Reviews

pubs.acs.org/acschemicalbiology

Bacterial Histidine Kinases as Novel Antibacterial Drug Targets

Agnieszka E. Bem,† Nadya Velikova,‡ M. Teresa Pellicer,§ Peter van Baarlen,† Alberto Marina,‡,∥
and Jerry M. Wells*,†
†
Host−Microbe Interactomics, Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands
‡
Instituto de Biomedicina de Valencia-Consejo Superior de Investigaciones Cientificas (IBV-CSIC), Jaume Roig 11, 46010-Valencia,
Spain
§
R&D Department Interquim, Ferrer HealthTech, Joan Buscalla 10, 08137-Sant Cugat del Valles Barcelona, Spain
∥
Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBER-ISCIII), Jaume Roig 11, 46010-Valencia, Spain

ABSTRACT: Bacterial histidine kinases (HKs) are promising

targets for novel antibacterials. Bacterial HKs are part of
bacterial two-component systems (TCSs), the main signal
transduction pathways in bacteria, regulating various processes
including virulence, secretion systems and antibiotic resistance.
In this review, we discuss the biological importance of TCSs
and bacterial HKs for the discovery of novel antibacterials, as
well as published TCS and HK inhibitors that can be used as a
starting point for structure-based approaches to develop novel
antibacterials.

■ THE GROWING PROBLEM OF ANTIMICROBIAL

DRUG RESISTANCE
reference to those HKs that are part of bacterial two-
component systems (TCSs).
In 2004, the Infectious Diseases Society of America (IDSA)
published an alarming report entitled “Bad Bugs, No Drugs”,1
discussing antimicrobial drug resistance (AMR) and the lack of
■ APPROACHES TO THE DISCOVERY OF NOVEL
ANTIBACTERIAL DRUGS
therapeutic alternatives to current antibiotics that have become For an antibacterial drug to be designated as novel, it should
fulfill at least one of the following criteria:6
less effective because of AMR. Since then, infections caused by
drug-resistant bacteria have increased worldwide. Unless action (1) belong to a novel chemical class and act on a new target;
is taken to halt this problem we are facing the prospect of (2) work via new mechanism of action or binding site even if
returning to a preantibiotic era.1 the drug target itself is not new;
Nearly all classes of antibiotics were discovered in the 1950s. (3) be biochemically modified to overcome bacterial
Resistance to these antibiotics was usually reported within a few resistance to the original drug;
years after their discovery, sometimes even years before their (4) inhibit the same target as the original drug but have new
introduction to the market.2 Only two novel classes of physicochemical properties within its class permitting
antimicrobials, the oxazolidinones and cyclic lipopeptides3 administration via a new route.
have entered the market in the last 30 years. Concurrently, Novel antibiotics ideally should be broad-spectrum and
pharmaceutical companies appear to have closed antibiotics amenable to structural changes that allow for optimization of
research and development (R&D) programs and instead focus potency, specificity and absorption, distribution, metabolism,
their financial and research efforts on medicines for chronic elimination and toxicity (ADMET) properties.7
pathologies such as cancer. Drug discovery projects on The most successful phenotypic screens for inhibition of
infectious diseases represent around 16% of all academic bacterial growth, usually determined via minimal inhibitory
concentration (MIC) assays, have used chemical libraries or
projects but comprise only 9% of pharmaceutical industry
natural compounds from extracts of soil or marine ecosystems
projects.4 In general, low investments in antibiotics research
including bacterial secondary metabolites. Typically, the
have been reported for Europe. During the period 2008−2013, putative inhibitory compounds, also known as lead compounds,
less than 2% of the European Union and U.K. total research
budget was committed to microbiology research, and less than
1% was committed to antibiotics research.5 In the following Special Issue: New Frontiers in Kinases
sections, we outline which strategies have so far been used in Received: September 6, 2014
antibiotics research and emphasize how novel antibiotics may Accepted: December 1, 2014
be targeted toward histidine kinases (HKs), with special Published: December 1, 2014

