Dual Escherichia coli DNA Gyrase A and B Inhibitors with Antibacterial Activity

Article abstract of DOI:10.1002/cmdc.201900607

The emergence of multidrug-resistant bacteria is a global health threat necessitating the discovery of new antibacterials and novel strategies for fighting bacterial infections. We report first-in-class DNA gyrase B (GyrB) inhibitor/ciprofloxacin hybrids that display antibacterial activity against Escherichia coli. Whereas DNA gyrase ATPase inhibition experiments, DNA gyrase supercoiling assays, and in vitro antibacterial assays suggest binding of the hybrids to the E. coli GyrA and GyrB subunits, an interaction with the GyrA fluoroquinolone-binding site seems to be solely responsible for their antibacterial activity. Our results provide a foundation for a new concept of facilitating entry of nonpermeating GyrB inhibitors into bacteria by conjugation with ciprofloxacin, a highly permeable GyrA inhibitor. A hybrid molecule containing GyrA and GyrB inhibitor parts entering the bacterial cell would then elicit a strong antibacterial effect by inhibition of both the GyrA and GyrB subunits of DNA gyrase and potentially slow bacterial resistance development.

Full text of DOI:10.1002/cmdc.201900607

Accepted Article
Title: Dual Escherichia coli DNA Gyrase A and B Inhibitors with
Antibacterial Activity
Authors: Danijel Kikelj, Benedetta Fois, Žiga Skok, Tihomir Tomašič,
Janez Ilaš, Nace Zidar, Anamarija Zega, Lucija Peterlin
Mašič, Petra Szili, Gábor Draskovits, Ákos Nyerges, and
Csaba Pal
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10.1002/cmdc.201900607
ChemMedChem
Dual Escherichia coli DNA Gyrase A and B Inhibitors with Antibacterial
Activity
Benedetta Fois,[a,b] Žiga Skok,[a] Tihomir Tomašič,[a] Janez Ilaš,[a] Nace Zidar,[a] Anamarija Zega,[a] Lucija
Peterlin Mašič,[a] Petra Szili,[c,d] Gábor Draskovits,[c] Ákos Nyerges,[c] Csaba Pál[c] and Danijel Kikelj*[a]
Abstract: The emergence of multidrug-resistant bacteria is a global
health threat necessitating the discovery of new antibacterials and
novel strategies for fighting bacterial infections. We report first-in-
class DNA gyrase B (GyrB) inhibitor / ciprofloxacin hybrids that
display antibacterial activity against Escherichia coli. Whereas DNA
gyrase ATPase inhibition experiments, DNA gyrase supercoiling
assays and in vitro antibacterial assays suggest binding of the
hybrids to the E. coli GyrA and GyrB subunits, an interaction with the
GyrA fluoroquinolone-binding site seems to be solely responsible for
their antibacterial activity. Our results provide a foundation for a new
concept of facilitating entry of nonpermeating GyrB inhibitors into
bacteria by conjugation with ciprofloxacin, a highly permeable GyrA
inhibitor. A hybrid molecule containing GyrA and GyrB inhibitor parts
entering the bacterial cell would then elicit a strong antibacterial
effect by inhibition of both the GyrA and GyrB subunits of DNA
Figure 1. Ciprofloxacin and GyrB inhibitors 1a-e.
gyrase and potentially slow bacterial resistance development.
