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130
Current Topics in Medicinal Chemistry, 2014, 14, 130-151
Prospects for Developing New Antibacterials Targeting Bacterial Type IIA
Topoisomerases
Tihomir Tomai and Lucija Peterlin Mai*
University of Ljubljana, Faculty of Pharmacy, Department for Medicinal Chemistry, Akereva 7, 1000 Ljubljana,
Slovenia
Abstract: The modulation of DNA topology by DNA gyrase and topoisomerase IV, both of which are type IIA topoisomerases and found in most bacteria, is a function vital to DNA replication, repair and decatenation. Despite the potential
for resistance development, DNA gyrase and/or topoisomerase IV have been proven to be and remain highly attractive
targets in antibacterial drug discovery due to their potential for dual targeting. The search for new GyrA and/or ParC inhibitors that can overcome the increasing spread of multidrug-resistant bacteria has been successfully focused in the last
decades on the modification of the known fluoroquinolone scaffold as primarily guided by ligand-based design via classical structure-activity relationship studies and the optimisation of physicochemical properties. This focus has resulted in
several novel fluoroquinolones that have been introduced into clinical practice since 2000, and several of these new compounds are currently in different phases of clinical trials. Due to increasing resistance to fluoroquinolones, a significant
part of DNA gyrase research has shifted to the discovery of new GyrB and/or ParE inhibitors, which are commonly identified through fragment-based design as well as virtual screening techniques and structure-based hit optimisation programs.
This research often results in lead compounds with potent inhibitory activity and promising antibacterial activity profiles.
Nevertheless, it is important to understand how different physicochemical properties (e.g., logD and total polar surface
area) and different structural motifs influence the compounds’ permeability to ensure the efficient discovery of potent,
small-molecule antibacterials particularly against Gram-negative strains.
Keywords: Antibacterial, ATP, DNA gyrase, dual inhibitors, structure-based design, topoisomerase.
INTRODUCTION
The increasing emergence of bacteria resistant to antibacterial drugs is a worldwide problem not only in hospitals,
where these drugs are heavily used, but also in community
settings [1]. In nosocomial infections, methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant S.
aureus and Enterococcus faecium (VRE) are among the most
problematic pathogens, whereas in community settings, serious infections are caused by Streptococcus pneumoniae,
Haemophilus influenzae and their resistant strains [2]. Particularly problematic are the infections caused by the antibiotic-resistant “ESKAPE” pathogens (E. faecium, S. aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species), which cause
significant morbidity and mortality [3]. Although the rate of
bacterial resistance to antibacterials currently in use is increasing, there are only a few successful examples of new
antibacterial drugs with novel modes of action that can circumvent the resistance problem (e.g., linezolid and daptomycin) [4]. To avoid cross-resistance with currently used
antibacterial drug classes, new drugs acting via novel
mechanisms of action or against previously unexploited
binding sites on existing targets are needed. However, given
the overall low probability of success associated with the
*Address correspondence to this author at the University of Ljubljana, Faculty of Pharmacy, Department for Medicinal Chemistry, Akereva 7, 1000
Ljubljana, Slovenia; Tel: +386-1-4769635; Fax: Fax: +386-1-4258031;
E-mail: lucija.peterlin@ffa.uni-lj.si
1873-5294/14 $58.00+.00
development of an antibacterial drug that can be approved
for clinical use, the use of novel approaches is associated
with even higher attrition and longer development timelines
[5]. The difficulty in finding an appropriate antibacterial lead
compound has been indicated by the disclosure of the activities at GlaxoSmithKline [6].
Bacterial topoisomerases are enzymes that catalyse
changes in DNA topology and are well-established targets
for antibacterial drug discovery [7]. These proteins can be
divided into type I and II topoisomerases, which catalyse
reactions involving the transient break of one or both strands
of DNA, respectively [8]. The modulation of DNA topology
by DNA gyrase and topoisomerase IV, which are type IIA
topoisomerases, is a function vital to DNA replication, repair
and decatenation. Therefore, these enzymes are essential for
cell viability and offer the possibility of the development of
antibacterial drugs that can bypass the existing resistance
mechanisms developed by bacteria through interactions with
unique binding sites, new compound chemical classes or
novel mechanisms of inhibition. The possibility of dual targeting in most bacteria, except mycobacteria, which may
reduce resistance development, makes type IIA topoisomerases attractive targets for discovery of novel antibacterials [9].
DNA gyrase introduces negative supercoils in DNA in
front of the replication fork and thereby relieves torsional
strain during replication, whereas topoisomerase IV is involved in the control of DNA supercoiling and in the decate© 2014 Bentham Science Publishers
Prospects for Developing New Antibacterials Targeting
nation of daughter chromosomes after DNA replication. Eukaryotic type II topoisomerases are large single-unit enzymes
that are active as homodimers, whereas prokaryotic enzymes
are composed of two subunits. DNA gyrase is a heterotetrameric protein consisting of two GyrA and two GyrB
subunits (A2B2), whereas topoisomerase IV is a DNA gyrase
paralogue composed of two ParC (GrlA – gyrase-like, in
Gram-positive bacteria) and two ParE (GrlB) subunits (C2E2 )
that are homologous to GyrA and GyrB, respectively. The
GyrA and ParC subunits are involved in DNA transit,
whereas the GyrB and ParE subunits contain ATPase domains [10].
The complexity of reactions in DNA replication catalysed by DNA gyrase and topoisomerase IV offers multiple
opportunities for intervention. Both enzymes are targets of
fluoroquinolones (e.g., ciprofloxacin, moxifloxacin and
gemifloxacin), a clinically important class of antibacterial
drugs that interact mostly with the GyrA and/or ParC (GrlA)
subunits and stabilise the enzyme-DNA complex [11], and
aminocoumarins (e.g., novobiocin and clorobiocin), which
bind to GyrB and ParE (GrlB) [12]. Bacterial resistance to
fluoroquinolones is associated particularly with mutations in
the quinolone resistance-determining region (QRDR) of the
genes encoding GyrA and/or ParC (GrlA) [13]. Resistance to
aminocoumarins has also been observed and is limited to
mutations in GyrB and/or ParE (GrlB) [14]. Despite the potential for resistance development, DNA gyrase and/or topoisomerase IV are still very attractive targets in the field of
antibacterial drug discovery because their inhibitors have
potential for dual targeting, which prolongs the onset of resistance. Several novel fluoroquinolones targeting these enzymes have been deployed in clinical practice since the year
2000, and some are currently in different phases of clinical
trials [15].
The present review discusses the binding sites of DNA
gyrase and topoisomerase IV from different Gram-positive
and Gram-negative bacterial strains that have been studied
using the ProBiS web server [16]. The rationale for the design of selective DNA gyrase and topoisomerase IV inhibitors, as well as dual inhibitors, which most likely have less
potential for developing bacterial resistance than compounds
selectively inhibiting one of the enzymes, is provided [17].
The binding sites were compared with those of eukaryotic
topoisomerases and other ATP-dependent enzymes (e.g.,
protein kinases) to highlight the possible selectivity problems of the ATP-competitive inhibitors of bacterial type IIA
topoisomerases.
Recent successful examples (mostly dating from 2007 to
the present) of DNA gyrase and/or topoisomerase IV inhibitors are presented in terms of their potency against the enzymes and their antibacterial activity with a focus on resistant strains that cause nosocomial infections. The problem
and present examples of transforming potent inhibitors of
DNA gyrase and/or topoisomerase IV with poor antibacterial
activity into lead compounds with promising antibacterial
activity against a range of Gram-positive and Gram-negative
bacterial strains are highlighted. At the end, we also
highlight prospects for DNA gyrase as a target for the development of new compounds against Mycobacterium
tuberculosis.
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
131
STRUCTURE OF TYPE IIA TOPOISOMERASES
DNA gyrase is a tetramer composed of two GyrA and
two GyrB subunits that form an A2B2 complex in the active
enzyme. Similarly, topoisomerase IV forms a C2E2 complex
composed of two ParC and two ParE subunits, which are
analogous to GyrA and GyrB, respectively. The sequence
alignment of the type IIA topoisomerases revealed that the
GyrB and ParE subunits correspond to the N-terminal region
of the eukaryotic enzymes, whereas the GyrA and ParC
subunits are homologous to the C-terminal region of the eukaryotic enzymes [10]. No crystal structure of a complete
DNA gyrase or topoisomerase IV tetramer has been published to date; however, several crystal structures of fragments of both DNA gyrase and topoisomerase IV subunits
have been determined [18-25]. These three-dimensional
structures, together with the crystal structure of the structurally homologous human topoisomerase II [26] have identified three gates, namely the N-gate, DNA-gate and C-gate
(Fig. 1), that open and close sequentially to allow the passage of double-stranded DNA at specific stages of the catalytic cycle. The N-gate is formed by the two GyrB subunits,
which bind ATP, whereas the N-terminal domains of the
GyrA subunits form the DNA-gate and C-gate. The GyrB
subunit has been crystallised in complex with the nonhydrolysable ATP analogue ADPNP (adenosine 5'-(,imido)triphosphate) [27] and several structural classes of
inhibitors [28-36], whereas the crystal structures of GyrA in
complex with inhibitors and DNA have been solved [37,38].
All of these structural data provides an excellent basis for the
structure-based design of DNA gyrase and topoisomerase IV
inhibitors, which will be discussed in the following sections.
As previously mentioned, bacterial type IIA topoisomerases are structurally homologous to human topoisomerase II, which is a target for the development of anticancer drugs [39]. Furthermore, these enzymes utilise ATP
hydrolysis as an energy source, which is common to many
human enzymes. Due to the possible similarity of inhibitorbinding sites between bacterial type IIA topoisomerases and
human enzymes, these inhibitors must display selectivity for
bacterial enzymes over their human homologues or proteins
possessing ATP-binding sites (e.g., protein kinases). We
recently reviewed the potential of ATP-binding sites of bacterial enzymes as targets for antibacterial drug design and
presented several successful examples in which ATPcompetitive bacterial enzyme inhibitors exhibit promising
antibacterial activity and display good selectivity profiles
against human targets [40].
Protein binding sites can be studied using the ProBiS
server [16], which enables the detection of structurally similar binding sites and pairwise local structural alignments. We
have used ProBiS to identify proteins that are structurally
similar to DNA gyrase in order to obtain insight into the possible selectivity problems of inhibitors that target this enzyme. The structure of the query enzyme was compared to
protein 3D structures in the database to identify proteins that
share local structural similarities with the query enzyme. The
results are ranked by the Z-score, which indicates by how
many standard deviations each alignment differs from the
mean.
