European Journal of Medicinal Chemistry xxx (2013) 1e8

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Mini-review

Antibacterial action of quinolones: From target to network

Guyue Cheng, Haihong Hao, Menghong Dai, Zhenli Liu, Zonghui Yuan*
National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory for the Detection of Veterinary Drug Residues in Foods,
Huazhong Agricultural University, Wuhan, Hubei 430070, China

a r t i c l e i n f o a b s t r a c t

Article history: Quinolones are widely used broad-spectrum antibacterials with incomplete elucidated mechanism of
Received 25 December 2012 action. Here, molecular basis for the antibacterial action of quinolones, from target to network, is fully
Received in revised form discussed and updated. Quinolones trap DNA gyrase or topoisomerase IV to form reversible drug-
23 January 2013
enzyme-DNA cleavage complexes, resulting in bacteriostasis. Cell death arises from chromosome
Accepted 26 January 2013
Available online xxx
fragmentation in protein synthesis-dependent or -independent pathways according to distinguished
quinolone structures. In the former pathway, irreversible oxidative DNA damage caused by reactive
oxygen species kills bacteria eventually. Toxineantitoxin mazEF is triggered as an additional lethal action.
Keywords:
Primary target
Bacteria survive and develop resistance by SOS and other stress responses. Enlarged knowledges of
Bacteriostatic action quinolone actions and bacterial response will provide new targets for drug design and approaches to
Lethal action prevent bacterial resistance.
Crystal structure Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.
Bacterial response

1. Introduction
Quinolones (QLs) are synthetic antimicrobials based on the
4-oxo-1,4-dihydroquinolone skeleton (Fig. 1). The first-generation
of QLs, such as nalidixic and oxolinic acids, acts against Gram-
negative bacteria and is used to treat urinary tract infections.
The second-generation of QLs, such as norfloxacin and ciproflox-
acin, is introduced a fluorine atom at position 6 and a bulky
piperidine at position 7, broadening the antimicrobial spectrum to
Pseudomonas species and some Gram-positive organisms, e.g.
Staphylococcus aureus. The third generation of QLs, with sub-
stitutions at the 7 as well as at the 8 position, enhances the ac-
tivity against Gram-positive bacteria. For instance, levofloxacin
and moxifloxacin are active against Streptococcus pneumoniae and
S. aureus that are pathogens responsible for respiratory tract in-
fections, acute otitis and meningitis [1]. Moreover, moxifloxacin is
active against Mycobacterium tuberculosis which lacks topoisom-
erase IV (topo IV) [2]. The fourth-generation of QLs is developed
with enhanced potency and a broader spectrum including
anaerobic bacteria. Gemifloxacin is currently one of the most
potent fluoroquinolones against community-acquired pneumonia
and acute bronchitis [3]. Trovafloxacin is used against intra-
abdominal and pelvic infections [4].
* Corresponding author. Tel.: þ86 27 87287186; fax: þ86 27 87672232.
E-mail addresses: yuan5802@mail.hzau.edu.cn, yuanzongh@126.com (Z. Yuan).
Now, it is quite clear that QLs interfere with chromosomal to-
pology by targeting bacterial type IIA topoisomerases, DNA gyrase
and topo IV, trapping these enzymes at the DNA cleavage stage and
preventing strand rejoining. As a result, the DNA replication ma-
chinery becomes arrested at the blocked replication forks, leading
to inhibition of DNA synthesis that immediately causes bacterio-
stasis [5]. Till now, several crystal structures have been resolved to
exhibit accurate structures of the drug-enzyme-DNA ternary
complexes, but data gained from these crystal structures demon-
strate some contradictions that need to be explained and unified.
QL-induced cell death is associated with the formation of double-
stranded DNA breaks (DSBs), resulting in chromosome fragmen-
tation and surge of reactive oxygen species (ROS) [5,6]. QLs differ
among various derivatives in rate and extent of killing, in the effect
of protein synthesis inhibitors on QL lethality and in the need for
aerobic metabolism to kill cells [7]. However, the molecular
mechanisms of these differences have not been clearly elucidated.
It is also known that chromosome-encoded toxin MazF which
causes programmed cell death (PCD) is involved in efficient QL
killing [8]. To fight against the drug action, SOS regulon and other
bacterial response networks are triggered in responses to QLs,
providing strategies for bacterial survival and development of
resistance.