design and preparation of the first dual inhibitors of Gyr A and
Bacterial DNA gyrase, a type IIa topoisomerase responsible
Gyr B that could open new avenues for DNA gyrase inhibition
for ATP-driven introduction of negative supercoils into DNA[1], is
and fighting bacterial resistance.[8b]
a well-established target of antibacterials.[2] Whereas fluoro-
Designed multiple ligands can be obtained by linking,
quinolones targeting the GyrA subunit of a heterodimeric A2B2
merging or fusing individual pharmacophores in a way tolerated
enzyme are widely used to treat infections with gram-positive
by respective targets.[9] Several 4-quinolone hybrids with
2d, 3]
and gram-negative bacteria,[2b,
GyrB inhibitors interfering
trimethoprim,[10a] linezolid,[10b] and tobramycin[10c] were obtained,
and these as well as other studies[11] demonstrated that moieties
of different sizes attached to the piperazine NH group of
ciprofloxacin are well tolerated, with retention of DNA gyrase
inhibition and antibacterial activity. Furthermore, exposure of the
terminal carboxylate group of our GyrB inhibitors to bulk water
observed in the crystal structure of the 4,5-dibromo-1H-pyrrole-
2-carboxamide inhibitor 1a bound to E. coli GyrB and simulated
by docking for a reversed inhibitor 1d (IC50 E.coli = 38 nM)[6]
should allow functionalization in this region without losing DNA
gyrase B inhibitory activity (positions in ciprofloxacin and in 1a-e
which should tolerate substitution without loss of DNA gyrase
with ATP binding to subunit B have not advanced into the clinic
despite intensive research over the last 50 years after the
discovery of novobiocin as the first ATP-competitive GyrB
inhibitor in the 1960s.[2f-2h] We recently reported several
structural types of low-nanomolar pyrrole-2-carboxamide GyrB
inhibitors[4-6] and established the binding mode of 2-((2-(4,5-
dibromo-1H-pyrrole-2-carboxamido)benzo[d]thiazol-6-yl)amino)-
2-oxoacetic acid (1a; IC50 E. coli = 58 nM),[6] a 4,5-dibromo-
pyrrole analog of 1b (IC50 E.coli = 43 nM),[7] to GyrB from E. coli
with X-ray crystallography. However, most of these inhibitors
were devoid of in vitro antibacterial activity because of insuf-
ficient permeation and/or extrusion by bacterial efflux pumps.[6, 7]
Dual targeting of GyrB and structurally similar topoisomerase
IV ParE subunits has been suggested to prolong the onset of
resistance in bacteria because mutations at both essential sites
are less probable than single mutations at GyrB or ParE ATP-
binding sites.[2g, 8a] This observation evoked our interest in the
[a]
Prof. Dr. D. Kikelj, Assoc. Prof. Dr. J. Ilaš, Assoc. Prof. Dr. T.
Tomašič, Assoc. Prof. Dr. N. Zidar, Assoc. Prof. Dr. A. Zega, Prof.
Dr. L. Peterlin Mašič, B. Fois, MPharm, Ž. Skok, MPharm, Faculty of
Pharmacy, University of Ljubljana, Aškerčeva cesta 7, 1000
Ljubljana, Slovenia; E-mail: Danijel.kikelj@ffa.uni-lj.si
B. Fois, MPharm, Faculty of Biology and Pharmacy, University of
Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
P. Szili, MSc, G. Draskovits, MSc, Dr. Á. Nyerges, Dr. Csaba Pál,
Synthetic and Systems Biology Unit, Hungarian Academy of
Sciences, Szeged, H-6726 Hungary
[b]
[c]
[d]
P. Szili, MSc, Doctoral School of Multidisciplinary Medical Sciences,
University of Szeged, Szeged, H-6720 Hungary
inhibition are colored red; Figure 1).
Figure 2. GyrA/GyrB inhibitor hybrids 2a-b and 3a-b.
Supporting information is given via a link at the end of the document.
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10.1002/cmdc.201900607
ChemMedChem
The presence of carboxylate groups in 1a, 1b and 1d did not
seem critical for GyrB inhibition since the N-acetyl analog of 1b
(1c; IC50 E. coli = 9 nM) and its reversed analog N-(2-acetamido-
benzo[d]thiazol-6-yl)-3,4-dichloro-5-methyl-1H-pyrrole-2-carbox-
amide (1e; IC50 E. coli = 66 nM) also showed good inhibition of
DNA gyrase. Because DNA gyrase A-inhibiting fluoroquinolones
and our pyrrole-2-carboxamide GyrB inhibitors 1a-e do not share
common structural features that would allow fusion of both phar-
macophores, we decided to use a merging strategy to combine
the GyrA inhibitor ciprofloxacin and our GyrB inhibitors in the
same molecule and obtain hybrid compounds 2 and 3 containing
both GyrA- and GyrB-inhibiting pharmacophores (Figure 2).