132 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Tomai and Mai
Fig. (1). Crystal structure of a) E. coli GyrB2 in complex with ADPNP (in CPK model) (PDB entry: 1EI1) and b) E. coli GyrA2 in complex
with simocyclinone D8 (in CPK model) (PDB entry: 2Y3P).
To study the ATP-binding site of GyrB, we used the
crystal structure of the N-terminal 43-kDa fragment of GyrB
from E. coli in complex with ADPNP (PDB entry: 1EI1)
(Fig. (1)) [27], whereas for GyrA, the crystal structure of the
N-terminal 59-kDa fragment of GyrA from E. coli in complex with the antibiotic simocyclinone S8 (PDB entry:
2Y3P) (Fig. (1)) [38] was used as the query. Currently, there
are 326 structures (Z-score > 1) similar to the ATPase domain of DNA gyrase. Among the top ranked structures (Zscore > 3) are GyrB proteins from different bacterial strains
(e.g., S. aureus, Mycobacterium smegmatis and Thermus
thermophilus) and, as expected, the topoisomerase IV ParE
subunit from E. coli and S. pneumoniae. High structural
similarity to E. coli GyrB (Z-score > 2) is also characteristic
of the topoisomerase VI from Sulfolobus shibatae [41], human DNA topoisomerase II [42] (Fig. (2a)) and other members of the GHKL (Gyrase, Hsp90, Histidine Kinase, MutL)
ATPase/kinase superfamily [43], all of which share a unique
ATP-binding motif called the Bergerat fold. From the GHKL
superfamily of enzymes, ProBiS identified MutL, PhoQ histidine kinase and heat shock protein Hsp90 as proteins that
are similar to GyrB. Significant similarity in the ATPbinding site can also be observed between GyrB, human pyruvate dehydrogenase kinase [44] (Fig. (2b)) and rat
branched-chain -ketoacid dehydrogenase kinase [45]. Using
the GyrA structure as the query in ProBiS, 436 similar structures (Z-score > 1) were found, but only 13 with the Z-score
greater that 2. These include DNA gyrase and topoisomerase
IV structures from different bacterial strains, jellyfish myosin light chain kinase, bacterial carboxylethyl arginine synthase and thioredoxin reductase.
Taken together, the ProBiS results indicate that there is a
basis for the dual targeting of DNA gyrase and topoisomerase IV and for the broad-spectrum activity of selective
or dual inhibitors due to the structural similarity between
these two enzymes from different bacterial strains. These
characteristics are being broadly pursued in the design of
inhibitors, as will be presented in the following sections. It
has to be noted that such inhibitors may exhibit non-selective
inhibition by interacting with similar human proteins, particularly human topoisomerase II and pyruvate dehydrogenase kinase, which is a topic that has rarely been addressed
in previous studies of inhibitors. However, fluoroquinolones
and aminocoumarins have been demonstrated to be sufficiently selective at clinically relevant doses.
DNA GYRASE AND TOPOISOMERASE IV INHIBITORS
Bacterial type IIA topoisomerase inhibitors usually target
both DNA gyrase and topoisomerase IV but with different
affinities depending on the chemical class of inhibitors and
the bacterial strain [46]. For example, in Gram-negative bacteria, the primary target of fluoroquinolones is DNA gyrase,
whereas in Gram-positive organisms, the preferred target is
usually topoisomerase IV. Multi-target and dual activities,
for example that of fluoroquinolones, make the development
of resistance difficult. In general, multi-target inhibitors of
bacterial enzymes form the basis of a promising strategy to
combat bacterial resistance due to the fact that targetmediated resistance to such compounds is less likely to
evolve because mutations conferring resistance would have
to occur in at least two different target genes during a single
generation [17]. Sequence alignment [10] and our ProBiS
structural analysis revealed similarities between DNA gyrase
and topoisomerase IV in their catalytic sites, which are targeted by fluoroquinolones, as well as their ATPase domains,
which are the targets of aminocoumarin antibiotics. Furthermore, the sequence and structural similarities of these enzymes from different bacterial strains render them optimal
for the development of inhibitors with broad-spectrum antibacterial activity.
GYRA AND/OR PARC INHIBITORS
Fluoroquinolones
Fluoroquinolones (Fig. (3)) are the only DNA gyrase and
topoisomerase IV inhibitors that are currently used in clinical
practice. Their common structural motif is the
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
133
Fig. (2). Superposition of the E. coli GyrB in complex with ADPNP (in green, PDB entry: 1EI1) and a) human topoisomerase II in complex
with ADPNP (in red, PDB entry: 1ZXM) and b) human pyruvate dehydrogenase kinase in complex with ATP (in yellow, PDB entry: 1Y8P).
For clarity, only those amino acid residues within 10 Å around the ADPNP or ATP (in sticks) were selected for representation. (The color
version of the figure is available in the electronic copy of the article).
O
O
F
O
OH
N
O
F
N
OH
N
HN
NH2 O
O
F
N
OH
N
O
N
N
O
NH
a)
ciprofloxacin (1)
O
O
F
O
N
Cl
O
O
O
OH
N
HN
F
N
antofloxacin (3)
F
OH
N
HO
moxifloxacin (2)
OH
H2N
N
CN
N
N
O
O
H 2N
b)
F
delafloxacin (4)
finafloxacin (5)
O
F
O
N
c)
N
H
N
H
O
OH
N
N
nemonoxacin (6)
N
F
O
MBX-500 (7)
Fig. (3). Representatives of the quinolone class of type IIA topoisomerase inhibitors a) Drugs used in clinical practice b) compounds still in
clinical trials and c) a hybrid anilinouracil-fluoroquinolone compound.
6-fluoroquinolin-4-one moiety with a carboxyl group at position 3 of the quinoline ring and several possible substituents
at the ring nitrogen (N-1) and position C-7 [47]. Since their
discovery in the early 1960s, the structure of quinolones was
optimised in terms of potency against Gram-positive, Gramnegative and atypical bacteria, their pharmacokinetics and
pharmacodynamics, which altogether resulted in the widespread of their use for the treatment of urinary, systemic and
respiratory tract infections [48]. These compounds display
broad-spectrum antibacterial activity and exert bacteriostatic
and bactericidal effects [49] but usually exhibit lower target
potency (approximately 100 nM) in supercoiling assays than
recently studied ATP-competitive inhibitors [31]. Their bactericidal activity is believed to result not only from the inhibition of DNA replication but also from the downstream
events that it causes (DNA damage, synthesis of incomplete
proteins, induction of oxidative damage and triggering celldeath mechanisms) [9]. Fluoroquinolones exert their effects
through the stabilisation of the cleaved DNA covalently
bound to the tyrosine residue in the GyrA active site. Details
134 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
of the interaction of these drugs with DNA and type IIA
topoisomerases were recently revealed through crystal structures of quinolones in complex with DNA gyrase or topoisomerase IV and DNA [22-24, 37]. To successfully crystallise DNA gyrase or topoisomerase IV in complex with quinolone-based inhibitors, fusion proteins containing the Nterminal domain of GyrA/ParC and the C-terminal domain of
GyrB/ParE, which retain catalytic activity, had to be prepared. In the crystal structure of moxifloxacin (Fig. (3)) in
complex with DNA and topoisomerase IV from Acinetobacter baumannii, the aromatic rings of the quinolone form van
der Waals and - stacking interactions with the DNA bases
immediately preceding (-1) or following (+1) the DNA
cleavage site (Fig. (4)). Furthermore, an octacoordinated
magnesium ion mediates the interaction between moxifloxacin and ParC. Two of the Mg2+-coordinating water molecules form hydrogen bonds with Ser84 and Glu88 of ParC,
which are the two amino acid residues that are commonly
mutated in quinolone-resistant bacteria [22]. Recently, studies on topoisomerase IV from Bacillus anthracis have shown
that water-metal ion bridge facilitates a critical interaction
between quinolones and the enzyme. It was proposed that the
bridge is anchored by the conserved serine and acidic residues, which can also be observed in the moxifloxacin –
topoisomerase IV complex, and that resistance to quinolones
is caused by the loss of coordination of the bridge to the enzyme [50,51]. The elucidation of the binding mode of the
fluoroquinolone inhibitor class at the molecular level not
only confirms the hypothesis of their mechanism of inhibition through their intercalation into DNA and interaction
with the protein but also provides a basis for further rational
design of novel effective inhibitors.
Fig. (4). Moxifloxacin (shown as a ball and stick diagram, coloured
by atom type) in complex with DNA (in green) and the ParC
subunit of topoisomerase IV (in red) from Acinetobacter baumannii
(PDB entry: 2XKK). Mg2+ is represented as a green sphere, water
molecules are represented as red spheres, and hydrogen bonds are
marked as green dashed lines.
Resistance to fluoroquinolones is usually caused by the
mutation of the amino acid residues in the QRDR of the
genes encoding GyrA and/or ParC (GrlA) [49]. Recently,
plasmid-mediated genes encoding efflux pumps and enzymes that modify quinolones were discovered [52]. As pre-
Tomai and Mai
viously mentioned, the onset of resistance can be prolonged
due to the dual targeting of DNA gyrase and topoisomerase
IV because it requires the introduction of mutations in both
topoisomerases. In addition to the dual targeting of both type
IIA topoisomerases, hybrid antibiotics targeting DNA or
RNA polymerase together with DNA gyrase and/or topoisomerase IV were recently investigated. For example, the
anilinouracil-fluoroquinolone hybrid compound MBX-500
[53] (Fig. (3)), which targets two distinct steps of DNA replication, was determined to be a potent inhibitor of DNA
polymerase and type IIA topoisomerases with broadspectrum antibacterial activity. MBX-500 inhibits several
antibiotic-resistant Gram-positive and Gram-negative strains,
including fluoroquinolone- and anilinouracil-resistant bacteria. When S. aureus strains resistant to the individual parent
compounds were obtained, spontaneous resistance to the
hybrid compound MBX-500 was not observed [54]. MBX500 was also observed to possess in vitro and in vivo efficacy against toxigenic Clostridium difficile [55]. Similarly,
CBR-2092, a rifamycin-quinolone hybrid RNA polymerase
and DNA gyrase/topoisomerase IV inhibitor, was designed
as a potential antibacterial agent for the treatment of infections with Gram-positive cocci [56,57]. However, neither of
the two hybrid compounds is being further developed.