© 2014 American Chemical Society 213 DOI: 10.1021/cb5007135

ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

Figure 1. Two-component system signaling pathway and inhibitors of TCS mediated signal transduction. TCSs represent the most common form of
bacterial signal transduction, based on phosphotransfer. A phosphorus group is transferred from the CA-domain to a conserved His-residue of the
histidine kinase and from there at a conserved Asp-residue of the RR. This leads to dimerization of the phosphorylated RR and activates it to induce
the expression of its downstream target genes. Possible places of signal transduction inhibition are marked with stars.

are purified and followed by analytical chemistry and essential or conditionally essential functions required for
cytotoxicity assays. The chemical structure of the lead bacterial survival.12 To identify inhibitors of a specific bacterial
compound is optimized, and ultimately, its mode of action is target in RTDD, high-throughput screening (HTS) is
elucidated and its antibacterial spectrum determined. Nearly all performed with libraries of natural compounds as well as
antimicrobials on the market are based on (derivatives of) small synthetic chemical molecules. In RTDD, the ideal
antibiotics produced by Streptomyces8 or antibiotics from antibiotic targets should:
unculturable bacteria expressed in heterologous hosts such as (1) have no human homologues or structurally similar
S. lividans.9 proteins;
The above screens have been supplemented with rational, (2) be highly conserved among various bacterial species to
knowledge-intensive in vitro and in silico screens. In structure- ensure broad-spectrum of antibacterial action;
based virtual screening (SBVS), the 3D structure of the target (3) be strictly essential for bacterial viability such that their
protein is obtained experimentally or by homology modeling inhibition would lead to bacterial death but as targeting
and analyzed in silico for potential physicochemical interactions essentiality might increase resistance selection pressure,
of a virtual library of chemical compounds with the active site of virulence targets have been proposed as an alternative;
the target.10 Selected ligands, or hit compounds, are then tested (4) be “assayable”, that is, have easily measurable activity, in
in vitro to confirm their target inhibitory capacity. Hit assays amenable to high-throughput screening (HTS);
compounds that show biological activity (lead compounds) (5) have available structural data on the target and genetic
typically are further optimized by chemical modifications. tools to validate the target in a key species.12
In fragment-based drug discovery (FBDD), libraries of Bacterial HKs fulfill most of these requirements except that
thousands of small chemical compounds, called fragments, are certain mammalian kinases (or ATPases) have a similar protein
screened against purified protein representing the selected drug fold in the ATP domain.13,14
target. Fragments that bind weakly to different parts of the In the following sections we review progress toward the
selected target site may be chemically linked to enhance discovery of novel antibacterials targeting HKs of TCSs.15,16
selectivity and potency of inhibition.11 The advantage of this
approach is that a collection of few thousands fragments covers
a larger chemical space than HTS libraries of the same size due
■ TWO-COMPONENT SYSTEMS
TCSs are present in most bacteria and are the most common
to possibility of pairwise combinations of fragments. Another form of bacterial signal transduction. The number of TCSs
approach exploited in antibacterial drug research is rational found in bacterial species is correlated to the genome size and
target-based drug design (RTDD) using proteins that perform the range of environments in which the organism can grow and
214 DOI: 10.1021/cb5007135
ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

Figure 2. Domain organization and conservation of HKs. (A) Domain organization of S. mutans WalK (PDB: 4I5S). A HK consists of a periplasmic
sensor domain (Sp, not shown) and cytoplasmic sensor domains (Sc), dimerization and phosphotransfer domain (DHp), and catalytic and ATP-
binding domain (CA). Sc is variable and not present in all HKs, whereas DHp and CA are relatively more conserved than Sc and present in all HKs.
(B) Structure of the CA domain of T. maritima HK853 (PDB: 3DGE). The CA domain accommodates the nucleotide ligand (shown in sticks) in a
well-defined binding pocket (shown as semitransparent surface). The nucleotide binding-pocket includes the conserved N-, G1-, G2-, G3-, and F-
boxes (yellow, light green, dark green, pale green, and purple, respectively) and the variable ATP-lid (red). (C) Sequence alignment of CA domains
of WalK from different Gram-positive bacteria. Homologues of HKs present in different bacterial pathogens suggest broad spectrum antibacterial
effect of putative HK-inhibitors. (D) Sequence alignment of CA domains of different E. coli HKs. Sequence similarities of the CA domain of HKs
suggest that a putative HK inhibitor would inhibit multiple targets (polypharmacology) which is expected to slow down antimicrobial resistance
development.

