Review

Multitarget ligands in
antibacterial research: progress
and opportunities
1. Introduction
Stephen P East† & Lynn L Silver
2. DNA replication †
Evotec (UK) Ltd., Oxfordshire, UK
3. Cell wall inhibitors
4. Miscellaneous multitarget Introduction: Resistance to current antibacterial therapies is an inevitability
ligands that represents a significant global health concern. Bacteria have the capacity
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13

5. Future opportunities to render all current drug treatments ineffective, which places a demand on
the drug discovery community to constantly develop new antibacterial
6. Expert opinion
agents. Compounds that inhibit multiple biological targets, often referred
to as multitarget ligands, are an inviting prospect in antibacterial research
because, although they will not solve the issue of resistance, they might
help to delay the onset.
Areas covered: This review covers some of the recent progress in identifying
new ligands that deliberately interact with more than one essential biological
target in bacteria. The two principal areas covered are inhibitors of DNA
replication and cell wall biosynthesis.
Expert opinion: Antibacterial programs for the design of multitarget ligands
present an important opportunity for production of antibacterial agents.
For personal use only.

Their longevity, due to slow development of resistance, is comparable to

that seen with other successful agents -- but is much improved over single-
targeted agents for which resistance can appear in vitro overnight. The pre-
clinical development of these agents will have to overcome the standard
problems of antibacterial discovery. Such problems include optimization of
characteristics favoring cell entry and particularly the demonstration of selec-
tivity of inhibition of the desired multiple targets without inhibition of other
bacterial or any mammalian functions.

Keywords: antibacterials, bacterial topoisomerases, cell wall biosynthesis, multitarget inhibitors,

resistance, structure-based drug design

Expert Opin. Drug Discov. (2013) 8(2):143-156

1. Introduction

Research into new antibacterial drugs is of paramount importance because of the

innate ability of bacteria to develop resistance to all existing therapies [1]. Walsh
neatly summarized the scale of the problem in 2000 when he commented
‘development of resistance is not a matter of if but only a matter of when’ and in
doing so he highlighted the continuing need for the identification of new anti-
bacterial agents [2]. One aspect of antibacterial research that is of current interest
is concerned with multitargeted therapies or polypharmacology [3]. This strategy is
an inviting prospect in the search for new antibacterial agents because, although it
would not solve the issue of resistance long term, it is expected to delay the onset
of resistance as the bacteria would have to develop viable mutations on more than
one biological target before the drug or combination of drugs are rendered ineffec-
tive. Spontaneous resistance mutations on single targets generally emerge at frequen-
cies between 10-6 and 10-9 [1] and so a multitarget approach might mean that
resistance develops at significantly lower frequencies than this.

10.1517/17460441.2013.743991 © 2013 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X 143
All rights reserved: reproduction in whole or in part not permitted
 S. P. East & L. L. Silver
Article highlights.
. Established strategies and new opportunities for
multitarget ligands as antibacterials with the potential
to slow the onset of resistance in bacteria.
. Impact of X-ray crystallography for lead identification
through computationally led virtual screens but also
for optimization of novel multitarget ligands using
structure-based drug design techniques.
. New preclinical antibacterials targeting the enzymes
gyrase and topoisomerase IV enzymes that are involved
in DNA replication.
.
Recent early stage advances in the search for novel
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multitarget inhibitors of enzymes in the cell wall
biosynthetic pathway.
. Optimization of compound physicochemical properties
as well as potency on multiple enzyme targets are
necessary to achieve good antibacterial activity.
This box summarizes key points contained in the article.
Multitargeted chemotherapy [4,5] can be broadly divided
into two categories: i) combination therapies, that is, two or
more compounds that when taken together, either as a cock-
tail or a multicomponent drug, work synergistically but typi-
cally elicit their pharmacological response by acting on
For personal use only.
independent biological targets [6] and ii) single compound
therapies, also termed multiple ligands [4,7] or multitarget
ligands [8], that is, one compound that modulates the pharma-
cological response of two or more relevant biological targets.
Multitarget ligands can be a product of two or more discrete
molecules which, when separate, interact independently with
quite different biological targets, but they have been joined
(via a linker) or fused together to form a single compound.
Indeed this approach has been used successfully in the search
for new antibacterial agents and reviewed recently [9]. Alterna-
tively, multitarget ligands can contain specific structural fea-
tures that are important in the formation of key binding
interactions with not just one but multiple biological targets
of interest. Morphy and Rankovic [4] have referred to these
types of ligands as being ‘merged’. In the scope of antibacterial
research such multitarget ligands have been identified, most
typically in cases where the biological targets are structurally
similar at the ligand-binding site and/or they have closely
related substrates and it is these ligands that are the focus of
this review. A possible advantage of these merged ligands
over the joined or fused ligands is that the resulting drugs
might be expected to have a lower molecular weight and con-
sequently might occupy more attractive chemical space in
terms of their physicochemical and DMPK properties [10].
So the questions are: How have we identified multitarget
ligands in antibacterial research programs in the past and
how might we do it better in the future? For the former
question, the leading classes of multitarget antibacterials are
the b-lactams that inhibit penicillin-binding proteins (PBPs)
(e.g., carbapenems [11]) and the quinolones that inhibit
DNA topoisomerases [12], but the discovery that these drugs
144 Expert Opin. Drug Discov. (2013) 8(2)
inhibit multiple biological targets has largely been determined
retrospectively. Since the elucidation of the bacterial genome,
advances in screening and improvements in protein structure
determination, the rational design of multitarget inhibitors
of antibacterial enzymes have been possible. Screening techni-
ques have identified new natural products (historically a fertile
hunting ground for antibiotics) that are multitarget ligands.
The emergence of structural information through X-ray
crystallography and NMR has facilitated structure-based
design opportunities for high-throughput screening hits as
well as providing the necessary tools to conduct computation-
ally led virtual screens. This review will discuss some of the
ongoing research programs aimed at identifying multitarget
ligands of essential bacterial enzymes. The focus is on chemi-
cal matter reported in recent publications and presentations
(2010 -- 2012).
2. DNA replication
Enzymes that function as part of the bacterial DNA synthesis
machinery play an essential role in bacterial cell survival [13].
Two enzymes that contribute to DNA replication are the
bacterial type II topoisomerases: DNA gyrase and topoisom-
erase (topo) IV. These enzymes are responsible for controlling
DNA topology at various stages of DNA replication. Both
enzymes form heterotetrameric complexes, which in the case
of DNA gyrase is an A2B2 complex and for topo IV is a
complex consisting of two ParC and two ParE subunits. As
a consequence of their similar structural homology, dual
inhibition of gyrase and topo IV has been enabled, albeit
serendipitously [14], and members of the quinolone (including
fluoroquinolones) class of antibiotics have validated this
approach clinically for antibacterial infections [12]. The quino-
lones inhibit the related GyrA and ParC subunits. The GyrB
and ParE subunits bind and process ATP and so the similarity
in the ATP-binding site of these proteins provides an addi-
tional opportunity for inhibition of two targets. Indeed dual
inhibitors of GyrB/ParE have been identified but currently
there are no approved GyrB/ParE drugs.
2.1 GyrA/ParC inhibitors
Despite a plethora of quinolone antibacterials in the market-
place, emerging resistance and issues with toxicity have meant
that the search for alternative GyrA/ParC inhibitors continues
unabated. Several compounds are progressing in the latter
stages of clinical development and these have been covered
elsewhere [15] but there are others that are at a preclinical stage
or that have recently entered Phase I, and some of these
developments will be covered here.
Pucci et al. [16] have detailed the in vitro and in vivo profile
of Achillion’s advanced isothiazoloquinolone ACH-702
(Figure 1). This compound is active in Staphylococcus aureus
topo IV decatenation assays (IC50 120 nM) and DNA gyrase
supercoiling (IC50 680 nM) assays and shows superior
dual-targeting inhibition compared with benchmark
 Multitarget ligands in antibacterial research: progress and opportunities
O O
F F
NH
N N S N
H2 N
H2N OCH3
ACH-702
OH
N O
S S F N
OH
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
Viquidacin
Figure 1. Quinolone-related dual GyrA/ParC inhibitors and other prototypical topoisomerase inhibitors.
fluoroquinolones moxifloxacin, gemifloxacin and ciprofloxa-
cin. It also retains some activity in vitro against single- and
double-mutated gyrases that show resistance to the same set
of control compounds. Broad-spectrum antibacterial activity
against clinical isolates was shown with ACH-702 having a
better profile than levofloxacin on Gram-positives including
activity on fluoroquinolone nonsusceptible organisms. On
For personal use only.
Gram-negative organisms, ACH-702 has a similar profile to
ciprofloxacin. Time-kill analysis revealed that ACH-702 is
bactericidal and resistant mutations were observed at frequen-
cies of < 10-10 at 4  MIC in strains of S. aureus and this
represents a ~ 2 log units improvement over ciprofloxacin.
Positive data on ACH-702 in two Gram-positive models of
infection have been reported. A dose-dependent effect in a
methicillin-resistance S. aureus (MRSA) mouse thigh infec-
tion model was observed, with 5 mg/kg leading to stasis in
infection after 24 h, and a 2.5-log reduction in colony
forming units (CFUs) was observed after the same timeframe
following a dose of 20 mg/kg. On the Achillion website [17],
ACH-702 is also reported to be active on DNA primase,
which is another enzyme involved in bacterial DNA replica-
tion. Although no data demonstrating activity of ACH-702
on this target are available, DNA primase and the topoisomer-
ases have been shown to share a homologous catalytic site, the
TOPRIM domain, which may underlie activity against all
three proteins [18]. Detailed structure--activity relationship
studies that led to the identification of ACH-702 have been
described by Kim et al. [19].
Evolva are progressing broad-spectrum bactericidal
2-pyridones (termed the EV-035 series) [20,21] that are isosteric
to the traditional quinolones and related in structure to
ABT-719 (Figure 1), a compound that reached Phase II
clinical trials [22]. In silico modeling has suggested that the
substitution pattern on the EV-035 series is crucial for
compounds to adopt an overlapping but different binding
mode to the quinolones. This subtle change in the ligands
interaction with DNA gyrase/topo IV is probably responsible
for the 2-pyridones showing good antibacterial activity against
Expert Opin. Drug Discov. (2013) 8(2)
O O O O
N OH N OH
N
H2N
ABT-719 GC-061
OCH3
O
N OCH3
H
S N
N
N
NC
GSK299423
fluoroquinolone-resistant organisms. For example, a represen-
tative compound in this series, GC-061 (Figure 1), is active in
S. aureus (MIC range < 0.015 -- 0.5 µg/ml), which includes
strains that are ciprofloxacin- and levofloxacin-resistant (i.e.,
MIC ‡ 16 µg/ml). In Gram-negative organisms, GC-061
has excellent activity against Haemophilus influenzae
(MICs < 0.015 µg/ml) but also shows some activity against
strains of Escherichia coli (median MIC 16 µg/ml), Pseudomo-
nas aeruginosa (median MIC 8 µg/ml) and other organisms.
Confirmation of dual-targeting with GC-061 was demon-
strated in gyrase and topo IV in vitro assays with
IC50s £ 1.42 and £ 2.33 µM reported. Spontaneous resis-
tance frequencies for compounds in this series are reported
to be low (i.e., ~ 10-11 in E. coli and S. aureus at 4  MIC)
which is suggestive but not conclusive of dual inhibition in
a cellular context. In vivo efficacy of GC-061 was achieved in
a mouse thigh MRSA infection model when dosed orally at
30 mg/kg with ~ 5-log reduction in bacterial tissue burden
being observed after 24 h. The same compound showed
a ~ 3-log units reduction in CFUs over the same time period
in a mouse E. coli thigh infection model. These data are at
least as good as ciprofloxacin (20 mg/kg i.v. dose) in the
same models. GC-061 was selective over human topoisomer-
ases (IC50 Topo II > 35 µM; IC50 Topo I > 100 µM), and
there are no apparent in vitro toxicity issues (e.g., hERG
IC50 60 µM, inactive at the highest concentration tested in a
HepG2 cell line, a micronucleus genotoxicity assay and
cytochrome p450 assays).
In addition to the quinolone and related classes of antibac-
terial agents, other novel bacterial topoisomerase II inhibitors
that target the GyrA/ParC subunits have been described in the
literature. A prototype compound was viquidacin (NXL101,
Figure 1) [23], and this antibacterial agent advanced into
Phase I studies before issues associated with QT prolongation
halted its progression in the clinic. Several research programs,
including GSK [24,25], AstraZeneca [26-28] and Achillion [29],
have all reported on chemotypes related to viquidacin with a
focus on addressing the cardiovascular toxicity side effects by
145
 S. P. East & L. L. Silver