Isothiazoloquinolones
The isothiazoloquinolone (ITQ) class of DNA gyrase inhibitors was first described by Abbot in 1988 [58]. These
compounds are quinolone analogues in which the carboxylic
acid group is successfully replaced by the isothiazol-3(2H)one moiety. These compounds most likely form stable ternary complexes with cleaved DNA and topoisomerases,
which is a mechanism of action similar to that of fluoroquinolones [49]. A-62824 (8, Fig. (5)), a direct ciprofloxacin (1,
Fig. (3)) analogue, and its analogues were observed to be
more potent against S. aureus than their quinolone counterparts [58]. Because these compounds possess selectivity and
toxicity problems, none were advanced into clinical development [59]. Recently, Achillion Pharmaceuticals identified
DNA gyrase and topoisomerase IV inhibitors of the ITQ
structural class [60-63]. The representative compound of
Achillion’s ITQs is compound 9 (Fig. (5)), which is a dual
inhibitor of DNA gyrase (IC50 = 1.6 M) and topoisomerase
IV (IC50 = 0.7 M) with potent in vitro antibacterial activity
against MRSA (MIC = 0.12 g/mL) [62] and other resistant
staphylococci and streptococci, as well as Gram-negative H.
influenzae, Moraxella catarrhalis and Neisseria sp. [64].
This compound also displays selectivity against human
topoisomerase II (EC2 = 100 M), low cytotoxicity against
the human cell lines Hep2 and HepG2 (CC50 > 100 M) [62]
and low mutation frequencies of S. aureus at concentrations
close to the MIC values [65].
ACH-702 (10, Fig. (5)) is a novel, advanced representative of ITQ derivatives with improved inhibition potency
against DNA gyrase (IC50 = 680 nM) and topoisomerase IV
(IC50 = 120 nM) compared with compound 9 and the fluoroquinolone moxifloxacin (2, Fig. (3)) [63]. ACH-702 also
retains some activity against single- and double-mutated
DNA gyrase and topoisomerase IV. It was also shown to be
active against bacterial DNA primase, which shares a homologous catalytic site with type IIA topoisomerases,
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
O
O
O
F
O
O
F
N
HN
NH
S
N
N
A-62824 (8)
O
F
NH
N
135
NH
S
O
N
S
N
O
H2N
9
ACH-702 (10)
Fig. (5). Representative isothiazoloquinolone-based inhibitors of type IIA topoisomerase.
designated a TOPRIM (topoisomerase-primase) domain. The
TOPRIM domain possesses two conserved motifs, one of
which centers at a conserved glutamate residue, whereas the
other one at two conserved aspartate residues [66]. ACH-702
possesses broad-spectrum antibacterial activity, covering
antibiotic-resistant Gram-positive strains, including methicillin-resistant (MRSA) and methicillin- and quinoloneresistant S. aureus (MQRSA). The compound displayed bactericidal activities against nondividing S. aureus and Staphylococcus epidermidis biofilm cells that are superior to that of
moxifloxacin, rifampin and vancomycin [67]. Promising
antibacterial activity was also observed against several anaerobes as well as Legionella pneumophila, Mycoplasma
pneumoniae and the Gram-negative H. influenzae, M. catarrhalis and Neisseria sp. but weaker activity was found
against E. coli and other members of the Enterobacteriaceae
[68]. ACH-702 displays in vivo efficacy against two Grampositive models of infection, namely, murine sepsis and
thigh infection, where it reduces the number of colony forming units equal to or greater than those observed for vancomycin treatment [68].
3-Aminoquinazolinediones
3-Aminoquinazolinediones represent a new class of antibacterial agents that inhibit bacterial DNA gyrase and topoisomerase IV and are structurally related to fluoroquinolones
but they lack the carboxylic acid group at position 3, similarly to ITQs. The most studied compound from this class is
Pfizer’s PD 0305970 (11, Fig. (6)), which inhibits E. coli
DNA gyrase with an IC50 value of 200 nM [69,70] and displays potent antibacterial activity against multidrug-resistant
staphylococci, streptococci and enterococci with a low level
of spontaneous resistance development in S. aureus and S.
pneumoniae [71]. This compound is also efficient in murine
systemic infection models and a pneumococcal pneumonia
mouse infection model [69, 71]. Although compounds from
this structural class possess remarkable similarity to the
structure-activity relationship of fluoroquinolones [72], it has
been demonstrated that these compounds interact differently
with the enzyme breakage-reunion and TOPRIM domains,
DNA and Mg2+ [73]. Mutations that affect the inhibitory
activity of compound 11 map mainly to the region of the
TOPRIM domain of GyrB/ParE and not to GyrA, as observed for the fluoroquinolones. The resistance-determining
region of compound 11 overlaps the GyrB quinolone resistance-determining region and the region binding Mg2+ [73].
Crystal structures of compound 11 and levofloxacin in complex with the S. pneumoniae ParC breakage-reunion domain
and the ParE TOPRIM domain in the presence of doublestranded DNA were recently determined [23]. Although the
overall architectures of both complexes appear similar, there
are differences in the interactions of inhibitors with the ParC
and ParE subunits of the enzyme. The oxo group at position
2 of the quinazolinedione ring of compound 11 forms hydrogen bonds with the side chain of Arg117 of ParC but is not
in contact with the Ser79 and Asp83 residues that confer
quinolone resistance. The group at position C-7 of compound 11 interacts with the ParE residues Arg456, Glu474,
Glu475 and Asp435, which are altered in mutants resistant to
compound 11. In contrast, levofloxacin is in contact with the
ParC residues Ser79 and Arg117 through hydrogen bonds
with a water molecule but does not interact with the ParE
Glu474 and Glu475 residues. Mutation of Ser79 may lead to
destabilisation of the drug binding, which leads to quinolone
resistance. These differences in interactions explain the observed lack of cross-resistance between the quinazolinedione
and quinolone classes of type IIA topoisomerase inhibitors
[23].
O
F
H
N
N
N
NH2
O
H 2N
PD 0305970 (11)
Fig. (6). Quinazoline-2,4-dione-based type IIA topoisomerase inhibitor.
Aminopiperidines and Related Compounds
Piperidines (12, 18 and 20, Fig. (7)), 4-aminopiperidines
(13, 15 and 17, Fig. (7)) and cyclohexylamines (14 and 16,
Fig. (7)) are novel non-fluoroquinolone bacterial type II
topoisomerase inhibitors (NBTIs) that target the GyrA/ParC
subunits of DNA gyrase/topoisomerase IV and are not affected by target mutations that cause resistance to fluoroquinolones. A prototype compound is the piperidine viquidacin
or NXL101 (12, Fig. (7)), introduced by Novexel, that was
advanced into Phase I clinical studies but was discontinued
due to significant QT interval prolongation [74,75]. Viquidacin displayed potent antibacterial activity against Grampositive bacteria, including methicillin- and fluoroquinoloneresistant strains. It is a more potent inhibitor of topoisomerase IV (IC50 = 2.2 M in decatenation assay and 0.2
M in relaxation assay) than DNA gyrase (IC50 = 11 M in
supercoiling assay and 23 M in relaxation assay) from E.
coli. For S. aureus enzymes, the inhibition preference is opposite because DNA gyrase is inhibited with IC50 values of
1.0 M and 0.4 M in supercoiling and relaxation assays,
respectively, whereas the IC50 values against topoisomerase
IV are 18 M and 0.3 M in decatenation and relaxation assays, respectively. The isolation of S. aureus mutants
136 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Tomai and Mai
O
S
OH
O
S
N
N
N
F
O
O
N
N
S
CN
HO
GSK299423 (13)
viquidacin, NXL101 (12)
O
O
N
N
NH
N
NH
N
OH
OH
O
N
N
O
NH
NH
N
O
F O
O
F
14
15
CN
O
N
F
OH
N
N
N
O
O
N
NH
NH
O
O
O
17
16
O
O
O
HO
N
HO
N
N
N
O
N
N
H
N
F
18
Se
H
N
O
S
19
OH
O
S
N
O
N
HO
20
Fig. (7). Piperidine- and cyclohexylamine-based type IIA topoisomerase inhibitors.
resistant to viquidacin led to the identification of point mutations within or close to the quinolone resistance-determining
region of GyrA. However, these mutations were different
than those leading to resistance to quinolones, which indicates that the binding sites of viquidacin and fluoroquinolones are distinct but similar [74].
Several pharmaceutical companies have been investigating antibacterial agents with chemotypes related to viquidacin with a focus on addressing its cardiovascular toxicity
side effects. Until recently, when the crystal structure of
GlaxoSmithKline’s GSK299423 (13, Fig. (7)) in complex
with S. aureus DNA gyrase and DNA was published [37],
the design of NBTIs related to viquidacin (12) mainly relied
on classical SAR studies, such as pharmacophore modelling
and the optimisation of the physicochemical properties of
compounds [76]. GSK299423 (13) displays potent inhibition
of supercoiling by DNA gyrase from S. aureus (IC50 = 14
nM) and E. coli (IC50 = 100 nM) and broad-spectrum antibacterial activity against Gram-positive and Gram-negative
pathogens. The binding mode of compound 13 to DNA gyrase differs from that of fluoroquinolones, which provides an
explanation for why compound 13 and its analogues circumvent resistance to fluoroquinolones. The crystal structure
reveals that the inhibitor interacts with DNA on one side and
binds to a transient non-catalytic pocket on the two-fold axis
at the GyrA dimer interface on the other side. The
GSK299423-binding site is close to the active and fluoroquinolone-binding sites. In detail, the left-hand-side (LHS) cyanoquinoline moiety of the inhibitor intercalates between
DNA base pairs, whereas the right-hand-side (RHS) ox-
Prospects for Developing New Antibacterials Targeting
athiolopyridine moiety binds to the formed non-catalytic
binding pocket, where it forms van der Waals interactions
with the Ala68, Gly72, Met75 and Met121 residues of GyrA.
The basic amine of the linker between the LHS and RHS
units is mostly solvent-exposed but also interacts with
Asp83. The key role of the central piperidine unit is to ensure the correct positioning of the LHS and RHS units.
Therefore, it provides an opportunity to modulate the physicochemical properties without compromising the target potency. The elucidation of the GSK299423-binding mode at
the molecular level provides rationalisation for the observed
SAR and a basis for the further optimisation of novel NBTIs
[37, 76].