Docking of compounds 2b and 3b to E. coli GyrB (PDB:
4DUH)[12a] and to S. aureus GyrA (PDB: 5CDQ)[12b, 12c] (data not
shown) suggested that they could bind to both subunits since
there was no steric clash with GyrA or GyrB binding site residu-
es. We further anticipated that merging our GyrB inhibitors with
highly permeable ciprofloxacin would facilitate entry of the hybrid
GyrA/GyrB inhibitors and make them active against bacteria.
The hybrids 2a and 2b were prepared (Scheme 1) by
acylation of the amines 7a and 7b with chloroacetyl chloride and
subsequent substitution of chlorine in 8a and 8b with
ciprofloxacin. Amine 7a was obtained by selective N2 acylation
of the diamine 6 with 4,5-dibromopyrrole-2-yl trichloromethyl
ketone, whereas the amine 7b was prepared by acylation of 6-
nitrobenzo[d]thiazol-2-amine (4) with 3,4-dichloro-5-methyl-
pyrrole-2-carboxylic acid chloride and subsequent reduction of
the nitro group in the obtained 5b.
For the synthesis of the isomeric hybrids 3a and 3b (Scheme
2) bearing a 1H-pyrrole-2-carboxamido moiety in position 6, 4
was acylated with chloroacetyl chloride[13], and the obtained nitro
derivative 10 was reduced to the 6-aminobenzothiazole
derivative 11 by catalytic hydrogenation over 10% Pd on
charcoal in ethyl acetate. Attempts to reduce the nitro group of
10 with tin(II) chloride in ethanol or with sodium sulfide
nonahydrate were not successful since, in both cases, reduction
of the nitro group was accompanied by amide bond cleavage,
and benzo[d]thiazole-2,6-diamine was isolated as the main
product. The amine 11 was coupled with 4,5-dibromo-pyrrole-2-
carbonyl chloride or 3,4-dichloro-5-methyl-pyrrole-2-carbonyl
chloride in the presence of triethylamine in dioxane, and the
resulting intermediates 12 underwent nucleophilic substitution
with unprotected ciprofloxacin to produce the target inhibitors 3a
and 3b. The final compounds 2a-b and 3a-b were purified on
Sephadex LH-20 to remove traces of ciprofloxacin.
Scheme 2. (a) ClCH2COCl, Et3N, dichloromethane, rt, 12 h; (b) H2, Pd/C
(10%), EtOAc, rt, 24 h; (c) 4,5-dibromo-1H-pyrrole-2-carboxylic acid chloride
or 3,4-dichloro-5-methyl-1H-pyrrole-2-carboxylic acid chloride, Et3N, dioxane,
rt, 18 h; (d) ciprofloxacin, KI, Na2CO3, CH3CN, reflux, 12 h.
A DNA gyrase supercoiling assay[4-6] demonstrated weaker
inhibition of E. coli DNA gyrase by the hybrids 2a-b and 3a-b
(IC50 values from 0.17 to 6.2 M) than by the GyrB inhibitors 1a-e
(IC50 values from 9 to 66 nM) and slightly weaker inhibition than
by the GyrA inhibitor ciprofloxacin (Table 1). The hybrids 2a and
2b with ciprofloxacin bound to position 6 of a benzothiazole core
were better (submicromolar) inhibitors than 3a and 3b that
Scheme 1. (a) 3,4-dichloro-5-methyl-1H-pyrrole-2-carbonyl chloride, toluene,
reflux, 18 h; (b) H2, Pd/C (10%), 1.5 bar, EtOH, rt, 18 h; (c) SnCl2  2 H2O,
EtOH, reflux, 24 h; (d) 4,5-dibromopyrrole-2-yl-trichloromethyl ketone, K2CO3,
DMF, 80 °C, 20 h; (e) ClCH2COCl, Et3N, dichloromethane, rt, 24 h; (f) acryloyl
chloride, K2CO3, THF, ice bath, 0.5 h; (g) ciprofloxacin, KI, Na2CO3, CH3CN,
reflux, 4 h; (h) ciprofloxacin, CH3CN/AcOH, reflux, 72 h.
