9947-9960.
A.-K.D.-K., A.R., J.W.S., and E.B. conceived and designed exper-
iments. T.J.S., C.M.B., J.W.S., E.J.W., A.P. and R.H. performed
experiments. A.-K.D.-K., A.R., C.M.B., J.W.S., and E.B. co-wrote
manuscript.
Funding Sources
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Prof. A-K. Duhme-Klair and Dr. A. Routledge would like to thank
the UK Engineering and Physical Sciences Research Council
(EPSRC) for studentships for Dr. T. J. Sanderson (EP/K503216/1),
Dr. E. J. Wilde (EP/L505122/1) and C. M. Black (EP/N509802/1),
and the University of York, Department of Chemistry for a Teach-
ing Studentship for J. W. Southwell.
ACKNOWLEDGMENT
A.-K. Duhme-Klair and A. Routledge thank J. E. Thomas-Oates for
helpful discussions, K. Heaton and H. Robinson for acquisition of
mass spectrometry data, P. Aguiar, B. Coulson and H. Fish for
NMR experiments and G. McAllister for elemental analyses. A.
Pandey and E. Boros acknowledge Dr Peter Tonge for access to the
E. coli MG1655 strain.
(20) Negash, K. H., Norris, J. K. S., and Hodgkinson, J. T. (2019)
Siderophore–antibiotic conjugate design: New drugs for bad bugs?
Molecules, 24, 3314-3330.
(21) To the best of our knowledge, at least four siderophore-based
Trojan Horse antibiotics have entered clinical trials: cefiderocol (S-
649266), GSK-3342830, cefetecol and BAL-30072 (see Ref 4 and
22/23). A number of others have failed at the pre-clinical stage.
(22) Butler, M. S., Blaskovich, M. A., and Cooper, M. A. (2013)
Antibiotics in the clinical pipeline in 2013. J. Antibiot. 66, 571-591.
(23) Page, M. G. P. (2019) The role of iron and siderophores in in-
fection, and the development of siderophore antibiotics. Clin. Infect.
Dis., 69, Supplement 7, S529-S537.
(24) FDA approves new antibacterial drug to treat complicated uri-
nary tract infections as part of ongoing efforts to address antimicro-
bial resistance, 2019; https://www.fda.gov/news-events/press-an-
nouncements/fda-approves-new-antibacterial-drug-treat-complicated-
urinary-tract-infections-part-ongoing-efforts
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