In this review, a diverse body of knowledge was drawn into the
mode of action of QLs from target to network levels. This mainly
includes the following details. First, targeting of QLs on DNA gyrase
or topo IV resulting in bacteriostatic actions was briefly described

0223-5234/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.057

Please cite this article in press as: G. Cheng, et al., Antibacterial action of quinolones: From target to network, European Journal of Medicinal
Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.01.057
2 G. Cheng et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
Fig. 1. The chemical structures of QLs.
and crystal structures of drugeenzymeeDNA ternary complexes
were updated. Then, different chromosome fragmentation path-
ways and subsequent events leading to cell death were depicted in
details. Last but not least, the bacterial responses induced by QL
treatment were summarized. Understanding the mechanisms
underlying QL actions and the corresponding bacterial responses
could provide new targets for drug design, enhance drug efficiency
and prevent bacterial resistance.
2. Primary targets of QLs: DNA gyrase and topoisomerase IV
The first QL, nalidixic acid, was discovered with antimicrobial
properties in the by-product distillates in the manufacture of the
anti-malarial chloroquine [9], then ten times more potent QL,
oxolinic acid, was synthesized five years later [10]. Goss et al. [11,12]
first showed that nalidixic acid was a selective, immediate and
reversible inhibitor of bacterial DNA synthesis. Soon after the dis-
covery of bacterial gyrase in 1976 [13], both Gellert and Cozzarelli
[14,15] demonstrated that the supercoiling activity of the purified
gyrase, extracted from wild-type cells but not from resistant nalA
mutants, was inhibited by nalidixic acid and oxolinic acids. In 1978,
Higgins et al. [16] confirmed that subunit A of DNA gyrase (GyrA)
was the product of the gene controlling sensitivity to nalidixic acid.
These results suggest that DNA gyrase is the primary target of QLs.
Gyrase, belonging to type IIA topoisomerases, functions as a
heterotetramer composed of two GyrA subunits and two GyrB
subunits [17]. Gyrase’s enzymatic activity is essential for the
Please cite this article in press as: G. Cheng, et al., Antibacterial action of quinolones: From target to network, European Journal of Medicinal
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regulation of DNA superhelicity, bacterial replication, transcription
initiation and elongation. Gyrase generates a pair of single-
stranded breaks (SSBs) in the region of DNA wrapped around the
enzyme. One DNA gate is opened through which another stretch of
DNA can pass. After that, the gate closes and hydrolysis of ATP re-
sets gyrase for another round.
In 1990, Kato et al. [18] discovered a homolog of gyrase called
topoisomerase IV (topo IV). Like gyrase, topo IV is composed of two
ParC subunits and two ParE subunits [19]. Soon after the discovery of
topo IV, it became clear that gyrase was not the only intracellular
target of QLs [20,21]. In Escherichia coli, mutations mapped in parE
[22] or near parC [23] were identified conferring a high level of
resistance. Since QL resistance alleles in parE or parC did not confer
resistance by themselves, topo IV must be a secondary target in E. coli
[22e25]. This is also the same case for Neisseria gonorrhoeae [26].
However, things are rather different for some Gram-positive
strains. Clinical isolates of S. aureus that were resistant to a mod-
erate level of ciprofloxacin contained a mutation in parC (grlA)
while isolates that were resistant to a high concentration of cip-
rofloxacin exhibited an additional mutation in gyrA [27]. Purified
topo IV was inhibited by QLs from a variety of assays, including
measurement of DNA cleavage, decatenation and relaxation activity
[25,28,29]. Gyrase and topo IV from both S. aureus and E. coli were
purified to study the fluoroquinolone sensitivity [30]. The super-
coiling activity of S. aureus gyrase was at least 500-fold less sensi-
tive to ciprofloxacin than that of E. coli gyrase and about 6-fold less
sensitive than the decatenating activity of S. aureus topo IV. These
observations suggest that topo IV, rather than gyrase, is the primary
target of ciprofloxacin in S. aureus. The same phenomenon occurs in
S. pneumoniae [31,32].
In summary, it is now quite clear that bacteria contain two
topoisomerase targets for QLs. In Gram-negative bacteria the pri-
mary target is gyrase, while in Gram-positive bacteria the primary
target is generally topo IV. Since the two enzymes have different
functions, it is likely that bacteria differ in their response to QLs
according to which enzyme is the primary target.