The next series of NBTIs from GlaxoSmithKline contained a cyclohexylamide moiety instead of an alkyl
piperidine central core [77]. The representative compound 14
(Fig. (7)) displays potent broad-spectrum antibacterial activity against Gram-positive bacteria and less potent activity
against Gram-negative bacteria, which was achieved through
inhibition of DNA gyrase with an IC50 value of 0.013 g/mL
as measured in a gyrase-dependent DNA replication assay.
Compound 14 displayed hERG inhibition with an IC50 of 24
M and demonstrated efficacy and promising pharmacokinetic attributes in a rat respiratory tract infection model [77].
hERG inhibition was further reduced with the next aminopiperidine series, which is represented by compound 15 (Fig.
(7)). This compound also displays potent DNA gyrase inhibition, a promising antibacterial activity profile against relevant pathogens and oral efficacy in a rat lung infection
model [78].
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
137
from S. pneumoniae with IC50 values of 8.84 M and 8.37
M, respectively, and displayed selectivity against human
topoisomerase II (IC50 > 200 M) and moderate hERG inhibition (IC50 = 24.9 M). Promising in vitro antibacterial activity against resistant S. aureus strains was also confirmed
in in vivo systemic infection models with S. aureus and S.
pyogenes in mice [83].
The structural characteristic of NBTIs produced by Johnson & Johnson is a tetrahydroindazole-based central core
between the LHS and RHS units [84,85]. These compounds,
such as compound 19 (Fig. (7)), have been demonstrated to
possess antibacterial activity against multidrug-resistant
Gram-positive pathogens, which is a result of their dual activity against DNA gyrase and topoisomerase IV [85].
At Achillion Pharmaceuticals, a selenophene analogue
(20, Fig. (7)) of viquidacin (12) was developed. This compound displayed improved DNA gyrase inhibition and similar antibacterial activity against a panel of MRSA clinical
isolates but importantly, displayed reduced hERG inhibition
compared with compound 12 [86].
Miscellaneous GyrA/ParC Inhibitors
QPT-1, previously named PNU-286607 (21, Fig. (8))
was discovered by screening at Pharmacia and Upjohn for
compounds possessing whole-cell antibacterial activity [87].
DNA gyrase and topoisomerase IV were identified as targets
by a reverse chemical genomics approach [88]. The active
isomer of QPT-1 is its (-)-enantiomer. The compound displayed activity against a range of pathogenic, antibioticresistant Gram-positive and Gram-negative bacteria and
showed oral efficacy in a murine infection model. QPT-1
selectively inhibited bacterial topoisomerases over human
topoisomerase II. It has been suggested that its mode of action is related to but distinct from the characteristic mechanism of action of fluoroquinolones [88].
NBTIs structurally related to viquidacin and GSK299423
have also been investigated by AstraZeneca [79-81]. It has
been demonstrated that compound 16 (Fig. (7)) inhibits
DNA-dependent ATP hydrolysis by E. coli DNA gyrase and
also blocks cleaved complex stabilisation by ciprofloxacin.
These observations led to the conclusion that compound 16
acts allosterically and increases the apparent Km of DNA
gyrase for ATP. Compound 16 thus acts in the catalytic cycle of DNA supercoiling prior to ATP binding, which is consistent with the observation that compound 16 and other
piperidine-based DNA gyrase inhibitors do not stabilise the
DNA-cleaved complex with the enzyme as observed with the
fluoroquinolones [82]. Efforts by AstraZeneca to reduce
hERG activity while maintaining potent inhibition of topoisomerases and broad-spectrum antibacterial activity resulted
in compound 17, which was advanced into Phase I clinical
trials. A reduction in the hERG activity of compound 17
(IC50 of 233 M) was achieved by lowering the pKa through
the introduction of an electron-withdrawing fluoro group on
the piperidine ring [81]. Compound 17 displays E. coli
topoisomerase IV inhibition with an IC50 value of 48 nM.
The MIC values against S. aureus wild-type and resistant
strains were between 0.06 and 0.125 g/mL, whereas the
MIC value for S. pneumoniae was 0.13 g/mL. The compound displayed weaker activity against Gram-negative P.
aeruginosa and E. coli with MICs of >8 g/mL and 4 g/mL,
respectively. The compound was active in vivo against
MRSA in a neutropenic mouse thigh infection model [81].
Gyramide A (22, Fig. (8)) was also discovered using
whole cell-based screening to find compounds that inhibit E.
coli division [89]. DNA gyrase was identified as the primary
target of compound 22 because it exhibited an IC50 value of
3.3 M in a DNA supercoiling assay [90]. The isolation of E.
coli mutants resistant to compound 22 resulted in the identification of mutations in GyrA, which were previously not
observed with other classes of inhibitors that target type IIA
topoisomerases. Thus, gyramides are predicted to target
DNA gyrase by binding to a previously unidentified region
of the active site. Gyramides B (23, IC50 = 0.7 M) and C
(24, IC50 = 1.1 M) are more potent DNA gyrase inhibitors
than gyramide A. The MIC values of compounds 22-24
against E. coli, P. aeruginosa, Salmonella enterica, S. aureus
and S. pneumonia range from 2.5 to 160 M [90].
Quinoline derivatives similar to viquidacin have also
been investigated by Pfizer (18, Fig. (7)). Their prototype
compound 18 inhibited DNA gyrase and topoisomerase IV
Simocyclinone D8 (26, Fig. (8)) is a hybrid antibiotic
composed of aminocoumarin and polyketide moieties produced by Streptomyces antibioticus Tü 6040. The compound
Novel DNA gyrase inhibitors interacting with GyrA were
identified by virtual screening of the National Cancer Institute bank of compounds. Compound 25 was determined to
target the previously unexplored structural pocket formed at
the dimer interface of the GyrA subunit with an IC50 value of
72 M and did not act as a DNA intercalator [91].
138 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Tomai and Mai
O
HN S
O
O
HN
O
O2N
NH
N
N
H
N
O
O
F
N
HN
R
(-)-PNU-286607, QPT-1 (21)
O
gyramide A (22), R = Oi-Pr
gyramide B (23), R = i-Bu
gyramide C (24), R = Ph
25
O
O
HO
O
OH
HO
O
H
N
O
O
O
O
O
O
O
O
OH
OH
OH
OH
Cl
simocyclinone D8 (26)
Fig. (8). Topoisomerase inhibitors of different structural types.
inhibits bacterial DNA gyrase and human topoisomerase II
but not bacterial topoisomerase IV. Its antibacterial activity
is limited to Gram-positive microorganisms [92,93]. Despite
its structural similarity to aminocoumarin-based DNA gyrase
inhibitors, which display ATP-competitive inhibition by
binding to GyrB, simocyclinone D8 has been observed to
bind to a site distinct from but adjacent to the quinolonebinding site. It interacts with two binding pockets, one of
which accommodates the aminocoumarin moiety and the
other accommodates the polyketide part of the molecule
[38]. The crystal structure of the simocyclinone D8-DNA
gyrase complex enables the design of novel inhibitors that
can target these newly identified binding pockets of the enzyme.
GYRB AND/OR PARE INHIBITORS
As previously described, the discovery of novel GyrA
and/or ParC inhibitors was mainly guided through ligandbased design via classical structure-activity relationship studies and the optimisation of physicochemical properties.
However, this may change in the near future because the
crystal structures of representatives of the major drug classes
in complex with DNA gyrase or topoisomerase IV have been
recently solved. In contrast, GyrB and/or ParE inhibitors are
commonly identified through fragment-based design as well
as virtual screening techniques, and structure-based hit optimisation often results in lead compounds with potent inhibitory activity and promising antibacterial activity profiles.
Design is often inspired by natural products that were observed to inhibit bacterial type IIA topoisomerases [12].
The ATPase domains of DNA gyrase and topoisomerase
IV were crystallised with the non-hydrolysable ATP analogue ADPNP [27,94]. In the E. coli GyrB43-ADPNP complex, the adenine ring forms hydrogen bonds with Asp73,
Gly77 and Thr165. Inhibitors often bind to the adeninebinding pocket, where they form two hydrogen bonds with
the Asp73 side chain, one of which is formed directly, and
the other is formed through a structurally conserved water
molecule. Therefore, inhibitors must possess a hydrogen
bond donor, which interacts with the carboxylate group of
Asp73 and an adjacent hydrogen bond acceptor, which interacts with the conserved water molecule. In addition, hydrogen bonds are generally formed with the Arg136 side chain,
which is the residue that is not in contact with the ATP
molecule. The described binding mode of the majority of
GyrB/ParE inhibitors provides some basis for the selectivity
against other ATP-utilising enzymes.
Natural Products
Many successful antibacterial agents have been identified
from empirical screening of natural products. Some of the
first bacterial enzyme inhibitors identified that occupy the
ATP-binding site were the aminocoumarins novobiocin (27)
and clorobiocin (28) (Fig. (9)), which are produced by Streptomyces species. The crystal structures of novobiocin [94-96]
and clorobiocin [29,97] in complex with GyrB and ParE indicate that their binding site partially overlaps the ATPbinding site, which prevents ATP binding. The L-noviose
sugar interacts with Asp73, whereas the coumarin ring is in
contact with the Arg136 side chain. Due to its potent antibacterial activity against Gram-positive bacteria, novobiocin
was used for the treatment of infections with MRSA but was
later withdrawn from clinical use because of its toxicity and
the development of resistance [12]. A naturally occurring
mutation of Arg136 of the E. coli GyrB leads to resistance to
aminocoumarins [14,98]. Novel derivatives of aminocoumarins are still being designed and synthesised to improve
their antibacterial activity and physicochemical properties
and to avoid resistance development [99,100].
The cyclothialidines 29 and 30 (Fig. (9)) are another
class of ATP-competitive DNA gyrase inhibitors derived
from nature. These are potent inhibitors of GyrB but possess
only weak antibacterial activity, most likely because of poor
penetration through the bacterial cell wall [101]. The crystal
structure of compound 30 in complex with GyrB24 reveals
that the adenine ring of ATP and the resorcinol ring of
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
139
Cl
O
O
O
O
O
OH
O
O
O
N
H
OH
O
O
OH
NH2
S
HN
O
HO
O
29, R = OH
30, R = H
O
N
H
OH
OH
28, clorobiocin
O N
O
OH
O
OH
O
HN
27, novobiocin
O
N
H
O
O
O
N
H
O
OH
OH
HN
OH
N
O
O
HN
O
Br
NH2
R
N
S
O
O
31
OH
O
H
O
HO
O
NH
HO
HN
HO
H
O
OH
H OH
O
O
O
N
Cl
O
O
O
Cl
O
32, kibdelomycin, R = Me
33, kibdelomycin A, R = H
O
OH O O
O
O
O
O HO HO
NH2
F
O
F
34
Fig. (9). Structures of naturally occurring DNA gyrase inhibitors and their derivatives.
compound 30 are involved in similar hydrophobic and hydrogen bonding interaction networks with the protein [95].