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900607
ChemMedChem
possessed low micromolar E. coli gyrase IC50 values. Whereas negative P. aeruginosa ATCC 15692 and A. baumannii ATCC
increasing the size of the 6-(N-acetylamino) substituent of 1c, BAA1605 or against gram-positive S. aureus ATCC 700699 and
producing 8b (N-chloroacetyl), and 2b in each case resulted in a Enterococcus faecalis ATCC 29212 strains, the antibacterial
ca. 20-fold increase in IC50 irrespective of the size of the N- activity of the hybrids in the absence of an efflux pump inhibitor
substituent, increasing the size of the 2-(N-acylamino) substituent was confirmed against gram-negative Shigella flexneri HNCMB
in the series 1e (N-acetyl) → 12b (N-chloroacetyl) → 3b clearly 20018, Shigella sonnei HNCMB 25021 (MICs between 1481 and
increased the IC50 values at each step, increasing in total 78-fold 3333 ng/mL) and against Klebsiella pneumoniae ATCC 10031
from 1e to 3b. This observation indicated that appending the (MICs between 293 and 658 ng/mL) as well as against gram-
ciprofloxacin moiety to position 6, resulting in the compounds 2a positive Listeria monocytogenes ATCC 19111 (MICs between
and 2b, is better tolerated than its attachment to position 2, 87 and 2863 ng/mL). These results indicate good penetration of
producing the compounds 3a and 3b. Because the observed the studied GyrA/GyrB-inhibiting hybrids through the bacterial
inhibition of E. coli DNA gyrase in the supercoiling assay could cell wall and provide evidence that their efflux in E. coli and in
not be undoubtedly attributed to inhibition of the GyrA or GyrB other tested bacteria is not intensive and not detrimental for their
subunit, a DNA gyrase ATPase assay (Table 1) was performed to anti-bacterial activity. However, the hybrid molecules 2a-b and
detect binding of the hybrid compounds to E. coli GyrB. Further, 3a-c did not show any reduction in antibacterial activity in the E.
MIC assays in the E. coli wild-type strain and in two strains with a coli K-12 MG1655 GyrB R136C mutant and suffered
a
mutated GyrB or mutated fluoroquinolone-binding sites were substantial loss of antibacterial activity toward the E. coli mutant
performed to assess the effect of the hybrids on E. coli (Table 2).
in which the fluoroquinolone-binding site was abolished by four
mutations (E. coli K-12 MG1655 GyrA S83L, D87N; ParC S80I,
E84G). This finding indicates that the observed antibacterial
activity of the hybrid molecules 2a-b and 3a-b is mainly due to
interaction with GyrA and/or ParC and not with the GyrB subunit.
Although the ATPase assay demonstrated that the hybrids 2a-b
and 3a-b inhibit the GyrB subunit, the inhibition is obviously too
weak (IC50 values between 0.055 and 0.35 M) to result in
antibacterial activity. Assuming that increasing the flexibility of
the linker between the GyrB and GyrA inhibitor moieties could
increase GyrB inhibition, we synthesized the hybrid 2c, a
homolog of the hybrid 2b that was most potent in the
supercoiling and ATPase assays, possessing an additional
methylene group between the ciprofloxacin and GyrB inhibitor
moieties (Scheme 1). To this end, the amine 7b was acylated
with acryloyl chloride in the presence of potassium carbonate in
tetrahydrofuran, and the obtained acrylamide 9 was reacted with
ciprofloxacin to produce the compound 2c with an elongated
linker. However, the hybrid 2c displayed behavior similar to that
of 2b in the supercoiling assay (IC50 = 0.16 M), ATPase assay
(IC50 = 0.054 M) and MIC assays in E. coli, demonstrating that
elongating the linker by one C-atom did not increase GyrB
inhibition and antibacterial activity against the wild-type and
mutated E. coli strains.