survive. TCSs are considered attractive antibacterial drug via the catalytic and ATP-binding (CA) domain, which binds
targets as multiple members are found in nearly all bacteria. ATP and phosphorylates the HK at a conserved histidine
Several TCSs have been shown to be essential for bacterial residue in the dimerization and histidine phosphotransfer
survival, and homologues have not been identified in mammals (DHp) domain. The CA and DHp domains are conserved and
including humans.17 TCS signaling involves autophosphor- present in all HKs, whereas the remaining sensor domains
ylation of a membrane-bound histidine kinase (HK), (periplasmic, PAS, GAF, HAMP) are variable and not present
phosphotransfer of the phosphoryl group to a cognate response in all HKs (Figure 2).18
regulator (RR), and ultimately modulation of the expression of The RR usually is a transcription factor that, upon
target genes (Figure 1).18 Appropriate phosphorylation levels of phosphorylation, undergoes conformational changes that
the RR are tightly regulated by the activity of the HK, the RR, promote dimerization or higher order oligomerization states20
or a partner protein.18,19 HK autophosphorylation is mediated favoring in the majority of cases higher affinity interaction
215 DOI: 10.1021/cb5007135
ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

between the RR and bacterial DNA and altered target gene Table 2. Regulation of Antibiotic Resistance by Two-
expression.21 Alternatively, the RR may act as an enzyme such Component Systems in Human and Animal Pathogens
as a methylesterase22 or an ATPase.23 TCS signaling is
TCS implicated
terminated by dephosphorylation of the RR, which can be in resistance
autoinduced or mediated by the cognate HK or by auxiliary pathogen gained resistance regulation
proteins.24,25 Usually, each HK possesses one cognitive RR with Staphylococcus aureus fluoroquinolone ArlS/R32
strict partner specificity.26 TCSs exhibit linear signal trans- bacitracin and nisin BraSR75
duction, which can be modulated by addition of extra modules vancomycin VraRS,GraRS,
to the conserved domain, as well as by addition of auxiliary VanSR31
proteins named “connector” proteins.18 TCS signaling can cationic antimicrobial peptides GraSR,Stk1/
Stp176
mediate rapid microbial adaptation in response to changes in
Klebsiella tetracycline, nalidixic acid, PhoBR34
the environment, including signals encountered in the environ- pneumoniae tobramycin, streptomycin and
ment of the host, by altering expression of specific genes21 that spectinomycin
may participate in virulence mechanisms, metabolic and chloramphenicol, erythromycin, LysR (oxyRKP)77
developmental pathways, antibiotic resistance, and regulation nalidixic acid and trimethoprim
of type II/III/IV/VI secretion systems27−29 (Table 1). β-lactams and chloramphenicol CpxAR78
Acinetobacter aminoglycosides, fluoroquinolones, AdeSR79
baumannii tetracycline, chloramphenicol,
Table 1. Pathways Regulated by Two-Component Systems in erythromycin, trimethoprim
Different Bacterial Pathogens Pseudomonas carbapenem CzcRS33
aeruginosa
regulated pathway regulating TCSs small colony variant aminoglycoside PhoPQ80
Nutrient Acquisition (SCV) of
Pseudomonas
nitrogen NtrBC aeruginosa
phosphorus PhoRB Mycobacterium multidrug MtrAB81