The modification of the cyclothialidine 29 structure to obtain
14-membered lactone derivatives led to analogues with antibacterial activity against S. aureus, S. pyogenes and E. faecalis [102]. The hydroxyl group adjacent to the thiomethyl
side chain of the phenyl ring is essential for potent inhibition
of the enzyme but is also a target for glucuronidation, which
leads to rapid inactivation and clearance from the plasma.
Recent efforts at cyclothialidine structure optimisation led to
bicyclic dilactam-lactone derivatives, such as compound 31
(Fig. (9)), which displays antibacterial activity against Grampositive strains and improved pharmacokinetic properties
that resulted in its in vivo efficacy in a septicaemia infection
model in mice [103]. Novel cyclothialidine derivatives have
also been developed through a combination of molecular
docking and quantitative structure-activity relationship
(QSAR) studies [104].
Kibdelomycin (32) [105], kibdelomycin A (33) [106]
(Fig. (9)) and structurally related amycolamycin [107] have
been observed to be inhibitors of the ATPase activity of
DNA gyrase and topoisomerase IV. Kibdelomycin exhibits
antibacterial activity against Gram-positive S. aureus, S.
pneumoniae, E. faecalis and B. subtilis with MIC values between 0.12 g/mL and 64 g/mL and Gram-negative H. influenzae with an MIC value of 2 g/mL but is inactive
against E. coli. The cellular target for compound 32 is DNA
gyrase, which is inhibited in a low nanomolar range (IC50
values of 60 nM and 9 nM for the E. coli and S. aureus enzymes, respectively). In contrast, this compound displays
weaker inhibitory potency for topoisomerase IV (IC50 values
of 29 M and 500 nM for the E. coli and S. aureus enzymes,
respectively). This natural product did not display crossresistance with other major classes of DNA gyrase inhibitors
[105]. Kibdelomycin A (33), a kibdelomycin derivative
without the methyl group on the pyrrole ring, displays
weaker DNA gyrase inhibition and consequently weaker
antibacterial activity [106]. The methyl group at position 5 of
the pyrrole ring appears to form additional hydrophobic interactions in the adenine-binding pocket of the enzyme,
which can also be derived from the activities of clorobiocin,
coumermycin A1 and the recently published GyrB inhibitors
developed by AstraZeneca [31]. Amycolamycin, which also
contains a 3,4-dichloro-5-methylpyrrole moiety in the structure, inhibits E. coli DNA gyrase and topoisomerase IV (IC50
values of 24.4 ng/mL and 6250 ng/mL, respectively) more
potently than novobiocin. It has also demonstrated very potent antibacterial activity against resistant strains of S. aureus
(MRSA, MSSA and QRSA), E. faecium, E. faecalis and S.
pneumoniae, including VRE and PRSP, with MIC values
between 0.125 g/mL and 2 g/mL. The compound has also
been demonstrated to moderately inhibit the growth of some
Gram-negative pathogens, including E. coli, P. aeruginosa
and K. pneumonia, with MIC values of 8 g/mL [107].
140 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Tomai and Mai
Quercetin diacylglycoside analogues, such as compound
34 (Fig. (9)), have been reported as dual inhibitors with IC50
values of 0.78 M and 9.2 M in E. coli DNA gyrase supercoiling and topoisomerase IV decatenation assays, respectively. Antibacterial activity was observed for Gram-positive
microorganisms with MIC values between 0.13 g/mL and
0.5 g/mL for MRSA, S. pneumoniae and VRE but no activity against Gram-negative strains was found. An ATPcompetitive mechanism of inhibition has been hypothesised
based on molecular docking studies; however, no data have
been obtained from ATPase assays [108].
High-Throughput Screening (HTS) and Structure-Based
Virtual Screening (VS)
HTS and VS are commonly used for the identification of
hit compounds, which are then optimised through structurebased design in the following iterative steps. The availability
of several crystal structures of the GyrB and ParE subunits in
complex with ADPNP and inhibitors and well defined, partially hydrophobic ATP-binding pockets provide an excellent
basis for structure-based optimisation.
Structural information on the binding of cyclothialidine
30 in complex with GyrB was used for the virtual screening
of DNA gyrase inhibitors [109,110]. In their first virtual
screening campaign, Brvar and co-workers constructed a 3D
pharmacophore based on a cyclothialidine 30 binding mode
using the LigandScout software [111]. Because the resorcinol moiety of compound 30 is involved in the formation of
hydrogen bonds with Asp73 in E. coli DNA gyrase, which
are crucial for affinity, approximately 20,000 compounds
containing the resorcinol, mono- and poly-substituted phenol
moieties were extracted from the ZINC library of commercially available compounds [112]. The enriched library was
then docked into the GyrB ATP-binding site, and twelve
compounds were selected for biological evaluation. Of these,
compound 35 (Fig. (10)) displayed the most potent inhibition
of E. coli DNA gyrase supercoiling activity with an IC50
value of 25 M [109]. In the next VS, the researchers retained the phenol moiety because it was demonstrated to be
sufficient for the formation of two hydrogen bonds with the
Asp73 side chain and searched for a replacement for the thiazole ring. From this virtual screening, 26 compounds,
mainly
based
on
thiazolidine-2,4-dione
and
2thioxothiazolidin-4-one (rhodanine) scaffolds, were selected
and tested for E. coli DNA gyrase inhibition. Four hit compounds with inhibitory activities between 64 M and 680 M
were identified. Compound 36 was the most potent and displayed weak activity against H. influenzae with an MIC
value of 32 g/mL [110].
structure of clorobiocin in complex with GyrB [29,98] was
used as a starting point. A three-dimensional pharmacophore
model was built and then used in a VS campaign that was
divided into two phases. In the first phase, a class of 4’methyl-(4,5’-bithiazole)-2,2’-diamine-based E. coli DNA
gyrase inhibitors was identified, and used as a starting point
for the second phase which involved a search for more potent members of this chemotype. Six E. coli DNA gyrase
inhibitors with IC50 values between 1.1 M and 125 M and
some antibacterial activity against H. influenzae (MIC values
between 32 g/mL and 128 g/mL) were identified. The
most potent VS hit, namely compound 37 (Fig. (11)), was
also evaluated through surface plasmon resonance (Kd = 6.6
M), differential scanning fluorimetry and microscale thermophoresis. The crystal structure of 37 in complex with the
GyrB24 fragment was determined which confirmed its binding to the ATPase domain of DNA gyrase. The amide hydrogen of the 2’-propionylamido group and the 3’-nitrogen
of the thiazole ring interact with the Asp73 side chain and
the conserved water molecule, respectively. The salt bridge
formed between the carboxylic acid group of the inhibitor
and the Arg136 guanidino group improves the potency of the
GyrB inhibition (Fig. (11)) [33].
H
N
O
N
O
S
OH
N
S
N
H
37
(a)
(b)
OH
S
OH
O
OH
NH
N
N
O
S
HO
35
O
O
S NH2
S
Cl
36
Fig. (10). Structures of resorcinol and phenol-based DNA gyrase
inhibitors identified through VS.
Substituted 4,5’-bithiazoles were identified through a VS
as novel E. coli DNA gyrase inhibitors [33]. The crystal
Fig. (11). a) Structure of the VS hit 37 and b) 2D interaction diagram of compound 37 in the ATP-binding site of E. coli DNA gyrase (PDB entry: 4DUH). The residues involved in electrostatic and
van der Waals interactions are coloured violet and green, respectively. (The color version of the figure is available in the electronic
copy of the article).
The benzimidazole urea class of DNA gyrase inhibitors
from Vertex was identified from 30,000 drug-like compounds using a high-throughput ATPase assay targeting the
GyrB subunit [113]. The crystal structure of 1-(4-acetyl-6-
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
pyridin-3-yl-1H-benzimidazol-2-yl)-3-ethylurea, a representative compound from this structural class of inhibitors, in
complex with E. coli ParE (PDB entry: 3FV5) displays that
the hydrogen bond donor NH groups of the ethyl ureas interact with the Asp69 side-chain carboxylate group in the adenine-binding pocket of the ATP-binding site. An additional
hydrogen bond is formed between benzimidazole N-1 and
the conserved water molecule that is in contact with Asp69.
An initial hit, namely compound 38 (Fig. (12)), inhibited S.
aureus and E. coli DNA gyrase with Ki values of 2 M and
20 M, respectively, and displayed poor antibacterial activity
against S. aureus, S. pneumoniae and H. influenzae. The initial structure-based optimisation of compound 38 resulted in
compound 39 (Fig. (12)), which exhibited improved inhibitory activity against E. coli and S. aureus DNA gyrase with
Ki values of 81 nM and 130 nM, respectively, and weak inhibition of E. coli topoisomerase IV (Ki = 2.3 M). The antibacterial activity of compound 39 was also better than that of
compound 38 because it displayed MIC values of 16 g/mL
and 2 g/mL against S. aureus and S. pneumonia, respectively. A combination of structure-based design, molecular
modelling and structure-activity relationship analyses led to
compound 40 (Fig. (12)) [114], which is a potent dualtargeting inhibitor of S. aureus and E. coli DNA gyrase and
E. coli topoisomerase IV (Ki values between <4 nM and 23
nM). The primary target of compound 40 in S. aureus, E.
faecalis and H. influenzae was determined to be GyrB,
whereas in S. pneumonia, the target appears to be ParE
[115]. Compound 40 also displayed selectivity against human topoisomerase II because the EC50 value was higher
than 25 M, as determined through decatenation assays. The
compound also possessed a promising antibacterial activity
profile with MIC values against S. aureus, S. pneumoniae
and H. influenzae of less than 1 g/mL. GyrB mutations conferring resistance to novobiocin in these pathogens displayed
little or no cross-resistance to compound 40 and the in vitro
spontaneous resistance frequencies were low for wild-type E.
faecalis [116]. In vivo, the compound showed efficacy in
rodent models of skin and skin structure infection and pneumonia when administered both intravenously and orally. All
of these features of the representative compound 40 from the
benzimidazole urea class of dual-targeting type IIA topoisomerase inhibitors highlights their great potential for becoming clinically viable antibiotics [114]. Vertex has recently reported the latest generation of dual-targeting ben-
zimidazole ureas, such as compound 41 (Fig.(12)), which
exhibits Ki values of 9 nM and 12 nM against S. aureus
DNA gyrase and topoisomerase IV, respectively, and displays good antibacterial activity against Gram-positive
pathogens and in vivo efficacy in a mouse S. aureus kidney
infection model and a S. aureus neutropenic rat thigh model
[117].