OCH3
tuning logD and pKa and monitoring hERG inhibition

N
in vitro. These chemotypes are of significant interest because

S. aureus (n = 7) 0.5 -- 2 µg/ml

OH X-ray crystal studies on GSK299423 (Figure 1) [30,31] using
S. aureus gyrase revealed that it binds to a site on GyrA and

Topo IV (S. aureus) 69 µM

Gyrase (S. aureus) 0.7 µM
O

OH
bridges to the bound DNA substrate such that it intercalates
between base-pairs, thus preventing DNA cleavage. This
represents an alternative binding mode to the quinolones [32]
N

and supports the potential usefulness in terms of their activity

on fluoroquinolone-resistant organisms. Some representative
Achillion
S

examples and data on recent inhibitors are shown in Table 1.

Although IC50 data on both DNA gyrase and topoisomerase
Se

[29]
are not provided in all of the publications, the implication is
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that these compounds are dual-targeting agents. For example,

Reck et al. [28] commented that there was a good correlation
between IC50s on E. coli topo IV activity and S. aureus
gyrase. Of the compounds shown in Table 1, the AstraZeneca
model at 40 mg/kg; 1 log unit drop in CFUs

compound is perhaps the furthest advanced as it was

Efficacy in mouse S. aureus thigh infection

reported to have moved into clinical trials. It should be noted

that IC50 data on both targets is not sufficient to prove
S. aureus (n = 5) 0.06 -- 0.125 µg/ml
CN

dual-targeting at the whole cell level and resistance studies

S. pneumoniae (n = 1) 0.13 µg/ml

P. aeruginosa (n = 1) > 8 µg/ml

(or other means) are necessary. Resistance studies supporting

N

dual-targeting, however, have been carried out in many of

O

Topo IV (E. coli) 0.048 µM

the referenced cases.

E. coli (n = 1) 4 µg/ml
N
F
For personal use only.

2.2 GyrB/ParE inhibitors

H
N

Natural product inhibitors of the ATPase activity of DNA

gyrase and topo IV, such as the aminocoumarin compound
N

novobiocin, have been well characterized biochemically, struc-

turally and pharmacologically; however, their clinical utility
O

[28]
O

AZ

stalled, owing to resistance and toxicity [33]. The cyclothiali-

dine class of natural product antibacterials have also been
widely studied as a result of their inhibition of bacterial
type II topoisomerases and synthetic analogs demonstrating
in vivo efficacy based on this chemical series have been
reported recently [34]. For example, compound 1 (Figure 2)
OCH3

was reported to have an ED50 of 3 mg/kg in a mouse S. aureus

N

Efficacy in rat S. pneumoniae lung

S. pneumoniae (n = 1) 0.06 µg/ml
Table 1. Recent bacterial topoisomerase II inhibitors.

septicemia model.
N

Gyrase (H. influenzae) 0.17 µM

H. influenzae (n = 1) 0.5 µg/ml

Phillips et al. [35] reported a new naturally occurring ATPase

OH

infection model at 50 mg/kg

S. aureus (n = 1) 0.03 µg/ml
F

inhibitor, kibdelomycin (Figure 2), which was isolated

[DNA replication assay]

from Kibdelosporangium sp. and shown to have activity on

N

Gram-positive pathogens (e.g., S. aureus MIC 0.5 -- 64 µg/ml;