3. Bacteriostatic actions of QLs
3.1. Formation of drug-enzyme-DNA complex
Formation of a QLeenzymeeDNA complex that contains broken
DNA is the central event in the interaction between the QLs and
gyrase or topo IV. Footprinting experiments suggested that the QLs
allowed gyrase to proceed to a conformational change in which
additional DNA was wrapped around gyrase [33]. The QLe
enzymeeDNA complex formation is readily reversible. Chromo-
some fragmentation was eliminated after the drug removed or by
mild heat treatment (60  C) [34]. Addition of a protein denaturant
such as sodium dodecyl sulfate (SDS) to the ternary complexes
would release DNA ends [14,15,29,34]. Nevertheless, the drugs
cannot cause generation of free DNA ends inside bacterial cells
since intact nucleoids containing negatively supercoiled DNA could
be isolated from oxolinic acid treated bacteria in a cell lysis pro-
cedure without SDS [34,35].
Comparison of the nucleotide sequences at the cleavage sites
reveals a loose consensus sequence [36e39]. Not all of the cleavage
sites identified on a given DNA molecule were cleaved when oxo-
linic acid is added to cells [34]. Cleavage is especially frequent at a
small number of specific sites called toposites on the chromosome
[40]. A large number of weaker sites also exist [41]. It is speculated
that the small number of strong interaction sites are used by gyrase
to maintain superhelical tension in the chromosome as a whole and
the weak and widely dispersed sites allow gyrase to provide local
swiveling needed for transcription and replication.
 G. Cheng et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8 3

3.2. Crystal structures of cleaved complexes
To date, a few of crystal structures of complexes between type
IIA topoisomerases and DNA have been resolved. Crystal structures
of a complex consisting of the DNA-binding core of Saccharomyces
cerevisiae Topoisomerase II and a gate-DNA segment [42] and a
cleaved complex composed of the DNA-binding core of
S. pneumoniae topo IV with broken DNA and either clinafloxacin or
moxifloxacin [43] have been comprehensively elucidated in the
review written by Drlica et al. [44]. However, the effects of several
amino acid substitutions that alter the antimicrobial activity and
drug structure variations cannot be explained by these structures.
Recently, a crystal structure of moxifloxacin in complex with
Acinetobacter baumannii topo IV has been reported, demonstrating
the wedge-shaped QL stacking between base pairs at the DNA
cleavage site and binding conserved residues in the DNA cleavage
domain through chelation of a noncatalytic magnesium ion [45]
(Fig. 2A). In this QL-bound noncatalytic magnesium model, two
moxifloxacin molecules intercalate four base pairs apart at the two
sites of DNA cleavage. The QL interaction with DNA is largely
through van der Waals, pep stacking interactions, as reported in
the crystal structure with S. pneumoniae topo IV [43], but this
higher-resolution structure reveals a different orientation and
binding mode for moxifloxacin, with a Mg2þ mediating the inter-
action between QL and the protein.
The QL-bound noncatalytic Mg2þ, some 14  A from Mg2þ at the
active site, is modeled with an octahedral coordination sphere with
two oxygens from the QL and four water molecules [45]. Two of the
Mg2þ coordinating water molecules make hydrogen bonds with
ParC Ser84 and Glu88, thus providing a structural explanation for
why mutations in these residues in DNA gyrase and topo IV confer
QL resistance in diverse bacterial species. Mg2þ coordination also
explains why the carbonyl at C4 of a QL is essential as well as why
only limited substitution is permitted at positions 3 and 4. The
bulky substituent at position 7 of moxifloxacin occupies a large and
open pocket between the DNA and the ParE subunit, consistent
with the C7 position being the most versatile position for substi-
tution in QLs. The C7 substituent is proximal to Arg418 from ParE. In
E. coli, mutation of the equivalent residue Lys447 to glutamate
enhances the activity of amphoteric QLs, such as moxifloxacin,
which has a basic substituent at the C7 position [46], providing a
structural rationalization for the effect of a ParE/GyrB mutation on
QL sensitivity. Nearly at the same time, a structure of S. pneumoniae
topo IV with DNA and levofloxacin was reported [47] (Fig. 2B),
presenting a similar fluoroquinolone orientation as in QL-bound
noncatalytic magnesium model [45], but without the QL-bound
Mg2þ ion.
In this QL-bound noncatalytic magnesium model, QL orientation
is likely to be conserved, as it explains the known involvement of
Mg2þ in QL action, QL structureeactivity relationships and resis-
tance mutations across orthologs [45]. Since the C-7 rings are still far
from GyrA helix IV in this model, it is not easy to explain the influ-
ence of the C-7 ring structure of fluoroquinolones on the increased
MIC of GyrA G89C (Mycobacterium smegmatis numbering, equiva-
lent to G81C in E. coli numbering) [48] or G81D (E. coli numbering)
variant [49]. C-7 ring structure may have effects on binding that are
not reflected by the static nature of the X-ray structures, since QL
binding is known to involve at least two steps, one that occurs before
DNA cleavage and one that occurs after [50,51].