Vertex recently also published on the novel pyrazolthiazole class of DNA gyrase inhibitors [30]. According to their
report, these compounds are generally more potent against E.
coli DNA gyrase than S. aureus DNA gyrase. For compound
42 (Fig. (12)), the Ki value was less than 4 nM against the E.
coli enzyme and 140 nM against its S. aureus orthologue.
Compound 42 was active against an E. coli tolC pump
knockout strain with an MIC value of 0.5 g/mL. The absence of activity against wild-type E. coli indicates that these
compounds appear to be effluxed from the bacteria. The difference in the inhibition of E. coli and S. aureus DNA gyrase
by pyrazolthiazoles was explained by the crystal structures
of the enzyme-inhibitor complexes. The superposition of the
3D structures revealed that there is less space in the hydrophobic pocket of the adenine-binding pocket in the S. aureus
enzyme than in its E. coli counterpart. The shape of the hydrophobic pocket of S. aureus GyrB is defined by Ile51,
Leu103 and Ile175 residues, whereas in E. coli, the GyrB
isoleucines are replaced by smaller valines, and leucine is
substituted by methionine. From this data, it can be concluded that compounds inhibiting E. coli GyrB show less
potent S. aureus GyrB inhibition due to less optimal interactions in the hydrophobic pocket of the adenine-binding
pocket.
Based on the success of the benzimidazole urea class of
dual-targeting type IIA topoisomerase inhibitors, several
novel structurally similar chemotypes have been investigated, focusing on the replacement of either the benzimidazole ring or the ethyl urea moiety. Evotec Ltd. reported a
class of imidazolo[1,2-a]pyridine- and [1,2,4]triazolo[1,5a]pyridine-based inhibitors targeting the GyrB and ParE
ATPase subunits. One of the most potent representatives of
the [1,2,4]triazolo[1,5-a]pyridine class is compound 43 (Fig.
(13)), which inhibits GyrB with an IC50 value of 54 nM and
ParE with an IC50 value of 25 M. The compound possesses
modest antibacterial activity against Gram-positive S.
aureus, S. pneumoniae and E. faecalis with an MIC value of
OH
OH
N
N
N
N
N
N
N
NH
HN
HN
HN
O
O
HN
HN
38
39
NH
O
HN
O
O
N
HN
NH
N
N
O
N
NH
NH
N
S
F
F
N
O
O
HN
40
141
41
42
Fig. (12). Benzimidazole urea class (38-41) and pyrazolthiazole class (42) of inhibitors of DNA gyrase and topoisomerase IV.
142 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
2 g/mL and against Gram-negative E. coli with an MIC
value of 4 g/mL. In contrast to [1,2,4]triazolo[1,5a]pyridine 43, imidazolo[1,2-a]pyridine 44 (Fig. (13))
showed balanced dual inhibition with Ki values against GyrB
and ParE of less than 4 nM and 14 nM, respectively, and a
significantly improved S. aureus MIC value of 0.031 g/mL.
The resistance frequency of the compounds was estimated to
be low (<1.8 10-9), which is consistent with dual-targeting
inhibition. Compound 43 was also found to be non-cytotoxic
in a HepG2 cell assay, which indicates that these compounds
are not general ATPase inhibitors [118].
Pfizer’s contribution to the field of dual-targeting DNA
gyrase and topoisomerase IV inhibitors similar to benzimidazole ureas are the imidazo[1,2-a]pyridines (Fig. (13))
[119]. In this series, GyrB inhibition has been demonstrated
to be generally more potent than ParE inhibition. In studies,
compound 45 (Fig. (13)) inhibited S. pneumoniae GyrB with
an IC50 value of 53 nM, whereas the inhibition of ParE was
five-fold weaker with an IC50 value of 250 nM. The compound displayed similar activity against wild-type S. aureus
and MRSA (MIC = 0.5 g/mL). In addition, it was active
against S. pyogenes (MIC = 0.5 g/mL) and wild-type (MIC
= 0.125 g/mL) and fluoroquinolone-resistant S. pneumoniae
(MIC = 0.125 g/mL). Compound 45 was also efficient in
vivo against murine S. pyogenes sepsis and S. pneumoniae
lung models.
N
N
N
N
N
HN
N
N
N
N
N
N
NH
HN
O
HN
N
HN
O
HN
43
N
N
O
HN
44
45
Fig. (13). [1,2,4]Triazolo[1,5-a]pyridine 43, imidazolo[1,2a]pyridine 44 and imidazo[1,2-a]pyridine 45 targeting GyrB and/or
ParE ATPase subunits.
Fragment-Based Drug Design
The advantage of fragment-based drug design (FBDD) is
that smaller compound fragments can feasibly probe a
greater proportion of the chemical space in the active site of
the protein under study. Through a process of structurebased fragment optimisation, it is possible to design and
build desirable physicochemical properties [120].
Because random screening of the F. Hoffman-La Roche
compound library discovered no suitable lead compounds in
the search for novel DNA gyrase inhibitors, an alternative
fragment-based approach was applied. In silico needle
screening, where a needle is defined as a low-molecularweight inhibitor that is able to penetrate into deep and narrow pockets of the active sites, was followed by a biased
high-throughput DNA gyrase screening. This strategy identified an indazole class of DNA gyrase inhibitors, which are
discussed elsewhere [12], as well as recently published phenol-based inhibitors interacting with GyrB [121]. The opti-
Tomai and Mai
misation of the initial hit 46 (Fig. (14)) resulted in the potent
E. coli DNA gyrase inhibitor 47 (Fig. (14)), which has a
maximum non-effective concentration (MNEC) of 0.13
g/mL and a promising antibacterial activity profile against
Gram-positive S. aureus, S. epidermidis and S. pyogenes
(MIC values between 0.5 g/mL and 32 g/mL).
Cl
OH
OH
O
N
H
N
N
O
O
Cl
46
47
Fig. (14). Phenol-based DNA gyrase inhibitors from F. Hoffman-La
Roche.
An AstraZeneca virtual screening of an assembled library
of fragments, which were previously observed as ATPcompetitive inhibitors of protein kinases, using the S. pneumoniae ParE structure, resulted in the discovery of azaindoles as dual GyrB and ParE inhibitors [122]. The initial hit
fragment 48 (Fig. (15)) was predicted to form interactions
with Asp78 and the conserved water molecule through its
azaindole moiety, whereas the phenyl ring at position 5 was
oriented toward Arg140, which is a residue crucial for potent
inhibition. Fragment 48 was first transformed into compound
49 (Fig. (15)), which inhibits S. pneumoniae ParE with an
IC50 value of 7.7 M. The crystal structure of compound 49
bound to S. pneumoniae ParE revealed the expected binding
mode and enabled further optimisation of the azaindole series. Compound 50 displayed very potent inhibition of S.
pneumoniae ParE and S. aureus GyrB with IC50 values of 5
nM but displayed weak antibacterial activity against S.
aureus and S. pneumoniae with MIC values higher than 25
M and 6.25 M, respectively. The crystal structure of S.
pneumoniae ParE in complex with compound 50 (Fig. (16))
revealed that the 2-aminocarboxamido azaindole moiety interacts with Asp78 and the conserved water molecule. An
additional electrostatic interaction was formed with Asn51,
and nicotinic acid formed a - stacking interaction with
Arg81 and a hydrogen bond with Arg140. Trifluoromethyl
pyrazole was observed to form hydrophobic contacts with a
small pocket formed by Met83, Pro84, Thr94 and Ile98. The
antibacterial activity did not correlate with improvements in
inhibitory activity, but a relationship between logD and the
antibacterial activity against Gram-positive strains was observed. An increase in the lipophilicity of compound 50
(logD = -0.99) resulted in compound 51 (logD = 2.91),
which retained inhibitory potency against S. pneumoniae
ParE and S. aureus GyrB with IC50 values of 3 nM and 2
nM, respectively, but exhibited remarkably improved antibacterial activity with a S. pneumoniae MIC value of less
than 0.02 M and MIC values against S. aureus and its methicillin- and quinolone-resistant strain of 0.098 M and 0.2
M, respectively [122]. The microbiological characterisation
of pyrimidine-based topoisomerase inhibitors sharing some
structural elements with azaindoles and possessing very potent antibacterial activity against Gram-positive pathogens
and their antibiotic-resistant strains was recently disclosed
by AstraZeneca [123].
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
H
N
N
H
N
N
NH
O
OH
48
49
H
N
N
NH
O
O
OH
50
N
N
F3C
N
H
N
N
143
O
O
N
51
N
N
F3C
N
HN
O
O
Fig. (15). Azaindole-based dual GyrB and ParE inhibitors investigated by AstraZeneca.
limits its Gram-negative potency [31, 124]. The crystal structure of the S. aureus GyrB-compound 54 complex confirmed
the general binding mode of GyrB inhibitors: hydrogen
bonding of the pyrrolamide moiety with Asp81 and the conserved water molecule, - stacking interactions between the
thiazole ring and Arg81, and a salt bridge between the carboxylate of compound 54 and the Arg144 side chain [31].
Fig. (16). 2D interaction diagram of compound 50 in the ATPbinding site of S. pneumoniae topoisomerase IV (PDB entry:
4EMV). The residues involved in the electrostatic and van der
Waals interactions are coloured violet and green, respectively.
Pyrrolamides are another series of DNA gyrase inhibitors
developed by AstraZeneca. These were identified through
fragment-based nuclear magnetic resonance screening [32].
The pyrrolamide series was optimised using iterative X-ray
crystallography, which quickly resulted in the identification
of compounds 52 and 53 (Fig. (17)). These compounds are
potent inhibitors of E. coli GyrB with IC50 values of 0.9 nM
and 25 nM, respectively. Although compound 52 displayed
the greatest potency against both Gram-positive and Gramnegative strains, compound 53 was selected for further profiling due to its combination of suitable potency and desirable solubility. Compound 53 exhibited bactericidal activity
against S. aureus, S. pneumoniae and H. influenzae with
minimal bactericidal concentrations (MBC) of 8 g/mL, 0.5
g/mL and 4 g/mL, respectively, and displayed efficacy
against S. pneumoniae in a mouse lung infection model [32].