Metabolism tuberculosis
electron transport system ArcAB Enterococcus faecalis ceftriaxone (β-lactam) CroRS82
catabolic machinery BarA/UvrY, TcDE, CitAB, MalKR Salmonella antimicrobial peptide PhoPQ,PmrAB
typhimurium ciprofloxacin BaeSR83
nitrogen metabolism NarXL
glucose metabolism UphBA Escherichia coli novobiocin and deoxycholate BaeSR8485
carbon metabolism BarA/UvrY multidrug ArcBA86
cell wall metabolism YycFG, VraSR, DesKR β-lactams and novobiocin BaeR87
Adaptation to Changes in the Environment Stenotrophomonas aminoglycosides, β-lactams, SmeSR88
maltophilia fluoroquinolones
osmolarity EnvZ/OmpR
light Cph/Rcp1, NblSR
chemotaxis CheYB located in the membrane of Gram-negative bacteria that control
temperature WalKR, DesKR outer membrane permeability of small molecules including
oxygen DevRS or DosRS carbapenem antibiotics,33 tetracycline, streptomycin, and
Developmental Pathways spectinomycin.34
sporulation SpoA
Many efflux pumps, transporter membrane proteins that
biofilm formation KinAB
actively pump toxic compounds out of bacteria, are regulated by
competence ComDE
TCSs and are responsible for multidrug resistance (MDR) of
Virulence
important human pathogens (Table 1). Efflux pumps recognize
their substrate mainly by physicochemical properties such as
CiaRH, MicAB (WalKR), RR04-HK04, RR06-HK06, RitR AgrAC, SrrAB,
SaeRS, AlRS, LytRS, PhoPQ, PmrAB, RscC-YojN-RscB, OmpR-EnvZ, hydrophobicity; due to their amphiphilic character several
SsrBA, SirA-BarA, DegU, VirRS, AgrAC, LisRK, FigRS, ArsRS, VirRS antibiotics are easily recognized and removed from bacterial
Others cells by these pumps. TCS-mediated regulation of the efflux
flagellar assembly CheA, FlrBC pumps of diverse important pathogens including K. pneumo-
secretion systems: niae25 and A. baumannii27 provides resistance to a wide range of
Type II TtsSR antibiotics (Table 2) highlighting the importance of TCSs as
Type III RhpRS, AlgZR, SpiR/SsrB, PhoPQ, OmpR/EnvZ attractive drug targets.
Type IV
Type VI
VirB/D4, PmrAB, CpxRA, LetAS, LqsRS
EnvZ/OmpR ■ TCS HKS ARE ATTRACTIVE ANTIBACTERIAL DRUG
TARGETS

■ LINK BETWEEN TCS AND ANTIBIOTIC RESISTANCE

TCSs may regulate antibiotic resistance by various mechanisms
The conserved features of the histidine kinase CA domain and
its essential role in TCS signal transduction make it an
attractive target for structure-based virtual screening and
(Table 2). The VanRS or VraSR TCS mediates changes in phenotypic screening of biochemical inhibitors. Moreover, the
bacterial cell wall metabolism causing resistance to vancomycin, high degree of sequence conservation in the CA catalytic site
an antibiotic targeting bacterial cell wall synthesis.30 Also, implies that inhibitors targeted against this site will possess
single-nucleotide polymorphisms (SNPs) in the promoter broad-spectrum antibacterial activity (Figure 2). Altogether,
region of WalKR, a TCS involved in cell wall synthesis, lead this makes the CA domain an attractive HK target site for the
to increased vancomycin resistance.31 TCSs may also regulate discovery and development of broad-spectrum antibacterials.
the activity of efflux pumps and porins.32 Porins are proteins Simultaneous inhibition of multiple targets (drug polypharma-
216 DOI: 10.1021/cb5007135
ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