Streptococcus pneumoniae 1 µg/ml) as well as H. influenzae
H
N

(MIC 2 µg/ml). The discovery of kibdelomycin followed an

N

examination of S. aureus fitness test profiles and comparisons

with known antibiotic profiles, where it exhibited a similar
GSK
O

[25]

mechanism of action to novobiocin. Macromolecular biosyn-

thesis assays confirmed that kibdelomycin inhibited DNA
replication by blocking the incorporation of 14C-thymidine;
however, at the MIC, synthesis of other macromolecules was
affected as well. Subsequently, dual inhibition of DNA gyrase
n: Number of isolates.

and topo IV was confirmed in catalytic ATPase in vitro

Organization

assays in E. coli (Gyr IC50 11 nM; Topo IV IC50 900 nM),

Comment
Structure

as well as gyrase supercoiling (IC50 9 nM) and topoisomerase

decatenation (IC50 500 nM) assays in S. aureus. Resistance
MIC
IC50

Ref.

frequencies were shown to be < 5  10-10 CFU/ml and little

146 Expert Opin. Drug Discov. (2013) 8(2)

 Multitarget ligands in antibacterial research: progress and opportunities

O N Interest in non-natural product inhibitors of the ATPase

N
activity of DNA gyrase and topo IV has been stimulated by
OH S
the availability of structural information on these targets.
HN O
Following pioneering work by Boehm et al. [39] on
O
HN
fragment-based screening, Charifson et al. were responsible
H3CO
Br O
for demonstrating the utility of synthetic GyrB/ParE inhibi-
O tors in vivo with a series of benzimidazole ethyl ureas of which
1 compound 3 in Figure 3 has been characterized extensively [40].
O
These inhibitors have been shown to be antibacterial solely
Cl
O OCH3 via their inhibition of GyrB and topo IV ParE [41]. Based on
Cl published crystallographic data [42], the crucial pharmaco-
O
O N OH NH phoric features for binding of this scaffold to ParE are the
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H
H2N O N donor NH groups on the ethyl ureas that interact with an
O O O O
Asp residue in the ATP pocket together with the N-1 of the
HO H H H OH
O benzimidazole that acts as an acceptor donor to a conserved
water molecule that also hydrogen bonds to the same Asp
Kibdelomycin residue. In a very recent patent disclosure [43], Vertex have
reported the latest generation dual-targeting benzimidazoles
OH with compound 4a (Figure 3) showing good enzymatic inhibi-
HO O
tion (S. aureus Gyr Ki 9 nM; S. aureus Topo IV Ki 12 nM)
OH and antibacterial activity particularly against Gram-positive
O
F strains (S. aureus MIC90 0.03 µg/ml (n = 67); S. pneumoniae
F OH O O
MIC90 0.015 µg/ml (n = 67); Enterococcus faecalis MIC90
O 0.06 µg/ml (n = 34)). The compound 4a is shown to be
For personal use only.

O
OH
O active in a mouse S. aureus kidney infection model and a
O OH
2 bes the prodrug 4b which was presumably investigated due to
Figure 2. Naturally occurring/derived dual DNA gyrase and
topo IV inhibitors.
or no cross-resistance with aminocoumarin-resistant bacterial
strains were observed. The lack of kibdelomycin-resistant
mutants found could be due to inhibition of other essential
functions above the MIC.
Hossion et al. described quercetin diacylglycosides as dual
DNA gyrase/topo IV inhibitors [36,37]. One of the most inter-
esting analogs, compound 2 (Figure 2), was reported to be active
in an E. coli gyrase supercoiling assay (IC50 0.78 µM) and
E. coli topo IV (IC50 9.2 µM) and S. aureus topo IV
(IC50 0.22 µM) decantenation assays. The compound is active
on Gram-positive organisms (MICs 0.13 -- 0.5 µg/ml on
S. pneumoniae, methicillin-resistant S. aureus and vancomycin-
resistant enterococci) but no activity on Gram-negative species
was observed (MICs > 128 µg/ml on P. aeruginosa and E. coli).
Molecular docking studies postulated that the compounds
bind to the ATP-binding site on GyrB, although no data in
ATPase assays were reported to confirm that this was the
site of action of this inhibitor class. It should be noted that
quercetin is a flavonoid that has been shown to inhibit a great
number of enzymes, eukaryotic, viral as well as bacterial [38]
and it is unlikely that gyrase/topoisomerase inhibition is the
sole or even major mechanism of action of quercetin in
Gram-positives.
S. aureus neutropenic rat thigh model. The patent also descri-
the low solubility of 4a and indeed 4b might be the preferred
delivery method for active ingredient 4a.
Recognizing the key inhibitor design features of the benz-
imidazole ethyl ureas, several other groups have published
research in which the benzimidazole scaffold has been inter-
changed with a number of other bicyclic and biaryl systems
that have the potential to retain the critical H-bond acceptor
as well as the ethyl urea moiety [44]. The benzothiazoles pre-
sented by Biota are the most recent refinement of the ethyl
urea series [45]. Fine tuning of the substituents in the 5- and
7-position of the benzothiazole scaffold provided compounds
such as example 5 in Figure 3 that has excellent activity on
Gram-positive pathogens (S. aureus MIC 0.06 µg/ml;
Streptococcus pyogenes MIC 0.06 µg/ml; E. faecalis MIC
0.02 µg/ml) and a good pharmacokinetic profile with
> 65% oral bioavailability being achieved in rodents. Some
Gram-negative activity has been observed (H. influenzae
MIC 0.5 -- 2 µg/ml); Moraxella catarrhalis (MIC
< 0.125 µg/ml) suggesting that the compound class could be
suitable for the treatment of respiratory tract infections. The
structures of the most advanced compounds have not been
revealed but spontaneous resistance frequencies for lead com-
pounds in this class were reported as being < 10-9 in S. aureus
and S. pneumoniae, which is consistent with a dual-
targeting mechanism of action. In vivo efficacy via oral
administration with a compound from this series was demon-
strated in models of S. aureus including a neutropenic thigh
model. In a S. pneumoniae mouse model at a 30 mg/kg dose

Expert Opin. Drug Discov. (2013) 8(2) 147

 S. P. East & L. L. Silver

RO
N N

N H N N H
N H N H
N N F N N
H O H
F O
N O

3 Vertex 4a R = H
4b R = P(=O)(ONa)2
HO2C

N N OCH3 N H
N N H
N N N
H
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N H
S N N O
O N N
O
N HN
CF3
O
5 Biota 6 AstraZeneca

O–
N+
S
N H
N N
N

N
Cl
H
For personal use only.

H2N
7 Trius

Figure 3. Dual GyrB/ParE inhibitors.