3.3. Replication fork arrest and bacteriostasis
Trapping topoisomerases on DNA can lead to a rapid inhibition
of DNA replication [11], since trapping gyrase in a QLegyraseeDNA
complex ahead of the replication fork would block fork movement.
Cleaved complexes also form with topo IV [29], but the rate at
which replication is inhibited is 50e100 times slower than with
gyrase in E. coli [25]. The difference is generally explained by topo
IV functioning behind replication forks [52], while gyrase is prob-
ably ahead of them [52,53]. In Gram-positive bacteria, such as
S. aureus, in vitro reactions with norfloxacin and gyrase fail to block
replication fork movement [54], explaining why norfloxacin in-
hibits DNA replication largely through topo IV in S. aureus [55].
The stability of QL-induced cleaved complexes contributes to
their ability to block replication fork movement. It was shown that
S. aureus gyrase wrapped DNA to catalyze DNA supercoiling and
arrest replication fork progression in vitro [54]. E. coli mutant gyrase
with lack of the entire GyrA CTD (GyrA59) cannot wrap DNA,
showing that gyrase-mediated DNA wrapping is required for
replication fork arrest [56]. Although topo IV does not wrap DNA,

Fig. 2. Mg2þ ion mediates QL interactions with topo IV. (A) Moxifloxacin (yellow) in complex with ParE28eParC58 (gray carbons) and DNA (green carbons) at 3.27 Å. Mg2þ (green
sphere) is coordinated by four water molecules (red spheres) and two oxygen atoms from the QL. (B) Cartoon/stick representation of the drug-binding pockets of topo IV-DNA in
complex with levofloxacin (magenta). The figure has been adapted with modifications from Refs. [45,47]. (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

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QL-induced cleaved complexes formed with topo IV are more stable

than those formed with GyrA59 gyrase [56]. Helix-4, part of the
DNA-binding domain of GyrA or ParC, is also important on complex
stability. Replacement of helix-4 of E. coli ParC with that of E. coli
GyrA [57] and replacement of an extended region around helix-4 of
E. coli GyrA with that of S. aureus GyrA [58] reveal that subtle dif-
ferences of helix-4 affect the QL sensitivity of a topoisomerase.
Thus, QL sensitivity of a topoisomerase correlates with the stability
of the cleaved complex.
Formation of cleaved complexes, inhibition of DNA synthesis,
and inhibition of growth are correlated [34,59]. However, they are
all reversible phenomena, while lethal action is not [12,60].
Nevertheless, blocked replication forks could stimulate secondary
events that kill cells slowly. When drug treatment is long (over-
night with E. coli), cell death can occur at QL concentrations that are
only twice MIC (rapid killing generally requires concentrations 5e
10 times the MIC). Slow killing, which is commonly expressed by
the parameter called the minimal bactericidal concentration (MBC),
is poorly understood mechanistically [5].
A few double-strand DNA breaks (DSBs) are generated by
collision of replication forks with cleaved complexes, since in a
plasmid model system where cleaved complex formation blocked
replication, double-strand breaks were observed [61]. It has been
suggested that DSBs arising after replication fork stalling are
generated by a recombination nuclease. One candidate is the
RuvABC complex, as RuvAB may reverse and dissociate the cleaved
complex at a stalled replication fork before RuvC cleaves DNA to
generate a DSB [61]. DNA cleavage could provide RecBC with access Fig. 3. Schematic representation of QL action. (a) Binding of gyrase to DNA.
to circular DNA, as required for the induction of SOS response [62]. (b) Reversible formation of QLegyraseeDNA complexes that rapidly block DNA repli-
cation. (c) Inhibition of replication leads to induction of the SOS response and cell
It is important to distinguish the few DNA breaks associated with filamentation. (d) Lethal chromosome fragmentation that requires ongoing protein
replication fork arrest from the extensive chromosome fragmen- synthesis in aerobic conditions, as seen with nalidixic acid treatment of E. coli.
tation associated with cell death (discussed below). (e) Lethal chromosome fragmentation that requires ongoing protein synthesis but not
aerobic conditions, as seen with norfloxacin treatment of E. coli. (f) Lethal chromosome
fragmentation that requires neither ongoing protein synthesis nor aerobic conditions,
4. Lethal actions of QLs
as seen for PD161144 with E. coli. (g) DNA breakage detected after treatment of cell
lysates with an ionic detergent such as SDS. (h) Chromosome fragmentation stimulates
4.1. Pathways of chromosome fragmentation a surge of ROS with hydroxyl radicals as the terminal product. (i) Formation of highly
toxic hydroxyl radicals, the end product of an ROS cascade, is blocked by 2,20 -bipyridyl,
Cell death arises from the release of DNA breaks resulting in an iron chelator, and thiourea, a hydroxyl radical scavenger, which prevent cell death.