Figure 3. The ATP-lid plays a critical role in ligand-binding and catalytic activity of members of GHKL-superfamily of proteins. The ATP-lid is a
flexible loop variable in length and in sequence. Features of the ATP-lid can be exploited to design histidine-kinase autophosphorylation inhibitors
with higher specificity to bacterial histidine kinases and reduced off-target effects to mammalian members of the GHKL-superfamily, that is, with
lower toxicity. Conservation scores were calculated using ConSurf. Ligands are shown in sticks.

cology) has been proposed as a strategy to slow down VanSR due to its role in antibiotic resistance, WalKR due to its
resistance development to drugs including novel antibacte- essentiality, and QseCB and DosRST due to their role in
rials.16,35−37 As bacteria possess multiple TCSs, inhibitors of the virulence. Progress toward developing inhibitors of these TCSs
highly conserved CA domain are likely to shut down multiple is discussed in the remainder of this review.
signaling pathways compromising the ability of the bacteria to Inhibition of VanSR TCS, Regulator of Vancomycin
rapidly adapt to environmental changes including those Resistance. Vancomycin, an extracellular-acting glycopeptide
encountered in the host during infection. For some bacteria antibiotic that inhibits bacterial cell wall synthesis, is now the
TCS inhibition may not be bactericidal but it is likely to only available antibiotic to treat methicillin-resistant Staph-
compromise efficient growth, thus reducing fitness. ylococcus aureus (MRSA).40 Vancomycin resistance has been
One potential downside to HKs is that the ATP-binding observed in Enterococcus faecalis and Enterococcus faecium.41,42
Bergerat fold that is present in the CA domain does also occur The genes conferring resistance to vancomycin are regulated by
in several human protein families and is present in crucial the VanSR TCS.43 It is suggested that VanS HK negatively
proteins such as Hsp90. The Bergerat fold includes four regulates VanR RR function in the absence of vancomycin.32
conserved motifs; N, G1, G2, and G3 boxes in the HK CA Due to its clinical relevance, VanSR was considered an
domain corresponding to Motif I, Motif II, Motif III, and Motif attractive drug target and several VanSR TCS inhibitors were
IV, respectively, in other members of the GHKL family (Figure discovered (Table 3) that function by uncoupling energy
2 and 3).18 The F box is a conserved motif preceding the G2 required for ATP synthesis.44 Although these compounds could
box and is characteristic for HK CA domains. Presence of the not be used readily as drugs due to their negative effect on
Bergerat fold in microbial and human ATP-binding protein mitochondrial respiration, they may provide a structural
domains might lead to off-target inhibitory effects of HK template for new inhibitors selective for VanSR TCS during
autophosphorylation inhibitors (HKAIs) toward human ATP- adjunct therapy.44
binding domains and, therefore, toxicity to mammalian cells. Inhibition of WalKR, an Essential TCS. WalKR (syn.
The similarity between the CA domain of bacterial TCS and YycF/G, MicA/B, or VicK/R) is a highly conserved TCS in
eukaryotic proteins containing the Bergerat fold is exemplified low-GC Gram-positive bacteria. It is the most widely
by the demonstration that the eukaryotic Hsp90 inhibitor distributed and obligate essential TCS found in many
radicicol is an inhibitor of PhoQ autophosphorylation and was pathogens belonging to the genera of Staphylococcus,
cocrystallized with the CA domain of PhoQ.38 However, other Streptococcus, Enterococcus, and Listeria. WalKR regulates
inhibitors with similar activity as radicicol could not be genes responsible for cell wall metabolism and cell wall
cocrystallized with PhoQ and did not inhibit PhoQ homeostasis.27,45,46 Additionally, WalKR regulates genes
autophosphorylation.38 The differences between radicicol and involved in metabolism, stress response, virulence, host-
the other Hsp90 inhibitors with respect to PhoQ inhibition microbe interactions, transport, and regulatory pathways
were attributed to differences in the putative interaction with (Table 4).28,47
the ATP-lid and the ATP-lid conformation in the CA domain. In most species, walR has been found to be essential for
The ATP-lid (Figure 3) is a variable loop that connects the G1 viability making it an attractive antibacterial target. Con-
and G2 boxes or the corresponding motifs in other GHKL struction of inducible knockouts of walR, as well as high
family members. The ATP-lid is crucially involved in structural conservation of WalR between species, led to the
autophosphorylation18,39 and contains the F box which is identification of a consensus WalR recognition sequence and
characteristic of bacterial HKs. Although HKs are structurally also of the regulon controlled by this TCS in S. pneumoniae.47
similar to some eukaryotic proteins, the biochemical study WalKR activation has also been linked to vancomycin
discussed above shows that it is possible to design inhibitors of resistance in S. aureus.48,49 It was shown that exposure to
microbial ATP-binding domains that have limited affinity for diverse antimicrobials induces vancomycin resistance pheno-
eukaryote ATP-binding protein domains and low toxicity to types by either single or multiple mutations localized in the
host cells.38 Indeed, despite sequence conservation of bacterial walKR operon.48,50 Vancomycin resistance of S. aureus may be
and eukaryote kinase domains, several research groups have comediated by the VraRS and GraRS TCSs that are involved in
focused on finding inhibitors for bacterial TCSs, including the cellular response of S. aureus to cell wall damage51 together
217 DOI: 10.1021/cb5007135
ACS Chem. Biol. 2015, 10, 213−224
 Table 3. Characteristics of Published Inhibitors of Two-Component Systems
inhibited two-
component
inhibitor system organisms tested drug discovery method mechanism/comments ref
thiazole derivatives Algr1/Algr2 P. aeruginosa HTS (natural and syn- inhibition of (a) phosphorylation/dephosphoryla- 89
thetic compounds) tion of Algr2; (b) DNA-binding activity of Algr2
thiazole derivatives VanR/S E. faecium HTS (aliginate biosynthe- inhibition of autophosphorylation 90
sis pathway targeted)
ACS Chemical Biology