(intravenous), 100% survival was noted in 9 days after inocu-
lation. In vitro toxicology assessment was good with no appar-
ent issues identified and compounds were reported to be
progressing through in vivo safety experiments.
Scaffolds that do not contain an ethyl urea group but have
been identified using structural information have also been
described recently. Manchester et al. [46] assembled a library
of fragments previously observed in ATP-competitive kinase
inhibitor programs at AstraZeneca. From a virtual screening
exercise using a S. pneumoniae ParE structure, they identified
an azaindole scaffold as satisfying the key molecular interac-
tions with the Asp and conserved water residue in ParE. This
scaffold was further elaborated to achieve low nanomolar
potency on both S. pneumoniae ParE and S. aureus GyrB
with example 6 in Figure 3 being one of the most potent ana-
logs described (ParE IC50 3 nM; GyrB IC50 2 nM; S. aureus
MIC 0.1 -- 0.2 µg/ml; S. pneumoniae MIC < 0.02 µg/ml).
Antibacterial activity did not correlate at all with enzymatic
activity but some relationship between logD and antibacte-
rial activity on Gram-positive organisms was observed.
The hypothesis presented was that compounds with
higher logD show improved permeability properties (e.g.,
compound 6, logD = 2.91) to traverse the bacterial cell
membrane. No activity for the azaindole compounds on
Gram-negative organisms was provided. Indeed Manchester
et al. have reported in a separate publication [47] that the
AstraZeneca experience of optimizing Gram-negative
antibacterial activity in new compounds classes is difficult.
Efficient efflux systems together with more complex
permeability barriers in Gram-negatives compared with
Gram-positives might be responsible for the apparent lack
of data for the azaindoles.
Trius have utilized a combination of structure-based virtual
screens and literature-led identification of new GyrB/ParE
dual inhibitor scaffolds. A key design feature in the Trius
program was to rationally design inhibitors of Gram-negative
pathogens as well as Gram-positive organisms. This was
achieved by building in some degree of flexibility to the
compounds to compensate for the differences in the Gram-
positive and Gram-negative protein structures, as well as opti-
mizing the compounds for Gram-negative entry and lack of
efflux. One of the advanced Trius compounds, 7 (Figure 3), is
from a series of pyrrolopyrimidines. This compound has
sub-nanomolar binding affinity on E. faecalis (Ki < 0.3 nM)
and Francisella tularensis GyrB (Ki < 0.3 nM) and single
digit nanomolar activity on ParE (E. faecalis Ki 8.6 nM;
F. tularensis Ki 1.7 nM; E. coli Ki 2.5 nM) [48]. The in vitro
antibacterial activity for compound 6 is good against

148 Expert Opin. Drug Discov. (2013) 8(2)

 Multitarget ligands in antibacterial research: progress and opportunities

Gram-positive (S. aureus MIC 0.03 µg/ml; S. pneumoniae

MurC (E. coli) 26%@500 µM

MurD (E. coli) 50%@500 µM
NH
MIC 0.25 µg/ml) and Gram-negative pathogens (H. influenzae

O
MIC 2 µg/ml; E. coli 2 µg/ml; P. aeruginosa 4 µg/ml), and it

Cl
also has good coverage of biodefense pathogens. The com-
S

N
pound was reported to show activity in a S. aureus septicemia

NH
N

N mouse model with a 100% survival at 12.5 mg/kg dose. Interes-

tingly, by performing macromolecular synthesis experiments,
H
N

Trius observed that structurally related compounds in their

[60]
11

nd
pyrrolopyrimidine series did not necessarily show all the same
specific effect on DNA synthesis in bacterial cells. This
experiment illustrates (as noted in several places above) that
antibacterial activity is not always a consequence of targeted
MurC (E. coli) 52%@100 µM
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inhibition in the cell and nonspecific effects might be at play.

MurE (S. aureus) 6 µM
NH

MurD (E. coli) 2 µM

Cell wall inhibitors

S

3.
MurF (E. coli) 2 µM
O

3.1 Mur ligases

No activity

Peptidoglycan is a crucial ingredient in bacterial cell walls of

OH

both Gram-positive and Gram-negative organisms. It

provides shape, integrity and the necessary protective barrier
[56]
HO

HO

10

to restrict permeability into and out of the bacterial cell.

Numerous antibacterial drugs, both naturally occurring and
synthetically derived, have been found to inhibit specific steps
NH

of peptidoglycan biosynthesis. For example, b-lactam antibi-

O
For personal use only.

otics inhibit transpeptidase enzymes that are responsible for

S

late-stage cross-linking of peptidoglycan. This class of drugs

thus compromises the rigidity of the cell wall leading to cell
lysis. Indeed, it was the identification of penicillin that
S. aureus (n = 2) 8 µg/ml
MurD (S. aureus) 6.4 µM

MurE (S. aureus) 17 µM

provided the basis for the current level of understanding of

MurD (E. coli) 8.2 µM

MurE (E. coli) 180 M

H
N

peptidoglycan assembly, a process that involves more than

E. faecalis 64 µg/ml

10 sequential enzymatic steps that are unique to bacteria.

Because peptidoglycan is essential for bacterial cell survival,
NH

its synthesis presents numerous opportunities for target-

CO2H

based inhibition and hence therapeutic solutions to bacterial

HO2C

infections [49].
[55]
9

The first six steps of peptidoglycan biosynthesis are

catalyzed by the enzymes MurA-F and result in the formation
of UDP-N-acetylmuramoyl (UDP-MurNAc)-pentapeptide.
Table 2. Multitargeting inhibitors of Mur ligases.

With the development of robust in vitro assays and high-

O

throughput screening, inhibitors of these enzymes have been

identified; however, with the exception of fosfomycin that
O
N

inhibits MurA, so far there are no other Mur inhibitors that

S

have advanced to the marketplace [50,51]. In terms of multitar-

S

MurE (S. aureus) 32 µM

MurD (E. coli) 270 µM

get therapies, the ligases MurC-F have been considered an

nd: No data available; n: Number of isolates.
O

attractive opportunity because they all catalyze the formation

CO2H

of a peptide bond and do so by binding ATP and the

UDP-MurNAc substrate. The emergence of structural
information on MurC-F suggested that the opportunity for
inhibitors that target all four enzymes might be limited
HO2C

[53]
nd

because, although the ATP-binding sites were well conserved,

8

it was revealed that UDP-MurNAc binds differently in

MurC/D compared with MurE/F [52] and so dual-targeting
Structure

rather than quad-targeting might be a more realistic outcome.

Cmpd #

Nevertheless, there have been some recent developments in

MIC
IC50

Ref.