Question marks indicate uncertainty about slow death and the nature of the DNA ends.
chromosome fragmentation from protein-mediated constraint The figure has been drawn based on Refs. [5,74].
existing in the cleaved complexes [23]. The QLs kill E. coli in diverse
pathways, distinguished by the effects of protein synthesis inhibitors
and anaerobiosis on QL lethality [7]. Nalidixic and oxolinic acids of with chloramphenicol [23]. Thus, it appears that an identified
the first-generation QLs are not lethal in the presence of chloram- protein factor is involved in releasing DNA ends from the QLe
phenicol or during anaerobic growth [20,23] (Fig. 3, pathway d). gyraseeDNA complexes in three suggested mechanisms: 1) prote-
Norfloxacin which is the second-generation QL fails to kill E. coli in ase digestion of gyrase, 2) nuclease-mediated cleavage on either
the presence of chloramphenicol, but at high concentrations it kills side of the cleaved complex, and 3) protein denaturation [44]. On
cells under anaerobic condition (Fig. 3, pathway e); while cipro- the other hand, lethal effect of chloramphenicol-insensitive mode
floxacin kills under both conditions but requires higher concentra- also arises from the release of DNA ends from QLegyraseeDNA
tions during anaerobiosis [20,23,63] (Fig. 3, pathway f). PD161144, a complexes [23]. An explanation for the protein synthesis-
third generation C-8-methoxy derivative, is affected little by chlor- independent mode emerged from the observation that the QLs
amphenicol or anaerobic growth for the lethal activity [63] (Fig. 3, stimulated a form of illegitimate recombination and deletion for-
pathway f). Since even the fourth-generation compounds are sensi- mation arising from subunit dissociationereassociation [67e71],
tive to chloramphenicol if QL concentrations are low enough, the suggesting that QL molecules might be able to force gyraseeDNA
choice of pathway depends on QL concentration. The different lethal complexes apart (Fig. 3, step f). An E. coli mutant with A67S in
pathways are also observed in mycobacteria [64,65]. GryA allowed nalidixic acid to kill E. coli and fragment chromo-
Much less was said about cell death arising from QL-topo somes in the presence of chloramphenicol [64]. Thus, the mutation
IV-DNA lesions. From the observation that killing of an E. coli gyrA appears to shift lethal activity from step d to step f in Fig. 3, perhaps
(Nalr) mutant was drastically reduced by rifampin [20] or chlor- by the alanine-to-serine change weakening hydrophobic in-
amphenicol [23], it is proved a protein synthesis-dependent mode. teractions between the GyrA subunits. Lethal effects arising from
In the case of C-8-methoxy fluoroquinolones (PD161148), the this chloramphenicol-insensitive mode of QL action become more
methoxy group increased lethality against wild-type S. aureus cells prominent as the QL concentration increases [23], perhaps by a
when protein synthesis was inhibited [66]. cooperative effect of drug stacking on gyrase subunit dissociation.
As for the mechanism of protein synthesis-dependent pathway, Furthermore, the effect of QL structure may explain the connection
it was shown that oxolinic acid failed to eliminate the topological between cleaved complex destabilization and chloramphenicol-
constraint needed for the maintenance of supercoils in cells treated insensitive killing [5].

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 G. Cheng et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
4.2. Oxidative damage cellular death pathway
Recent findings indicate that production of ROS is a common
mechanism of cell death induced by bactericidal antibiotics,
including QLs [6,72]. Hydroxyl radical concentrations were elevated
in E. coli following treatment with norfloxacin. Thiourea or 2,20 -
bipyridyl, agents that reduce the level of hydroxyl radical, inhibited
norfloxacin lethality [72] (Fig. 3, step i). When both sodA and sodB
were deficient, norfloxacin lethality was reduced, in consistence
with that superoxide dismutase normally promoted QL lethality by
stimulating formation of peroxide [73]. A deficiency in catalase/
peroxidase (katG) elevated the lethal activity of norfloxacin,
because a buildup of peroxide lead to accumulation of highly toxic
hydroxyl radicals [73].