(a) bisamidine indole (lead compound); (b) amidinobenzimid KinA/Spo0F B. subtilis, S. aureus, E. faecalis, E. faecium, MRSA HTS (TBDD; screening (a) −, (b) reducing DNA binding by RR 91
azoles (23 compounds) with purified proteins)
and chemical synthesis
tyramines derivative (RWJ-49815) KinA/Spo0F B. subtilis, E. faecium, S. pneumoniae, MRSA HTS (TBDD; screening 92
with purified proteins)
6-oxa isosteres of anacardic acids (11 compounds) KinA/Spo0F, S. aureus, E. faecalis, E. faecium, MRSA chemical synthesis of nat- 93
NRII/NRI ural compounds with
reported activity
salicylanilides (closantel and 14 compounds) KinA/Spo0F B. subtilis HTS (TBDD; screening negative effects on mitochondria 44, 94
with purified proteins)
thienopyridine, TEP (lead) HpkA and HTS (TBDD; screening competitive ATP inhibitor of HK autophosphor- 95
DrrA with purified proteins) ylation
hexapeptides (N-acetylated C-amidated D-amino acid), CheA E. coli, S. aureus, S. saprophyticus, S. epidermidis, E. faecium, S. HTS of combinatorial inhibition of mammalian kinase protein C 69
2 peptides pneumoniae, S. pyogenes, S. vividans, E. coli, Moraxella chemistry library
catarrhalis, K. pneumoniae, Proteus mirabilis, P. aeruginosa
cyanoacetoacetamide (CAA) HpkA77 S. pneumoniae, E. faecium HTS and SAR inhibition of autophosphorylation, phosphotransfer, 96
(WalK, noncompetitive inhibitors with ATP; protein
VanRS) aggregation; strong serum binding activity