the search for multitarget Mur ligase inhibitors and these

Expert Opin. Drug Discov. (2013) 8(2) 149

 S. P. East & L. L. Silver
CO2H Br
NH
O S
O O
O
12
O NO2 O
OH
N B
H H
OH
F
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
15
Figure 4. Non-covalent PBP inhibitors.
have utilized the available X-ray structural data to perform
pharmacophore-based virtual screens.
Several publications reported on the use of MurD structures
for the virtual screen experiments. Perdih et al. [53] identified
benzene dicarboxylic acids that were postulated to act as gluta-
mic acid mimics in the catalytic site. The most interesting com-
pound reported was compound 8 (Table 2), which shows
For personal use only.
in vitro activity on both MurD and MurE. Tomašić et al. [54]
identified glutamic acid containing MurD inhibitors and these
compounds have subsequently evolved into dual MurD/E
inhibitors [55] with compound 9 (Table 2), which bears some
structural resemblance to compound 8, showing inhibition of
ligase activity on E. coli and S. aureus MurD and MurE. The
antibacterial activity of compound 9 on S. aureus (including
an MRSA strain) and E. faecalis was modest and no activity
was seen on E. coli or P. aeruginosa up to 128 µg/ml.
A common feature of compounds 8 and 9 is the rhodanine
scaffold and Tomašić et al. [56] have also published on a set of
5-benzylidenethiazolidin-4-ones with the trihydroxy deriva-
tive, compound 10 (Table 2) showing activity on all four Mur
ligases although the compound does not display antibacterial
activity on Gram-positive or Gram-negative strains up to
128 µg/ml. Rhodanines and other five-membered multihetero-
cyclic scaffolds have been the subject of much debate in terms
of whether they are privileged structural elements or simply
promiscuous hitters [57-59]. In the case of the rhodanine
compound 9, the published ligand-protein X-ray structure [55]
is compelling evidence for a specific interaction on MurD.
In what seems to be a complementary virtual screen to
those described previously, Tomašić et al. [60] scrutinized the
X-rays structures of available E. coli and H. influenzae
MurC-F enzymes focusing on the residues in the ATP-
binding sites. They concluded that the binding sites were
similar and subsequently performed a virtual screen using
E. coli MurD. A selection of ligands was tested and weak
dual inhibitors of MurC and MurD were identified with
compound 11 (Table 2) being the best compound of this
initial set. No antibacterial activity was reported, although,
150 Expert Opin. Drug Discov. (2013) 8(2)
CO2H
O
NH
O OH
N
H
13 14
OH
N B
OH
16
based on the level of potency observed in the biochemical
assay, the compounds would not be expected to have a specific
effect on bacterial cells.
3.2 Penicillin-binding proteins
The b-lactam antibiotics were the first, and they are arguably
the most widely, studied of all of the clinically prescribed
antibacterial agents. They can also be considered as the arche-
typal multitarget inhibitors because they covalently modify
members of a family of enzymes known as the PBPs. PBPs
have a variety of functions in the latter stages of bacterial
cell wall biosynthesis including transpeptidase activity (as
mentioned above). Although b-lactam antibiotics have been
very successful, one of the resistance mechanisms that have
emerged to this class of compounds is as a consequence of their
inherent reactivity. Bacteria express a family of enzymes termed
b-lactamases that inactivate b-lactams via a similar mechanism
to the way that b-lactams inhibit PBPs. An established
method to overcome this path of resistance is to co-administer
a b-lactam antibiotic with a b-lactamase inhibitor; however,
this combination approach can complicate the clinical develop-
ment. Recently, there has been much interest in developing
reversible PBP inhibitors that do not contain the b-lactam func-
tionality and hence may not be as susceptible to b-lactamase
inactivation (although they might still bind to b-lactamases as
well as PBPs).
Turk et al. [61] have reported some biaryl sulfonamides and
biaryl amides as noncovalent binders to PBPs. Compounds
such as those shown in compounds 12 and 13 (Figure 4) were
identified from a limited compound screen and show weak
inhibition (IC50s ~ 100 -- 400 µM) of PBP2a (S. aureus),
PBP2x (S. pneumoniae) and PBP5fm (E. faecium). Some
antibacterial activity against Gram-positive organisms was
also reported (MICs 1 -- 256 µg/ml), although it was not
determined whether the cellular inhibition observed was a
consequence of PBP inhibition. The compounds were inactive
(MICs > 512 µg/ml) on Gram-negative organisms. Shilabin
et al. [62] have reported a series of 4-quinolones as pan-PBP
 Multitarget ligands in antibacterial research: progress and opportunities
Table 3. Miscellaneous multitargeting antibacterials.
Pathway Example compound
Fatty acid biosynthesis OH
O O
HO2C N
H H
OH
DNA replication H3CO
O
OCH3
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
N N N N
H H
OCH3
Folate biosynthesis OH
N NO2
H2N N NH
inhibitors. Compound 14 (Figure 4) has Kis of 5 -- 220 µM
against PBP 1a/1b, PBP2, PBP3, PBP4 and PBP5/6 from
E. coli. No antibacterial activity up to 1 mM on a strain of
E. coli was observed.
For personal use only.
Boronic acids have been investigated as PBP inhibitors.
Here the concept is that the boronic acid forms a tetrahedral
intermediate with a critical serine residue in the active site
of the PBPs. This reaction is similar to what happens with
b-lactam antibiotics and PBPs (and indeed b-lactamases)
but, in the case of the boronic acid inhibitors, the reaction is
reversible because the boron-oxygen bond that is formed can
be readily hydrolyzed. Structural information on how boronic
acids bind to PBPs is beginning to emerge and this should
facilitate improved reversible inhibitors in the future.
A series of N-alkyl boronates were reported by Woon
et al. [63] and Contrerase-Martel et al. [64]. Compound 15
(Figure 4) is one of the most interesting compounds in
this series and it was active on PBP1b (IC50 20 µM) and
also shown to have activity on PBP2, PBP2a, PBP3,
PBP4 together with sub-micromolar inhibition of the PBP
R39 (IC50 0.27 µM). Antibacterial activity on S. aureus
(MIC 32 µg/ml including MRSA strains) and other Gram-
positive organisms was demonstrated and X-ray structural
studies suggested that compounds of this type can adopt
two different binding modes. In a more recent paper,
Zervosen et al. [65] have described related glycine boronic
acids, for example, compound 16 (Figure 4) with activity on
S. pneumoniae PBP1b (IC50 26 µM), S. pneumoniae PBP2x
(IC50 138 µM) and Actinomadura R39 (IC50 0.6 µM).
Curiously, X-ray data on compound 16 crystallized with
R39 revealed that the tetrahedral intermediate forms by inter-
actions with two serine residues and a lysine residue in the
active site. This has been described as a tricovalent interaction;
however, the ligand-protein complex of compound 16 with
PBP1b revealed that only a monocovalent interaction is
Expert Opin. Drug Discov. (2013) 8(2)
Molecular targets Ref.
FabF and FabH [69]
DNA polymerases III: PolC and PolE [70]
OCH3
Dihydropteroate synthase (DHPS) [71]
and dihydrofolate reductase (DHFR)
observed. Despite these differences in the structural informa-
tion it is reasonable to assume that this compound is also a
reversible inhibitor.
3.3 Lipid A biosynthesis
Gram-negative bacteria have a layer of lipopolysaccharide
(LPS) on their outer membrane that functions as a protective
coating to the cell. A constitutive ingredient of LPS is
Lipid A which acts as the anchor for LPS to the cell mem-
brane but it also functions as an endotoxin, mobilizing the
innate immune system in humans and leading to sepsis.
Lipid A is crucial for the survival of Gram-negative bacteria
so an inhibitor of the one or more steps in the synthesis of
Lipid A could provide a new therapy. The first three steps
in Lipid A biosynthesis are catalyzed by the enzymes LpxA,
LpxC and LpxD. LpxC has garnered much attention from
the drug discovery community [66] with ACHN-975 recently