How do protein synthesis-dependent and -independent path-
ways fit with the observation that hydroxyl radical accumulation is
associated with QL lethality? It was recently demonstrated that
lethal activity of oxolinic acid was completely blocked by thiourea
plus 2,20 -bipyridyl and by chloramphenicol, but only chloram-
phenicol blocked both chromosome fragmentation and hydroxyl
radical accumulation [74]. Thus, the chromosome fragmentation
step occurs before the ROS step (Fig. 3, step h). In contrast, lethal
action of PD161144 by the subunit dissociation pathway (Fig. 3, step
f) was affected little by treatment with chloramphenicol or thiourea
plus 2,20 -bipyridyl or all three agents together [74]. With moxi-
floxacin, both chloramphenicol and thiourea plus 2,20 -bipyridyl
separately exhibited the same partial inhibition of QL lethality, and
no additivity in protection from moxifloxacin lethality was
observed when these three agents were combined [74]. These
studies indicated that hydroxyl radical action contributes to QL-
mediated cell death occurring via the chloramphenicol-sensitive
but not via the chloramphenicol-insensitive lethal pathway.
4.3. Toxineantitoxin and programmed cell death (PCD)
Toxineantitoxin modules, which appear to be part of the bac-
terial stress responses, also contribute to QL lethality. These mod-
ules act when elimination of a short-lived antitoxin allows the
cognate toxin to interfere with the bacterial transcriptione
translation machinery, leading to programmed cell death (PCD),
an irreversible series of events that causes the loss of colony-
forming ability even after removal of the stressor [75]. When cells
were treated with nalidixic acid at 500 to 1000 times the MIC for
10 min, a 90% decrease in the number of CFU followed unless the
chpAIK (mazEF) toxineantitoxin module was absent, suggesting
that triggering the E. coli chromosomal toxineantitoxin system
mazEF was an additional determinant in the lethal action of QLs [8].
Further study is needed to examine whether the action of
mazEF-mediated cell death in E. coli involve the production of ROS,
which is a common mechanism of cell death induced by bacteri-
cidal antibiotics [72]. It was found that antibiotics as DNA damaging
reagents (i.e. nalidixic acid) caused mazEF-mediated cell death in an
ROS-independent pathway [76]. Furthermore, a signaling molecule,
Extracellular Death Factor (EDF), participated in mazEF induction.
This mode of action implies that using synthetic EDF together with
QLs can increase the efficiency of bacterial killing. Besides, bacte-
riostatic antibiotics can be turned into bactericidal antibiotics by
using EDF to turn on the mazEF system.
5. Bacterial responses to QLs
5.1. SOS response of bacteria
One consequence of QL treatment is the induction of the SOS
regulon, a set of more than 30 lexA repressor-controlling genes
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5
involved in a variety of activities, including DNA repair, recombi-
nation and mutagenesis [77,78]. RecBCD complex exhibit a single-
strand (ss) DNA exonuclease activity associated with the genera-
tion of free DNA ends followed by endonucleolytic activities of the
RuvABC or SbcCD complex of E. coli [79]. Free ssDNA ends are bound
by RecA molecules to form nucleoprotein filaments, activating the
auto-self-degradation activity of the LexA repressor. As a conse-
quence, the complete SOS response is induced [80].
QLs appear to produce at least two types of lethal damages that
can be distinguished by protective elements of the SOS response.
For some QLs, such as nalidixic acid, survival depends largely on the
recombination function of the RecA and RecBCD proteins [81,82].
Moreover, nalidixic acid lethality is unaffected by the lexA mutation
in a gyrAþ background [64]. However, for the potent fluo-
roquinolones (e.g. norfloxacin and ciprofloxacin), rapid lethality is
increased by a lexA allele [83]. Since norfloxacin and ciprofloxacin
are able to attack topo IV, and recA and lexA mutations can increase
the ciprofloxacin susceptibility of gyrA mutants [23], topo IV-
mediated killing appears to be mitigated by both recA-dependent
recombination and SOS induction.
Induction of the SOS response not only causes QL-induced
mutagenesis, contributing to the acquisition of QL resistant muta-
tions, but also enhances bacterial survival in the presence of fluo-
roquinolones. sfiA encodes an inhibitor of cell division that function
to allow time for the repair of DNA damage, and recA encodes the
key factor in homologous recombination (HR) reactions [84].