218
Ethodin (ethacridine lactate) HpkA77 T. maritima SBVS protein aggregation 96
phenylocoumarin derivative DosRS M. tuberculosis (nonreplicating) SBVS 63
NSC9608 (8 compounds) PhoP/Q S. enterica SBVS inhibition of PhoP−DNA complex 97
DrrA peptide HpkA77 T. maritima inhibition of autophosphorylation; protein aggrega- 96
tion
(a) thiazolidinone derivatives (3 compounds), (b) benzamide WalK S. epidermidis SBVS (HTVS) binds to HK and inhibits its autophosphorylation 98
derivatives (2 compounds), (c) furan derivative (1
compound), (d) pyrimidinone derivative (1 compound)
sulfonoamide derivative, LED2009 QseBC E. coli 57
diaryltriazole analogs (15 compounds) KinA/Spo0F S. epidermidis, S. aureus, E. faecalis, E. faecium, VRE, MRSA chemical modifications of closantel 99
thioridazine VraRS MRSA reuse of known neuro- affected membrane fluidity, which disturbed signal 100,
leptic antipsychotic drug transduction; thioridazine can reverse resistance 101
to oxacilin (methicillin analogue)
Carolacton PknB (not S. mutans secondary metabolite from 102,
ComD/E, Streptomyces sp. 103
not VicK/R)
Walkmycin B WalK/R B. subtilis, S. aureus secondary metabolite from inhibition of autophosphorylation by binding to HK 53
Streptomyces sp. cytoplasmic domain
Waldiomycin WalK/R B. subtilis, S. aureus secondary metabolite from 67
Streptomyces sp.
Reviews

DOI: 10.1021/cb5007135
ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

with four other genes, a number of which are controlled by the

T), icaR (ica operon transcription regulator)

(chaperone, heat shock protein), hrcA (heat-

scription repressor), sarST (staphylococcal
grpE (heat shock protein), dnaJ (chaperone

grpE (chaperone, heat shock protein), dnaK

WalKR TCS.50

seaPRSQ (TCS), hrcA (heat-inducible tran-

accessory regulator-like protein, regulator

inducible transcription repressor), groEL

protein), radA (DNA repair protein)
Different drug discovery strategies have been used to identify
inhibitors of WalK. New lead chemical compounds inhibiting

Regulatory pathways
stress response

the CA domain of WalK histidine kinase in Staphylococcus

stress response
epidermidis (Figure 5) that were discovered using SBVS have
been validated experimentally (Table 3).52 In another study,

Regulatory pathways
antibiotic-producing Streptomyces strains were screened for their
ability to produce potential inhibitors of Streptococcus mutans

(chaperonin)
WalK.53 In this study, a new family of inhibitors termed
walkmycins came out as promising lead compounds and are

n/a
currently under preclinical development. Walkmycin C was
shown to inhibit biofilm formation and competence in
binding protein A), coa (staphylocoagulase precursor), hlb (beta-hemolysin), sbi (immunoglobulin G-binding protein),

phosphate synthetase), nrdF (ribonucleotide-diphosphate reductase), mreC (degradation of

metabolism), pur operon (purine synthesis), pyrD (pyrimidine synthesis), carB (carbamoyl-
hla (alpha-hemolysin precursor), emp (fibrinogen-binding protein), splABCDEF (serine proteases), f nAB (fibronectin-

Streptococcus mutans.54 During this study, signermycin B

pyrBC (aspartate carbamoyltransferases), carAB (carbamoyl-phosphate synthases), pyrEF (orotate phosphoribosyl-

emerged as a lead molecule targeting WalK dimerization.55

transferases), ilvA1 (threonine dehydratase), argGH (urea cycle), pyr and pur operons (pyrimidine and purine

fabDFGK (fatty acid metabolism), pspA (fatty acid metabolism), accABS (fatty acid
chp (chemotaxis-inhibiting protein CHIPS), spa (Immunoglobulin G binding protein A precursor), can; efb

Targeting Virulence Genes Regulation by QseCB.