The antibacterial activity of compound 53 was optimised in a
subsequent study to yield compound 54 (Fig. (17)) with an
IC50 of 4 nM against S. aureus DNA gyrase and MIC values
of 0.06 g/mL against S. pneumoniae, 2 g/mL against S.
aureus and its methicillin- and quinolone-resistant strain,
0.06 g/mL against M. catarrhalis and 0.25 g/mL against
E. faecium and H. influenza; however, this compound was
inactive against E. coli. Its activity against an effluxdeficient strain of E. coli suggested that an efflux mechanism
Pyrrolopyrimidine dual-targeting GyrB and ParE inhibitors were investigated by Trius [35,36]. These researchers
used a fragment-based crystallographic screening to identify
scaffolds with adjacent hydrogen bond donor and acceptor
moieties, which can participate in interactions with the aspartate residue and conserved water molecule in the adeninebinding pocket of the ATP-binding site. A key goal of the
rational design of topoisomerase inhibitors was to obtain
activity against both Gram-negative bacteria and Grampositive bacterial strains. This was accomplished by introducing some degree of flexibility into the structure to compensate for the differences between the enzyme structures of
Gram-positive and Gram-negative bacterial strains. The
compounds were also carefully optimised for physicochemical characteristics that promote Gram negative entry. One of
the optimised inhibitors (55, Fig. (18)) showed excellent
binding affinity for E. coli and Francisella tularensis with Ki
values of less than 0.3 nM and potent inhibition of E. coli
and F. tularensis ParE with Ki values of 1.7 nM and 2.5 nM,
respectively. Compound 55 displayed good antibacterial
activity against Gram-positive S. aureus (MIC < 0.06
g/mL) and S. pneumoniae (MIC = 0.25 g/mL) and Gramnegative E. coli (MIC = 2 g/mL), H. influenzae (MIC = 2
g/mL), A. baumannii (MIC = 4 g/mL) and P. aeruginosa
(MIC = 4 g/mL) [36].
DNA GYRASE AS A TARGET
TUBERCULAR DRUG DISCOVERY
FOR
ANTI-
The prospect of using DNA gyrase as a target for the development of new antituberculosis drugs and the role of
fluoroquinolones in tuberculosis were comprehensively reviewed by Mdululi and Ma [125]. The development of new
drugs that can overcome the spread of multidrug-resistant
tuberculosis (MDR-TB) and emerging extremely drugresistant tuberculosis (XDR-TB) can be approached from
two perspectives. The first approach, which is a genomederived, target-based approach, includes the identification
and validation of novel targets with no pre-existing mechanisms of resistance. This approach has had little success in
the antibacterial field in general. For many essential targets,
we are unable to identify potent and specific drug-like inhibitors, and very often we are unable to understand how to
convert an in vitro potent enzyme inhibitor into a compound
144 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
H
N
HO
O
O
HN
Cl
Tomai and Mai
N
H
N
S
N
HN
Cl
Cl
N
N
Cl
52
H
N
O
O
O
O
S
OH
HN
Cl
N
Cl
F
53
OH
N
54
Fig. (17). Representatives of the pyrrolamide series of GyrB inhibitors investigated by AstraZeneca.
with good antimycobacterial activity that can easily penetrate
the bacterial cell wall. The second approach, which has had
significant success in the past, includes remodelling of the
existing antibacterial drug classes and exploration of known,
clinically validated drug targets with the aim of preparing
new generations and new chemical classes based on older
drugs with better antimycobacterial activity and a lower possibility of cross-resistance with drugs for which resistance
has developed. DNA gyrase is a validated and pharmaceutically effective target for the drug discovery of new antitubercular drugs. Using the second approach, several newer
fluoroquinolones, such as moxifloxacin (2, Fig. (3)) and gatifloxacin, have demonstrated potent activity against M. tuberculosis, and this class of compounds holds great potential for
shortening the duration of treatment from the current 4 to 6
months and overcoming multidrug-resistant tuberculosis
[125,126].
H
N
N
Cl
O
N
S
N
N
N
H
H
NH2
55
Fig. (18). Pyrrolopyrimidine-based GyrB and ParE inhibitor investigated by Trius.
The M. tuberculosis genome encodes only homologues
of the E. coli DNA gyrase genes (gyrA and gyrB) and E. coli
topoisomerase I genes, but there is no homologue of the E.
coli topoisomerase IV genes (parC and parE). There is no
evidence that M. tuberculosis has homologues of the topoisomerase IV, parC and parE genes, which are present in
most bacteria. Therefore, DNA gyrase is the only topoisomerase II enzyme in M. tuberculosis and must be responsible for negatively supercoiling the DNA, relaxing positive
supercoils and decatenating replicated DNA [125].
The newer fluoroquinolones moxifloxacin and gatifloxacin were introduced into the market for use against a wide
variety of pathogens, and both exhibit good activity against
M. tuberculosis with MIC values of 0.12 g/mL and 0.5
g/mL, respectively [127]. The antimycobacterium activities
of both compounds correlate very well with their IC50 values
against M. tuberculosis DNA gyrase (the gatifloxacin IC50
value is 3 g/mL, and the moxifloxacin IC50 is 4.5 g/mL).
Both drugs are active against replicating as well as nonreplicating, persistent mycobacteria and are effective against
quinolone-resistant clinical isolates [128]. A number of
clinical trials have been conducted with both compounds in
several different drug combinations, and both moxifloxacin
and gatifloxacin are currently in Phase III clinical trials to
evaluate the multidrug usage and their tuberculosis treatment-shortening potential (HIV-HCV-TB Pipeline Report,
June 2013). However, resistance of M. tuberculosis to
fluoroquinolones is emerging and is caused by amino acid
substitution within the QRDR of the GyrA subunit as well as
substitutions in GyrB inside and outside the QRDR
[129,130].
Aubry and co-workers studied the correlation between
the antimycobacterium activity of quinolones and their activities against other common pathogens, such as E. coli and
S. aureus. These researchers concluded that the quinolone
structure-activity relationship against M. tuberculosis correlates poorly with the activities against other pathogens, such
as E. coli and S. aureus [131]. Solely based on their activities
against other pathogens, it is not possible to predict the potency of fluoroquinolones against M. tuberculosis. These
facts necessitate the need for specifically targeting M. tuberculosis by taking into account its unique and complex cell
wall structure and efflux systems and the structural differences in the active site of M. tuberculosis. For example, the
widely conserved Ser83 is the key residue for the interaction
with fluoroquinolones in E. coli, and the equivalent position
in M. tuberculosis is Ala90 [131].
Several groups have focused their efforts in establishing
structure-activity relationships for fluoroquinolones with
respect to M. tuberculosis. Recently, Gomez and co-workers
prepared a small library of gatifloxacin derivatives with different substituents on the piperazine ring of the gatifloxacin
molecule and evaluated these compounds against M. tuberculosis; however, all of the compounds were less active at
inhibiting M. tuberculosis growth compared with the existing
fluoroquinolones [132]. Malik and co-workers compared the
activity of a set of 8-methoxy quinazolinediones with cognate fluoroquinolones against several fluoroquinoloneresistant gyrA mutants of Mycobacterium smegmatis, which
lack the topoisomerase IV enzyme. To examine the relationships between drugs and gyrase structures in order to determine possible difference in the interactions, the MIC for
each drug was measured with three gyrA mutants and was
normalised to the wild-type MIC. These researchers found
that quinazolinediones exhibited higher MIC values than
fluoroquinolones; however, the MICs for fluoroquinoloneresistant gyrA mutants normalised to the MIC for wild-type
cells were lower. These researchers concluded that the 2,4quinazolinediones impart lethality to fluoroquinolone-like
molecules in the absence of protein synthesis, which is a
phenomenon that is not explained by the X-ray structure of
the drug-enzyme-DNA complex [133]. Minovski and coworkers presented a combination of chemometric and molecular modelling approaches and provided novel QSAR
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
guidelines that may aid the further development and optimisation of fluoroquinolones against M. tuberculosis [134,135].
essential for activity and forms two hydrogen bonds with
Asp79. Aryl or heteroaryl groups at site 2 form cation- interactions with the conserved Arg82. The basic side chain
attached to the aryl or heteroaryl group at site 2 is essential
for cellular potency, because its removal results in the loss of
mycobacterium activity. The crystal structure also revealed
additional novel interactions in the hydrophobic pocket beneath the active site loop. This unique hydrophobic pocket is
most likely responsible for the specificity of this series of
compounds, which are very weakly active or inactive against
DNA gyrase from other Gram-positive and Gram-negative
species. The most promising compounds demonstrated excellent mycobacterial killing under in vitro, intracellular and
hypoxic conditions [34].
The results of studies with moxifloxacin and gatifloxacin
(potent inhibitors of the GyrA subunit) suggest that DNA
gyrase may be a good target for reducing the length of tuberculosis treatment regimens. Increasing the resistance to
fluoroquinolones has driven interest in targeting the DNA
gyrase subunit B (GyrB). The inhibition of the GyrB subunit
still exerts the same phenotypic effects on bacterial viability
as fluoroquinolones. The ATPase domain of M. tuberculosis
DNA gyrase is thus an attractive but under-explored target
for the development of new classes of compounds as potential antimycobacterial agents. In 2011, Chopra and his coworkers evaluated two potent small-molecule inhibitors of
GyrB, novobiocin (27, Fig. (9)) and aminobenzimidazole 1
(AB-1, 44, Fig. (13)), to validate the targeting of GyrB as a
therapeutic strategy in the context of new tuberculosis treatments. The results revealed that the AB-1 GyrB inhibitor is
bactericidal with excellent activity against drug-resistant
tuberculosis strains, including fluoroquinolone-resistant
strains, with MIC values in the range of 0.312 to 2 mg/L. In
contrast, novobiocin exhibited lower potency with MIC values ranging from 0.156 to 8 mg/L. AB-1 does not exhibit
antagonism against rifampicin and isoniazid. The resistance
generated against AB-1 does not cross-react with fluoroquinolones. AB-1 was found to have potent activity in a LORA
assay, which is an in vitro assay for antimicrobial activity
against non-replicating persistent tuberculosis, with an MIC
of 1 mg/L. Additionally, AB-1 significantly reduced the
counts of lung colony-forming units in a dose-dependent
manner in a murine model of tuberculosis. These researchers
concluded that the AB-1 GyrB inhibitor exhibits many of the
characteristics required for consideration as a potential frontline antimycobacterial therapeutic [136].