Additionally, genes umuCD are activated to express low-fidelity
DNA polymerase V, which is involved in SOS-mediated mutagen-
esis by inducing the accumulation of replication errors in surviving
cells. dinB encoding DNA polymerase IV is activated to restart DNA
replication behind DSBs which is called translesion synthesis (TLS)
and repair independently from other DNA polymerases [85]. Cells
may benefit from this enhanced mutational activity by ensuring
long-term survival as well as evolutionary fitness [78]. It is shown
that the development of fluoroquinolone resistance is impaired in
the absence of SOS response [86]. Thus, the error-prone activity of
the SOS response is considered to be an intrinsic system of bacteria
for adaptive response to DNA damaging stress in those survival
cells.
Besides SOS response, which preferentially repairs DSBs via
error-free HR or tolerates them via error-prone TLS, alternative
pathways resulting in large chromosomal rearrangements also
exist. Illegitimate recombination is accomplished by subunit ex-
change between two type II topoisomerase holoenzymes bound in
two separate cleavage complexes, which results in the ligation of
DNA ends from the two separate DSBs [70]. Non-homologous
end joining (NHEJ) has also been reported in mycobacteria as an
alternative repair pathway for DSBs by religating modified DNA
ends [87].
5.2. Other QL-induced stress responses
Bacteria can survive antibiotic treatment without acquiring
heritable antibiotic resistance, but to induce stress responses that
protect the cell from lethal factors such as DNA damaging agents.
Several TA (toxin/antitoxin) genes were induced in E. coli in
response to ciprofloxacin, whose promoters contain a Lex box:
symER, hokE, yafN/yafO, and tisAB/istR, which is necessary for
persister formation (dormant cells formation) [88]. The mechanism
of this ciprofloxacin-induced persister formation was recently
investigated. It was shown that a knockout of tisAB/istR had a
sharply decreased level of persisters tolerant to ciprofloxacin, and
step-wise administration of ciprofloxacin-induced persister for-
mation in a tisAB-dependent manner. Moreover, cells producing
TisB toxin were tolerant to multiple antibiotics [89]. TisB is a
6 G. Cheng et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8
membrane peptide that functions to decrease proton motive force
and ATP levels, consistent with its role in forming dormant cells.
Lon protease degrades abnormal proteins and proteins pro-
duced in excess, influencing a variety of physiological phenomena
[90]. The role of Lon protease in chromosome maintenance was
firstly noticed when examining paradoxical survival of bacteria at
very high concentrations of QLs [91]. A deficiency of Lon protease
eliminated paradoxical survival, and plasmid-borne protease ac-
tivity of Lon restored the paradoxical behavior of QLs [92]. These
observations confirm that Lon is necessary for paradoxical survival.
It was also found that a Lon-deficient mutant, PA1803, exhibited
more than 4-fold-increased susceptibilities to fluoroquinolones,
and complementation of the lon mutant restored wild-type anti-
biotic susceptibility and cell morphology [93]. The data suggest that
the induction of Lon is involved in adaptive resistance of bacteria.
The precise roles of Lon protease are described in the review
written by Drlica et al. [44].
YrbB, a component of an ABC transport system that maintains
lipid asymmetry in the Gram-negative outer membrane by pre-
venting surface exposure of phospholipids, was found to be one of
the proteins involved in protecting E. coli from QL-mediated death
by genetic searching [94]. A yrbB mutation rendered E. coli cells
more susceptible to the lethal action of QLs under conditions in
which bacteriostatic susceptibility was unaffected. YrbB worked in
both protein synthesis-dependent and -independent lethal path-
ways of QL action. In the absence of YrbB, nalidixic acid could kill
cells independently of hydroxyl radical action, in which the E. coli
toxineantitoxin system ChpB was found to be involved. In addition,
proteases ClpP and Lon were also involved in the action of
YrbB [95].
For some time the fluoroquinolones have been suspected to
have a secondary mode of action involving membrane damage
[96,97]. By using Bacillus subtilis biosensors, both inhibitions of DNA
synthesis and membrane damage by ciprofloxacin were detected
[98]. The yorB promoter indicating DNA synthesis and the ypuA
promoter indicating cell wall synthesis/cell envelope stress were
induced by ciprofloxacin, supporting a weak secondary mode of
action involving membrane damage. Probably, ROS is one of the
factors contributing to the membrane damage. Other mechanisms
involved in membrane damage, if there is any, still need to be
examined.