being the first LpxC inhibitor to enter human trials [67].
Jenkins and Dotson [68] have suggested that LpxA and
LpxD might offer an opportunity for a dual-targeted
therapy. These two enzymes are both acyltransferases
and have been shown to be inhibited by the dodecapeptide
RJPXD33 with Kd values measured as 22 µM (E. coli
LpxA) and 6.5 µM (E. coli LpxD), respectively. Clearly the
development of a long peptide chain as a novel antibacterial
is likely to be challenging; however, improving the under-
standing of the minimum pharmacophoric features of the
peptide required for binding might present possibilities for
small molecule inhibition.
4. Miscellaneous multitarget ligands
Other multitarget ligands have been reported in the literature
and some of the salient examples are included in Table 3
[69-71]. As there is little new relevant information that
151
 S. P. East & L. L. Silver
merits inclusion in this review, the readers are directed to
other articles that contain the key references and a more
comprehensive summary of the work shown in Table 3 [1,3].
5. Future opportunities
Multitarget ligands need to modulate the pharmacological
response of multiple enzymes. Looking at this challenge
simplistically, an ideal ligand would inhibit the enzymes of
interest to a similar degree (perhaps within an order of magni-
tude [4]), as determined by in vitro biochemical experiments.
In reality, the situation is much more complex and there
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
will be many factors that ultimately determine what the pri-
mary target is in vivo and these are not easy to predict. For
example, ligand structure and enzyme-binding site structure
have both been reported to influence the primary target of
dual GyrB/ParE inhibitors [41] in different bacterial strains.
What is clear though is that true, multitarget ligands can
reduce in vitro spontaneous resistance frequencies (selected
at high levels of compound) to frequencies below those for
single mutations; sequential mutations in more than one
essential target would be necessary in order for the bacteria
to develop high-level resistance to a multitargeted drug. If
the inhibitor is well balanced, then the likelihood of single
For personal use only.
step resistance is decreased.
To search for new multitarget inhibitors in the future, the
most obvious opportunities are to identify enzymes that
have either identical substrates (e.g., ATP in the case of
GyrB and ParE) or structurally related substrates (e.g.,
UDP-MurNAc in the case of Mur C-F enzymes) where the
homology of the binding site is expected to be high. Protein
sequence homology is usually a reliable guide for similar pro-
teins, but similar binding sites can be formed by the same spa-
tial arrangement of similar residues in otherwise dissimilar
sequences. Once the proteins of interest have been identified,
then X-ray crystallography data on these related targets can
then potentially be collected to be sure that the three-
dimensional substrate binding sites of different enzymes are
truly similar. As soon as this is confirmed, then screening
methods in the form of HTS or virtual screens can be imple-
mented to identify new multitarget ligands. Some of the
examples presented in this review illustrates that structure-
led approaches like this have been successful in the recent
past and it seems reasonable that there are opportunities to
exploit this further in the future. For example there are other
bacterial ATPases that could be explored to identify new
classes inhibitors as suggested by Škedelj et al. [72].
Where X-ray crystal structures are not available, the chal-
lenge to identify new multitargeting opportunities requires
a more indirect approach. One strategy is to construct homol-
ogy models based on enzymes with related sequence and
substrate. An alternative strategy to the protein-centric
approaches described is to focus on known ligand similarity.
In silico tools have been utilized by Keiser et al. [73] and
Durrant et al. [74] where secondary biological targets to known
152 Expert Opin. Drug Discov. (2013) 8(2)
ligands have been identified for biological targets unrelated in
protein sequence or structure. The idea here is to find addi-
tional biological targets for ligands already known to inhibit
one enzyme of interest.
6. Expert opinion
Multitarget ligands represent an approach to discovery of
novel antibacterials that should yield drugs having a low
probability of selecting rapidly for high-level target-based
resistance. While all antibacterials will eventually fall to resis-
tance mechanisms, it is highly likely that antibacterial com-
pounds targeting single enzymes will immediately select for
pre-existing resistance mutations that arise at a measurable
rate per generation. Indeed, a standard method of identifying
the mechanism of action of a novel compound is to select for
such target-site mutations. Whether the rate of resistance seen
in the laboratory will translate into therapeutic failure
depends upon (among other things) that rate, the bacterial
burden during the infection, the fitness and virulence of the
resistant mutants and the possibility of reaching high enough
levels of compound at the site of infection to inhibit the
resistant organisms. While there are single-enzyme targeted
agents in clinical use, they are usually dosed as combinations
(as in therapy of Mycobacterium tuberculosis) or used topically
or in indications where high concentrations can be achieved
(such as urinary tract infections).
As discussed here, DNA gyrase/topo IV and the PBPs are
the multienzyme targets of clinically validated drugs and
they represent new options going forward with novel chemi-
cal classes: for example, dual inhibitors of GyrB and ParE as
well as reversible inhibitors of PBPs. Table 3 notes three
other inhibitors of different dual enzyme targets. Another
dual targeting opportunity, specifically for Gram-negatives,
is the two early transacylases of Lipid A synthesis, LpxA
and LpxD. Recently a small peptide, RJPXD33, has been
shown to bind to both enzymes (albeit to LpxD with greater
affinity than to LpxA), to be competitive with substrate
for binding to LpxD and to exhibit antibacterial activity
(when produced intracellularly) that is abrogated by over-
expression of LpxD [68]. Thus with further study, this
type of probe peptide may be useful in designing small mole-
cule inhibitors with the ability to enter the Gram-negative
cytoplasm (see below).
The recognition that polypharmacology can have positive
implications has been especially apparent in the area of kinase
inhibitors in cancer chemotherapy, with the caveat that such
multitargeting must be balanced, demonstrating selectivity
without promiscuity [75]. In bacteria, it has been recognized
that the successful systemic monotherapeutic antibacterials
are multitargeted, at least at the genetic level (in that targets
such as cell wall precursors, ribosomal RNA, membranes,
DNA are products of multiple genes or structures produced
by multiple genes), and it has been proposed that this
multitargeting prevents rapid resistance selection [3]. But the
 Multitarget ligands in antibacterial research: progress and opportunities
same caveats as for kinases would apply in the search for novel
antibacterial multitarget ligands.
Indeed, an important aspect of antibacterial discovery in
general, also applicable in the case of multitarget ligands, is
that the mechanism of action at the whole-cell level of
enzyme inhibitors cannot be inferred from their activity on
the isolated enzymes or, in the case of putative dual-targeting
agents, from the inability to obtain mutants. Independent
proof of mechanism is needed [1]. With specific pharmaco-
phoric features in ligands that target multiple enzymes, there
is a finite likelihood that other targets will be inhibited as
well. Such extra activities might have implications for toxic-
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
ity. The possibility of nonselectivity is illustrated by the find-
ings, noted above, with series of GyrB/ParE compounds in
studies by Trius [48]. Even small changes in structure that