Karkare and co-workers observed that naphthoquinone
diospyrin (58, Fig. (19)) is an inhibitor of the supercoiling
reaction catalysed by M. tuberculosis gyrase with an IC50
value of 15 M. This compound is a non-competitive inhibitor of the ATPase reaction, and these researchers proposed
that its binding site is within the N-terminal domain of GyrB
close to but not overlapping the ATP-binding site. This novel
binding mode may be a starting point for the development of
novel antimycobacterial agents [137].
IMPORTANCE OF PHYSICOCHEMICAL PROPERTIES FOR ANTIBACTERIAL ACTIVITY
Currently, it is well known that antibacterial compounds
occupy a unique chemical space with unique physicochemical properties that is different from the space occupied by
drugs following the Lipinski rule of 5 [138]. O’Shera and
Moser studied the physicochemical properties of antibacterial compounds and their accompanying chemical spaces and
proposed guidelines for distinguishing compounds with
probable Gram-negative activity. These researchers compared a set of 147 compounds with antibacterial activity to
drug-like compounds and found that those with Gramnegative activity are generally more polar, water- soluble
molecules with lower lipophilicity and increased total polar
surface area and possess a skewed distribution of molecular
weights tending toward but not exceeding 600 Da [139].
Recently, AstraZeneca published some nice examples of
broad-spectrum antibacterial activity optimisation of the
azaindole [122] and pyrrolopyrimidine [36] series of GyrB
inhibitors against Gram-negative pathogens through a combination of structure-based design and physicochemical
property optimisation. For the efficient discovery of broadspectrum antibacterial compounds, particularly against
A high-throughput screening of the AstraZeneca compound collection against M. smegmatis resulted in the discovery of an initial aminopyrazinamide hit compound 56
(Fig. (19)) with moderate enzyme activity (GyrB IC50 value
of 1.7 M) and moderate mycobacterium growth inhibition
(MIC of 16 g/mL). The established structure-activity relationship resulted in compound 57 (Fig. (19)), which exhibited an improved IC50 against GyrB and a potent mycobacterium MIC (GyrB IC50 value of less than 2 nM and MIC
value of 0.5 g/mL). The crystal structure of a 22-kDa fragment of the N-terminal domain of the mycobacterium GyrB
in complex with one of the compounds confirms the key
interactions at sites 1 and 2. The primary amide (site 1) is
O
N
O
N
O
O
N
N
NH
NH2
N
56
Fig. (19). Inhibitors of the mycobacterium GyrB enzyme.
NH
O
F
57
O
NH2
N
O
F
145
OH
O
58, diospyrin
146 Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Gram-negative pathogens, it is important to understand how
different physicochemical properties (e.g., logD and total
polar surface area) and different structural motifs influence
the permeability of each chemical class of compounds. In the
case of the GyrB enzyme, using a structure-based design and
structure-activity relationship studies inspired by the careful
analyses of the SAR of other series of GyrB inhibitors, it is
not difficult to obtain potent GyrB enzyme inhibitors. However, it is very often difficult, particularly in academic settings, to convert these inhibitors into efficient broadspectrum antibacterial compounds.
One of the resistance mechanisms common to all structural classes of antibacterial compounds is reduced cell entry
due to the reduced diffusion into the cell or efflux from it.
Manchester and co-workers recently described the molecular
determinants of AcrB-mediated bacterial efflux, which are
important for the discovery of new antibacterials, particularly against Gram-negative strains [140]. AcrB is a very
promiscuous transporter and is part of the most prevalent
efflux transporter among Gram-negative strains, such as E.
coli, P. aeruginosa and K. pneumoniae. This protein pumps
substrates, such as tetracyclines, aminoglycosides, fluoroquinolones and cephalosporins, from the periplasmic space
to the exterior of the bacterial cell. It has been postulated that
this is the single most significant hurdle to achieving therapeutic levels of antibacterial compounds in Gram-negative
cells [140]. AstraZeneca researchers evaluated compounds
from different discovery programs against Gram-negative
strains and found 3,066 compounds (at least 50 different
scaffolds) that were significantly effluxed via AcrB (an efflux ratio greater than 4, defined as MIC (parent) / MIC (mutant - strain H. influenzae lacking AcrB). The majority of the
compounds with high efflux ratios are distributed around a
mean molecular weight (MW) of 500 Da and a fractional
polar surface area (FPSA) of 25% (or a clogP of 2 for the
compounds in the set). Compounds with low efflux ratios are
significantly smaller, with a mean MW of 400 Da. In addition, nearly all of the compounds with efflux ratios less than
4 have a MW below 600 Da, which is consistent with the
porin size exclusion results. With respect to the polar surface
area, the most polar compounds belong to established classes
of antibiotics with Gram-negative activity that possess functional groups implicated in circumventing the efflux, such as
periplasmic targeting (-lactams), irreversible inhibition (oxaboroles, hydroxamates and -lactams), self-promoted uptake (aminoglycosides and fluoroquinolones), and ion trapping (compounds with weakly acidic functional groups). It
has been found that the targeting of specific ranges of physical properties is not sufficient to guarantee antibacterial activity against Gram-negative strains, and structural motifs
that can circumvent the efflux mechanisms should be incorporated in the design and/or selection of novel compounds
[140].
To understand the physicochemical space occupied by
antituberculosis compounds, Koul and co-workers studied 14
different physicochemical descriptors of first- and secondline antituberculosis drugs (including molecular weight,
lipophilicity and polar surface area) and compared these
properties to those of 1,663 marked non-antibacterial drugs
identified from the Prous Integrity database [126]. These
researchers observed that antituberculosis drugs are widely
Tomai and Mai
distributed within the chemical space of marketed drugs that
actually occupy a broad chemical space and do not fall into
any defined chemical area. For example, fluoroquinolones
are located among the drug bulk with more drug-like properties. In addition, we have to stress that most antituberculosis
drugs were discovered several decades ago without notable
consideration of optimal physicochemical properties. Because the optimal physicochemical space for antimycobacterial compounds is undefined, it has been postulated that the
chemistry for the discovery of new scaffolds should be more
diverse and less restricted [126]. For example, it is worth
mentioning that the antimycobacterial compound TMC207,
which is in Phase II clinical trials and has a logD of 5.14 at
pH 6.0, has potent bactericidal activities. Nevertheless, it is
important to understand how to convert a potent enzyme
inhibitor into a compound that can penetrate the highly impermeable mycobacterial cell wall with its high content of
lipids for the successful discovery of new chemically unique
antimycobacterial compounds [126].
CONCLUSION
Our ProBis structural analysis indicated that the catalytic
active sites of DNA gyrase and topoisomerase IV from E.
coli are structurally similar to those of DNA gyrase and
topoisomerase IV enzymes from different bacterial strains.
These findings serve as a basis for the development of selective or dual inhibitors with broad-spectrum antibacterial activity. In fact, the use of a drug with multi-target or dual activity makes the development of resistance more difficult
because mutations conferring resistance would have to occur
in at least two different target genes during a single generation. Fluoroquinolones, which are the only DNA gyrase and
topoisomerase IV inhibitors that are currently used in clinical
practice (although with significant resistance problems), target both DNA gyrase (GyrA domain) and topoisomerase IV
(ParC domain), usually with unbalanced affinities. We believe that a broad enzymatic spectrum and a balanced dual
target activity would make the development of resistance
more difficult. Mainly guided by ligand-based design via
classical structure-activity relationship studies and optimisation of physicochemical properties, pharmaceutical companies have studied and optimised several other classes of
compounds, such as isothiazoloquinolones, 3-aminoquinazolinediones, piperidines (e.g., viquidacin), 4-aminopiperidines and cyclohexylamines. Some of these broadspectrum antibacterial compounds have advanced to clinical
trials. However, because the crystal structures of representative compounds from the different structural classes in complex with DNA gyrase and topoisomerase IV have been recently resolved, classical SAR studies may now change. In
addition to dual targeting of both DNA gyrase and topoisomerase IV, hybrid compounds targeting DNA or RNA
polymerase together with DNA gyrase or topoisomerase IV
are very effective against several resistant Gram-positive and
Gram-negative strains.
In contrast, due to the increasing resistance to fluoroquinolones, the focus of DNA gyrase research has partly moved
to the discovery of new GyrB and/or ParE inhibitors that are
commonly identified through virtual screening techniques
and structure-based hit optimisation programs. Because the
ATPase domain of GyrB is significantly similar to the hu-
Current Topics in Medicinal Chemistry, 2014, Vol. 14, No. 1
Prospects for Developing New Antibacterials Targeting
man pyruvate dehydrogenase kinase and human topoisomerase II, it is important to address this selectivity challenge in the studies of new GyrB inhibitors. Based on the
available structural information, it is not difficult to design
potent enzyme inhibitors of GyrB and/or ParE. Nevertheless,
to convert these compounds into potent broad-spectrum antibacterials, it is important to understand how different physicochemical properties (e.g., logD and total polar surface
area) and different structural moieties influence the compounds’ permeability for the efficient discovery of potent
small-molecule broad-spectrum antibacterial compounds.
[7]
DNA gyrase, which is the only topoisomerase IIA in M.
tuberculosis, is a validated and pharmaceutically effective
target for the discovery of new antituberculosis drugs that
can overcome the spread of multidrug-resistant tuberculosis
(MDR-TB) and emerging extremely drug-resistant tuberculosis (XDR-TB). The fluoroquinolones moxifloxacin and
gatifloxacin have demonstrated potent activity against M.
tuberculosis and are currently in Phase III clinical trials to
evaluate the multidrug usage and tuberculosis treatmentshortening potential. The increasing resistance to fluoroquinolones has increased interest in the targeting of the GyrB
subunit of the M. tuberculosis DNA gyrase, which exerts the
same phenotypic effects on bacterial viability as fluoroquinolones. The ATPase domain of M. tuberculosis DNA gyrase is thus an attractive but under-explored target for the
development of new classes of compounds as potential antimycobacterial agents.
[12]
CONFLICT OF INTEREST
The author(s) confirm that this article content has no conflicts of interest.
[8]
[9]
[10]
[11]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
ACKNOWLEDGEMENTS
This work was supported by the Slovenian Research
Agency (Grant No. P1-0208), and by European Union FP7
Integrated Project MAREX: Exploring Marine Resources for
Bioactive Compounds: From Discovery to Sustainable Production and Industrial Applications (Project No. FP7-KBBE2009-3-245137).
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