The diverse set of genes protecting against the lethal effects
of QLs may serve as potential targets for future screening of
small-molecular antimicrobial potentiators, which can be used as
enhancers for QLs.
6. Conclusions and prospective
The QLs are of an important class of antimicrobial agents. It is
clear that formation of drugeenzymeeDNA complexes is the cen-
tral event in QL action. QL lethality can be described as a two step
process in which the first step is reversible formation of cleaved
complexes (bacteriostatic). This step blocks bacterial DNA replica-
tion, induces the SOS response, and leads to cell filamentation
(Fig. 3, pathway c). Although these events do not appear to be
rapidly lethal, their involvement in slow death has not been ruled
out. The second lethal step requires higher QL concentrations, and
DNA breaks are released from constraint by two pathways, one
requires protein synthesis (Fig. 3, steps d and e) and the other does
not (Fig. 3, step f). Each pathway to cell death depends on QLs
structures. The protein synthesis-dependent pathway involves
removal of gyraseedrug complexes from DNA and liberation of
lethal double-strand DNA breaks, while the protein synthesis-
independent pathway involves gyrase subunit dissociation and
release of DNA ends with the gyrase subunits attached. DNA breaks
Please cite this article in press as: G. Cheng, et al., Antibacterial action of quinolones: From target to network, European Journal of Medicinal
Chemistry (2013), http://dx.doi.org/10.1016/j.ejmech.2013.01.057
induce irreversible oxidative DNA damage by ROS that occurs in a
protein synthesis-dependent manner. Besides, the toxin MazF is
required under certain conditions for efficient killing by QLs with
EDF participating in mazEF induction. In contrast to the killing ef-
fect of QLs, bacteria can survive and acquire resistance by SOS
response and other stress responses.
Nevertheless, our knowledges about QL actions are far from
complete and needed to be explored. For example, (1) the already
resolved crystal structures of the drugeenzymeeDNA complexes
still cannot explained several amino acid substitutions and drug
structure variations, additional studies should be required to
explain what is likely to be a multistep binding [99]; (2) Nor-
floxacin appears to represent an intermediate situation (Fig. 3, step
e), how norfloxacin-mediated replication fork breakage fits into
these categories is not currently understood; (3) Proteins involved
in steps d and e (Fig. 3) need to be identified to show how DNA
breaks are released from the protein-mediated constraint, and
cell-free tests for step f need to be extended to plasmid systems
that can be readily studied; (4) It is clear that death ultimately
arises from the accumulation of hydroxyl radicals with the older
QLs, but how DNA breaks promote a cascade of ROS is still un-
known; (5) Blocked replication forks could stimulate secondary
events that kill cells slowly with QL concentrations that are only
twice MIC, and the mechanism of slow killing is poorly under-
stood; (6) The paradoxical loss of lethality at very high QL con-
centrations need to be explained in molecular terms; (7) Whether
Lon-mediated repair involves direct removal of the complexes or
an indirect effect due to rapidly removing an unidentified lethal
factor involved in fragmentation of DNA is not known; (8) Those
genes and proteins involved in bacterial response contributing to
survival and resistance should be identified and studied in mo-
lecular terms, which would serve as potential targets for designing
enhancers for QLs.
Although QLs are broad-spectrum agents, they have encoun-
tered resistance that makes new topoisomerase inhibitors attrac-
tive. Among the newer QLs under development, delafloxacin,
nemonoxacin and JNJ-Q2 are with enhanced activity against Gram-
positive bacteria, including MDR (multiple drug resistance)
S. pneumoniae and ciprofloxacin-resistant MRSA (Methicillin-
resistant S. aureus). However, the molecular mechanism underlying
the anti-resistant activity of these newer QLs has not been eluci-
dated yet. As reviewed here, the available crystal structures of the
QL-type IIA topoisomeraseeDNA complexes have explained some
determinants for resistance, and it is of great interest to study the
anti-resistant activity of these newer QLs on the structural basis.
Moreover, the biological events in bacteria under QL pressures
contribute to the cell death and development of resistance of bac-
teria, providing plenty of novel targets for drug design to improve
activity and tackle resistance. As expected, the growing under-
standing of mechanisms of QL actions will give insights into new
clinical treatment and approaches for fighting threat from QL
resistant pathogens.
Declaration of interest
The authors report no declarations of interest.
Acknowledgments
This work was supported by National High Technology
Research and Development Program of China (grant number
2011AA10A214).
 G. Cheng et al. / European Journal of Medicinal Chemistry xxx (2013) 1e8 7

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