had little effect on topoisomerase inhibition led to altera-
tions in whole cell activity which proved to be due to inhibi-
tion of macromolecular synthesis in addition to DNA. For
the GyrB/ParE inhibitors kibdelomycin [35] and querce-
tin [38], the available data does show inhibition of the topoi-
somerases but does not exclude (and even demonstrates)
inhibition of other cellular processes at concentration near
the MIC. With the rhodanine inhibitors of Mur ligases,
mentioned above, while there is crystallographic data
For personal use only.
supporting specific enzyme interaction, the selectivity (anti-
bacterial activity being due solely to inhibition of the ligases)
remains unproven [56,57].
A major obstacle to target-based antibacterial discovery is
the necessity for enzyme inhibitors to reach their target
site [1]. This is not as critical for activity against extracytoplas-
mic targets of Gram-positives (such as b-lactams and
glycopeptides), but is important for periplasmic targets in
Gram-negatives and cytoplasmic targets of all pathogens.
Expert Opin. Drug Discov. (2013) 8(2)
For Gram-negatives, the double membrane with its orthogo-
nal sieving properties presents a formidable barrier for which
no set of chemical rules for its passage have been formulated.
Thus, although target structure can guide rational design of
inhibitors, the physicochemical properties of the compound
enabling cell entry must be considered for antibacterial
activity -- preferably during the optimization process. For
example, it is highly likely that inability to find membrane
permeant compounds has stymied the discovery, despite
much effort, of validated antibacterial inhibitors of the Mur
ligases, mentioned above as potential multitarget enzymes,
since their common binding sites are for UDP-MurNAc
and ATP substrates, and may thus require nonpromiscuous
permeant diphosphate isosteres.
Thus, antibacterial programs for the design of multitarget
ligands present an important opportunity for production of
antibacterial agents with longevity due to slow development
of resistance comparable to that seen with other successful
agents, such as the fluoroquinolones -- but much improved
over single-targeted agents for which resistance can appear
overnight (at least in the laboratory).
Acknowledgments
The authors would like to thank R Law, J Heim, J Manchester,
J Finn, J Palmer and D Haydon for their assistance preparing
this manuscript.
Declaration of interest
SP East is an employee of Evotec (UK) Ltd, while LL Silver is
an independent consultant in antibacterial discovery at LL
Silver Consulting, LLC.
153
 S. P. East & L. L. Silver
Bibliography
Papers of special note have been highlighted as
either of interest () or of considerable interest
() to readers.
1. Silver LL. Challenges of antibacterial
discovery. Clin Microbiol Rev
2011;24:71-109
2. Walsh C. Molecular mechanisms that
confer antibacterial drug resistance.
Nature 2000;406:775-81
3. Silver LL. Polypharmacology as an
emerging trend in antibacterial discovery
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
in Polypharmacology in drug discovery.
John Wiley & Sons, Inc., Hoboken,
New Jersey; 2012. p. 167-202
4. Morphy R, Rankovic Z. Designed
multiple ligands. An emerging drug
discovery paradigm. J Med Chem
2005;48:6523-43
.. Key perspective article on strategies
employed in multitargeted drug
discovery programs, classification of
designed multiple ligands and
historical example of compounds
interacting with multiple
For personal use only.
biological targets.
5. Metz JT, Hajduk PJ. Rational
approaches to targeted
polypharmacology: creating and
navigating protein-ligand interaction
networks. Curr Opin Chem Biol
2010;14:498-504
6. Fischbach MA. Combination therapies
for combating antimicrobial resistance.
Curr Opin Microbiol 2011;14:519-23
. A compact review article describing
different approaches for combination
therapies with antibacterial drugs.
7. Morphy R, Kay C, Rankovic Z. From
magic bullets to designed multiple
ligands. Drug Discov Today
2004;9:641-51
8. Morphy R, Rankovic Z. Design of
multitarget ligands in Lead generation
approaches in drug discovery.
John Wiley & Sons, Inc., Hoboken,
New Jersey; 2010. p. 141-64
9. Pokrovskaya V, Baasov T. Dual-acting
hybrid antibiotics: a promising strategy
to combat bacterial resistance. Exp Opin
Drug Discov 2010;5:883-902
. Comprehensive account of
dual-targeting ligands that are a
combination of two discrete
antibacterial compounds
joined together.
154
10. Morphy R, Rankovic Z. The
physicochemical challenges of designing
multiple ligands. J Med Chem
2006;49:4961-70
11. Papp-Wallace KM, Endimiani A,
Taracila MA, Bonomo RA.
Carbapenems: past, present, and future.
Antimicrob Agents Chemother
2011;55:4943-60
12. Drlica K, Hiasa H, Kerns R, et al.
Quinolones: action and resistance
updated. Curr Top Med Chem
2009;9:981-98
13. Sanyal G, Doig P. Bacterial
DNA replication enzymes as targets for
antibacterial drug discovery. Expert Opin
Drug Discov 2012;7:327-39
14. Collin F, Karkare S, Maxwell A.
Exploiting bacterial DNA gyrase as a
drug target: current state and
perspectives. Appl Microbiol Biotechnol
2011;92:479-97
15. Butler MS, Cooper MA. Antibiotics in
the clinical pipeline in 2011. J Antibiot
2011;64:413-25
16. Pucci MJ, Podos SD, Thanassi JA, et al.
In vitro and in vivo profiles of
ACH-702, an isothiazoloquinolone,
against bacterial pathogens.
Antimicrob Agents Chemother
2011;55:2860-71
17. http://www.achillion.com/ACH_702
18. Aravind L, Leipe DD, Koonin EV.
Toprim -- a conserved catalytic domain
in type IA and II topoisomerases,
DnaG-type primases, OLD family
nucleases and RecR proteins.
Nucleic Acids Res 1998;26:4205-13
19. Kim HY, Wiles JA, Wang Q, et al.
Exploration of the activity of
7-pyrrolidino-8-
methoxyisothiazoloquinolones against
methicillin-resistant Staphylococcus
aureus (MRSA). J Med Chem
2011;54:3268-82
20. Heim J. Safe drugs for bad bugs:
EV-035, a new topoisomerase inhibitor.
Available from: http://www.evolva.com/
pharmaceuticals/ev-035
21. Evolva SA. 2-Pyridone antimicrobial
compositions. WO2012047487; 2012
22. Li Q, Mitscher LA, Shen LL. The
2-pyridone antibacterial agents: bacterial
topoisomerase inhibitors. Med Res Rev
2000;20:231-93
Expert Opin. Drug Discov. (2013) 8(2)
23. Black MT, Stachyra T, Platel D, et al.
Mechanism of action of the antibiotic
NXL101, a novel nonfluoroquinolone
inhibitor of bacterial type II
topoisomerases.
Antimicrob Agents Chemother
2008;52:3339-49
24. Miles TJ, Barfoot C, Brooks G, et al.
Novel cyclohexyl-amides as potent
antibacterials targeting bacterial type
IIA topoisomerases. Bioorg Med
Chem Lett 2011;21:7483-8
25. Miles TJ, Axten JM, Barfoot C, et al.
Novel amino-piperidines as potent
antibacterials targeting bacterial type
IIA topoisomerases. Bioorg Med
Chem Lett 2011;21:7489-95
26. Geng B, Comita-Prevoir J,
Eyermann CJ, et al. Exploring
Left-hand-side substitutions in the
benzoxazinone series of
4-amino-piperidine bacterial type IIa
topoisomerase inhibitors. Bioorg Med
Chem Lett 2011;21:5432-5
27. Reck F, Alm R, Brassil P, et al. Novel
N-linked aminopiperidine inhibitors of
bacterial topoisomerase type II:
broad-spectrum antibacterial agents with
reduced hERG activity. J Med Chem
2011;54:7834-47
28. Reck F, Alm RA, Brassil P, et al. Novel
N-linked aminopiperidine inhibitors of
bacterial topoisomerase type II with
reduced pKa: antibacterial agents with an
improved safety profile. J Med Chem
2012;55:6916-33
29. Wiles JA, Phadke AS, Bradbury BJ, et al.
Selenophene-containing inhibitors of type
IIA bacterial topoisomerases.
J Med Chem 2011;54:3418-25
30. Bax BD, Chan PF, Eggleston DS, et al.
Type IIA topoisomerase inhibition by a
new class of antibacterial agents. Nature
2010;466:935-40
. X-ray evidence illustrating the binding
mode of the non-quinolone bacterial
topoisomerase II inhibitor
GSK299423 with DNA gyrase, thus
rationalizing the activity of this
compound on fluoquinolone resistant
bacterial strains.
31. Widdowson K, Hennessy A. Advances in
structure-based drug design of novel
bacterial topoisomerase inhibitors.
Future Med Chem 2010;2:1619-22