Table 1. Inhibition of E. coli DNA gyrase supercoiling and ATPase activities
Compound
MW
Supercoiling
ATPase
IC50 (M)
IC50 (M)
1a
485.85
413.23
383.25
383.25
787.45
712.58
726.61
787.45
712.58
492.57
417.70
492.57
417.70
612.62
313.35
0.0580.031
0.0430.034
0.00950.0025
0.0660.008
0.910.37
0.170.03
0.160.01
6.21.7
n.d.
1b
n.d.
1c
n.d.
1e
n.d.
2a
0.270.01
0.0550.005
0.0540.025
0.350.18
0.320.19
n.d.
2b
2c
3a
3b
5.22.0
8a
2.30.2
8b
0.180.02
0.430.02
0.330.02
0.170.01
0.120.02
n.d.
12a
0.380.09
n.d.
Table 2. MIC values (ng/mL) of the hybrids 2a, 2b, 2c, 3a and 3b.
12b
Bacterium
2a
2b
2c
3a
3b
CP[a]
novobiocin
ciprofloxacin
0.160.05
n.d.
E. coli ATCC 25922 + PaβN
130 130 500
130
130
4.4
6.6
9
E. coli K-12 MG1655 + PaβN 439 439 250
293
293
[n.d.] not determined
E. coli ATCC 25922
E. coli K-12 MG1655
2222 1481 439
3333 1481 658
1481
1481
n.a.
1481
2222 12
All four hybrid compounds (2a-b and 3a-b) displayed potent
activity against the E. coli strains ATCC 25922 and K-12
MG1655 in the presence of the efflux pump inhibitor PaβN (MIC
values between 130 and 439 ng/mL). MIC values in the absence
of PaβN were in the range 1481 - 3333 ng/mL in the strains
ATCC 25922 and K-12 MG1655, which indicates that the
hybrids 2a-b and 3a-b are not intensively effluxed in E. coli.
Whereas the hybrids were not active against either gram-
A. baumannii ATCC BAA1605 n.a. n.a. n.a.
n.a.
n.a.
<173
<173
P. aeruginosa ATCC 15692
S. flexneri HNCMB 20018
S. sonnei HNCMB 25021
n.a. n.a. n.a.
3333 1481 658
3333 1481 658
n.a.
1481
1481
2222 12
2222 12
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10.1002/cmdc.201900607
ChemMedChem
K. pneumoniae ATCC 10031 658 293 195
293
293
4
Acknowledgments
E. cloacae ATCC 13047
S. aureus ATCC 700699
5000 2222 1481
n.a. n.a. n.a.
n.a. n.a. n.a.
2222
n.a.
3333 26
This work was supported by the Slovenian Research Agency
(grant P1-0208), COST Action CA15135, European Research
Council H2020-ERC-2014-CoG 648364 ‘Resistance Evolution’
(to C.P.), the Wellcome Trust (to C.P.), GINOP (MolMedEx
TUMORDNS) GINOP-2.3.2-15-2016-00020, GINOP (EVOMER)
GINOP-2.3.2-15-2016-00014 (to C.P.), EFOP 3.6.3-VEKOP-16-
2017-00009 (to P. Sz.), UNKP-18-3 and UNKP-19-3 New
National Excellence Program of the Ministry for Innovation and
Technology (to P. Sz.), the ‘Lendület’ Program of the Hungarian
Academy of Sciences (to C.P.), and a PhD fellowship from
Boehringer Ingelheim Fonds (to Á.N.).
n.a.
n.a.