32. Wohlkonig A, Chan PF, Fosberry AP,
et al. Structural basis of quinolone
inhibition of type IIA topoisomerases
and target-mediated resistance.
Nat Struct Mol Biol 2010;17:1152-3
33. Oblak M, Kotnik M, Solmajer T.
Discovery and development of ATPase
inhibitors of DNA gyrase as antibacterial
agents. Curr Med Chem
2007;14:2033-47
34. Angehrn P, Goetschi E, Gmuender H,
et al. A new DNA gyrase inhibitor
subclass of the cyclothialidine family
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
based on a bicyclic dilactam-lactone
scaffold. Synthesis and antibacterial
properties. J Med Chem
2011;54:2207-24
35. Phillips JW, Goetz MA, Smith SK, et al.
Discovery of kibdelomycin, a potent new
class of bacterial type II topoisomerase
inhibitor by chemical-genetic profiling in
Staphylococcus aureus. Chem Biol
2011;18:955-65
36. Hossion AM, Otsuka N, Kandahary RK,
et al. Design, synthesis, and biological
evaluation of a novel series of quercetin
For personal use only.
diacylglucosides as potent
anti-MRSA and anti-VRE agents.
Bioorg Med Chem Lett
2010;20:5349-52
37. Hossion AM, Zamami Y,
Kandahary RK, et al. Quercetin
diacylglycoside analogues showing dual
inhibition of DNA gyrase and
topoisomerase IV as novel antibacterial
agents. J Med Chem 2011;54:3686-703
38. Cushnie TP, Lamb AJ. Antimicrobial
activity of flavonoids. Int J
Antimicrob Agents 2005;26:343-56
39. Boehm HJ, Boehringer M, Bur D, et al.
Novel inhibitors of DNA gyrase: 3D
structure based biased needle screening,
hit validation by biophysical methods,
and 3D guided optimization.
A promising alternative to random
screening. J Med Chem
2000;43:2664-74
.
Important fragment-based screening
paper in the identification of novel
ligands modulating the effect of an
essential bacterial enzyme.
40. Charifson PS, Grillot AL, Grossman TH,
et al. Novel dual-targeting benzimidazole
urea inhibitors of DNA gyrase and
topoisomerase IV possessing potent
antibacterial activity: intelligent design
and evolution through the judicious use
of structure-guided design and
Multitarget ligands in antibacterial research: progress and opportunities
structure-activity relationships.
J Med Chem 2008;51:5243-63
. Medicinal chemistry summary of the
benzimidazole ureas including a
hypothesis on the importance of the
conformation of the small molecule for
dual GyrB/ParE inhibition and in vivo
efficacy data.
41. Grossman TH, Bartels DJ, Mullin S,
et al. Dual targeting of GyrB and ParE
by a novel aminobenzimidazole class of
antibacterial compounds.
Antimicrob Agents Chemother
2007;51:657-66
42. Wei Y, Letiran A. PDB accession code:
3FV5
43. Vertex pharmaceticals incorporated.
Pyrimidine gyrase and topoisomerase IV
inhibitors. WO2012097269; 2012
44. East SP, Czaplewski LG, Haydon DJ.
Ethyl urea inhibitors of the bacterial
type II topoisomerase DNA gyrase
(GyrB) and topoisomerase IV (ParE) in
designing multi-target drugs.
RSC Publishing, Cambridge;
2012. p. 335-52
45. Palmer J. Dual-targeting
DNA supercoiling inhibitors for the
treatment of bacterial infections.
Presented at the challenges of
antibacterial drug development;
San Diego; 2012
46. Manchester JI, Dussault DD, Rose JA,
et al. Discovery of a novel azaindole class
of antibacterial agents targeting the
ATPase domains of DNA gyrase and
topoisomerase IV. Bioorg Med
Chem Lett 2012;22:5150-6
47. Manchester JI, Buurman ET,
Bisacchi GS, McLaughlin RE. Molecular
determinants of AcrB-mediated bacterial
efflux implications for drug discovery.
J Med Chem 2012;55:2532-7
48. Finn J. The discovery of potent dual
targeting pyrrolopyrimidine inhibitors of
bacterial DNA gyrase B and
topoisomerase IV with broad spectrum
antibacterial activity. Presented at the
challenges of antibacterial drug
development; San Diego; 2012
49. Bugg TD, Braddick D, Dowson CG,
Roper DI. Bacterial cell wall assembly:
still an attractive antibacterial target.
Trends Biotechnol 2011;29:167-73
50. Gautam A, Vyas R, Tewari R.
Peptidoglycan biosynthesis machinery:
Expert Opin. Drug Discov. (2013) 8(2)
a rich source of drug targets.
Crit Rev Biotechnol 2011;31:295-336
51. Silver LL. Does the cell wall of bacteria
remain a viable source of targets for
novel antibiotics? Biochem Pharmacol
2006;71:996-1005
52. Smith CA. Structure, function and
dynamics in the mur family of bacterial
cell wall ligases. J Mol Biol
2006;362:640-55
53. Perdih A, Kovač A, Wolber G, et al.
Discovery of novel benzene
1,3-dicarboxylic acid inhibitors of
bacterial MurD and MurE ligases by
structure-based virtual screening
approach. Bioorg Med Chem Lett
2009;19:2668-73
54. Tomašić T, Zidar N, Rupnik V, et al.
Synthesis and biological evaluation of
new glutamic acid-based inhibitors of
MurD ligase. Bioorg Med Chem Lett
2009;19:153-7
55. Tomašić T, Šink R, Zidar N, et al. Dual
inhibitor of MurD and MurE ligases
from Escherichia coli and Staphylococcus
aureus. ACS Med Chem Lett
2012;3:626-30
56. Tomašić T, Zidar N, Kovač A, et al.
5-Benzylidenethiazolidin-4-ones as
multitarget inhibitors of bacterial Mur
ligases. Chem Med Chem 2010;5:286-95
57. Tomašić T, Peterlin Mašič L. Rhodanine
as a scaffold in drug discovery: a critical
review of its biological activities and
mechanisms of target modulation.
Expert Opin Drug Discov
2012;7:549-60
58. Mendgen T, Steuer C, Klein CD.
Privileged scaffolds or promiscuous
binders: a comparative study on
rhodanines and related heterocycles in
medicinal chemistry. J Med Chem
2012;55:743-53
59. Baell JB, Holloway GA. New
substructure filters for removal of pan
assay interference compounds (PAINS)
from screening libraries and for their
exclusion in bioassays. J Med Chem
2010;53:2719-40
60. Tomašić T, Kovač A, Klebe G, et al.
Virtual screening for potential inhibitors
of bacterial MurC and MurD ligases.
J Mol Model 2012;18:1063-72
61. Turk S, Verlaine O, Gerards T, et al.
New noncovalent inhibitors of
penicillin-binding proteins from
155
 S. P. East & L. L. Silver
penicillin-resistant bacteria. PLoS One
2011;6:e19418
62. Shilabin AG, Dzhekieva L, Misra P,
et al. 4-Quinolones as noncovalent
Inhibitors of high molecular mass
penicillin-binding proteins. ACS Med
Chem Lett 2012;3:592-5
63. Woon ECY, Zervosen A, Sauvage E,
et al. Structure guided development of
potent reversibly binding penicillin
binding protein inhibitors. ACS Med
Chem Lett 2011;2:219-23
Expert Opin. Drug Discov. Downloaded from informahealthcare.com by University of Sussex Library on 01/20/13
64. Contreras-Martel C, Amoroso A,
Woon EC, et al. Structure-guided design
of cell wall biosynthesis inhibitors that
overcome b-lactam resistance in
Staphylococcus aureus (MRSA).
ACS Chem Biol 2011;6:943-51
65. Zervosen A, Bouillez A, Herman A, et al.
Synthesis and evaluation of boronic acids
as inhibitors of penicillin binding
proteins of classes A, B and C.
Bioorg Med Chem 2012;20:3915-24
66. Zhang J, Zhang L, Li X, Xu W. UDP-3-
O-(R-3-hydroxymyristoyl)-N-
For personal use only.
acetylglucosamine deacetylase (LpxC)
inhibitors: a new class of antibacterial
156
agents. Curr Med Chem
2012;19:2038-50
67. Achaogen announces all objectives met in
Phase 2 plazomicin complicated urinary
tract infections study and start of
first-in-human study with ACHN-975.
Available from: http://www.achaogen.
com/news/148
68. Jenkins RJ, Dotson GD. Dual targeting
antibacterial peptide inhibitor of early
lipid A biosynthesis. ACS Chem Biol
2012;7:1170-7
69. Wang J, Kodali S, Lee SH. Discovery of
platencin, a dual FabF and FabH
inhibitor with in vivo antibiotic
properties. Proc Natl Acad Sci USA
2007;104:7612-16
70. Ali A, Taylor GE. Development of
DNA polymerase IIIC inhibitors for the
treatment of Gram-positive bacterial
infections. Expert Opin Ther Patents
2005;15:947-53
71. Bennett BC, Xu H, Simmerman RF,
et al. Crystal structure of the anthrax
drug target, Bacillus anthracis
dihydrofolate reductase. J Med Chem
2007;50:4374-81
Expert Opin. Drug Discov. (2013) 8(2)
72. Škedelj V, Tomašić T, Mašič LP,
Zega A. ATP-binding site of bacterial
enzymes as a target for antibacterial drug
design. J Med Chem 2011;54:915-29
73. Keiser MJ, Roth BL, Armbruster BN,
et al. Relating protein pharmacology by
ligand chemistry. Nat Biotechnol
2007;25:197-206
74. Durrant JD, Amaro RE, Xie L, et al.
A multidimensional strategy to detect
polypharmacological targets in the
absence of structural and sequence
homology. PLoS Comput Biol
2010;6:e1000648
75. Morphy R. Selectively nonselective kinase
inhibition: striking the right balance.
J Med Chem 2010;53:1413-37
Affiliation
Stephen P East†1 & Lynn L Silver2
†
Author for correspondence
1
Evotec (UK) Ltd., 114 Milton Park,
Abingdon, Oxfordshire OX14 4SA, UK
E-mail: stephen.east@evotec.com
2
LL Silver Consulting, LLC,
955 S. Springfield Ave.,
Unit C403 Springfield,
NJ 07081, USA