878
15000
Enterococcus faecalis
ATCC 29212
Listeria monocytogenes
ATCC 19111
n.a.
390
585
390 87
195
2863
E. coli GyrB R136C + PaβN
439 439 250
293
293
6.6
E. coli K-12 MG1655
GyrA S83L, D87N;
ParC S80I, E84G + PaβN
5000 5000 16000 5000
20000 33000
Keywords: antibiotic • ciprofloxacin • drug discovery • dual
inhibitor • gyrase A • gyrase B • hybrid
[a] CP = ciprofloxacin; n.a. : MIC  15000 ng/mL.
To investigate bacterial evolvability toward the dual inhibitors, as
many as 1010 wild-type E. coli cells were exposed to a 4MIC
concentration of the tested compounds in a standard frequency
of resistance assay.[14] The two tested E. coli strains showed
similar potential to develop spontaneous resistance against the
hybrid molecules as they did against ciprofloxacin (Table 3).
This result supports other aforementioned results indicating that
the observed antimicrobial activity of the tested hybrid molecules
is due mainly to their interaction with GyrA and/or ParC,
whereas GyrB inhibition by the hybrid molecules is limited.
Therefore, resistance can arise in the form of canonical
mutations against ciprofloxacin derivatives on GyrA and/or ParC.
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2c
3a
3b
CP[a]
[5]
[6]
E. coli K-12 MG1655
1.30 2.60 3.13
11.2 4.17 11.9
3.07
4.88
4.43 1.56
13.9 6.31
E. coli ATCC 25922
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[a] CP = ciprofloxacin
[7]
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M. Barančokova, D. Kikelj, J. Ilaš et al., unpublished.
In conclusion, the first dual DNA gyrase A and B inhibitors
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not intensively effluxed, and display a strong antibacterial activity
due to the interaction of the hybrids with the GyrA and/or
topoisomerase IV ParC subunits. As demonstrated by DNA
gyrase ATPase and MIC assays, inhibition of GyrB by the
presented hybrids, although present, is not strong enough to
provide a substantial contribution to the observed antibacterial
activity. In perspective, hybrids combining a benzothiazole DNA
gyrase B inhibitor and the DNA gyrase A inhibitor ciprofloxacin in
the same molecule connected by a cleavable linker, are a logical
extension of the presented concept that could result in strong
inhibition of both the DNA gyrase A and B subunits in the
bacterial cell and in potent antibacterial activity.
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Experimental Section
[12] a) M. Brvar, A. Perdih, M. Renko, G. Anderluh, D. Turk, T. Šolmajer, J.
Med. Chem. 2012, 55, 6413-6424. b) P. F. Chan, V. Srikannathasan, J.
Huang, H. Cui, A. P. Fosberry, M. Gu, M. M. Hann, M. Hibbs, P. Homes,
Experimental procedures are available in the supporting information that can be
accessed via a link ……
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10.1002/cmdc.201900607
ChemMedChem
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10.1002/cmdc.201900607
ChemMedChem
COMMUNICATION
The first DNA gyrase B (GyrB)
inhibitor / ciprofloxacin hybrids that
display antibacterial activity against
Escherichia coli are reported. They
provide a foundation for a new
concept of facilitating entry of non-
permeating GyrB inhibitors into
bacteria by conjugation with
ciprofloxacin, a highly permeable
GyrA inhibitor and eliciting a strong
antibacterial effect by inhibition of both
the GyrA and GyrB subunits of the
bacterial DNA gyrase.
Benedetta Fois, Žiga Skok, Tihomir
Tomašič, Janez Ilaš, Nace Zidar,
Anamarija Zega, Lucija Peterlin Mašič,
Petra Szili, Gábor Draskovits] Ákos
Nyerges, Csaba Pál and Danijel Kikelj*
Page No. – Page No.
Dual Escherichia coli DNA Gyrase A
and B Inhibitors with Antibacterial
Activity
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