QseCB is a TCS involved in recognizing host-derived
(fibrinogen-binding protein), dltD ((D-alanine transfer protein), capDGGJMP (capsule biosynthesis)

adrenergic signals and the bacterial quorum sensing signal

autoinducer AI-3; AI-3 has been shown to trigger expression of
virulence genes in several bacterial species.56 Homologues of
QseC are present in at least 25 important human and plant
pathogens; therefore, a QseC inhibitor is expected to be a
promising drug for antivirulence therapy against a wide range of
Virulence and Host-Microbe Interaction

metabolism

pathogenic bacteria.57 The QseC protein bears a periplasmic

domain that recognizes both host adrenergic signals as well as
Virulence and Host−Microbe Interaction

the microbial AI-3 molecule.56 Drugs targeting the QseC

metabolism

periplasmic domain have an advantage compared to drugs with

metabolism), genes responsible for amino acids biosynthesis

intracellular targets as they only need to pass the outer

membrane to reach the target. High-throughput screens to
Table 4. Categories of Genes That Are Regulated by WalKR in S. aureus and S. pneumoniae

search for a compound that inhibits activation of QseC by AI-3

pspA (iron transporter)

led to the identification of LED209 (Figure 5), an inhibitor of

the cell envelope)

AI-3 binding to QseC from different Gram-negative pathogenic

bacteria (Table 3).57 LED209 inhibited QseC autophosphor-
ylation and bacterial pathogenicity in vitro and in vivo but not
cell growth.57
Targeting M. tuberculosis Persistence through Inhib-
ition of DosRST. Tuberculosis (TB), the infectious disease
coccal surface protein A), plaBCDA, yocH (autolysin), GbpB (extracellular

caused by Mycobacterium tuberculosis, remains a major public

hydrolase), lytN, fabK (trans-2-enoyl-ACP reductase II), pspA (pneumo-
pscB (extracellular cell wall hydrolase; role in cell division), lytB (cell wall

health problem. In a study to identify mycobacterial targets,

DosS (named after dormancy survival) came out as a target for
dormant mycobacteria.58 It is worth mentioning that in this
study DosS had been classified as a high-confidence target, as it
had been identified after having excluded mycobacterial
proteins with high similarity to commensal gut flora and host
prsA (transport protein), opp3CDF (oligopeptide
f mtB(methicillin resistance determinant FmtB

cell wall metabolism

atlA, lytM, ssA (autolysins; AtlA role in biofilm

proteins.
formation), isaA, cceD (proteins with trans-

DosRST TCS comprises the DosR response regulator and

glycosylase domain, autolytic activity),

the DosS and DosT histidine kinases that play an essential role
cell wall metabolism

in triggering and maintaining dormancy and in enabling future

pia system (iron transport system)

mycobacterial proliferation.59−61 In infected cells, hypoxia and

Transport

toxic respiratory byproducts (i.e., NO, CO) trigger dormancy

and metabolic adaptation of M. tuberculosis via DosR. DosR is
cell wall hydrolase)

activated by phosphorylation at D5462 and by phosphorylation

at T198 and T205 by the serine-threonine kinase PknH. DosR
transport)
protein)

phosphorylation induces its dimerization and promotes its

Transport

binding to the promoter of the DosRST regulon.62

Several sets of small molecules have been assayed for their
inhibition of DosR activity63.64 Several small molecules have
Staphylococcus

been described as DosR inhibitors that bind DosR and inhibit

pneumoniae
Streptococcus

its binding to its target DNA sequence.63 These molecules have

aureus

also shown potential antibacterial activity against other

mycobacteria (Table 3).
219 DOI: 10.1021/cb5007135
ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

Figure 4. Antibacterial drug discovery and development pipeline. Different approaches can be used in antibacterial drug discovery (upper part of the
scheme). Key stages in novel drug discovery process are indicated as blue squares. Estimated amount of time of each phase of the antibacterial drug
discovery process are indicated on the left side of the scheme. New business models for R&D to facilitate the development of novel antibacterials are
listed in purple boxes. SBVS, structural based virtual screening; FBDD, fragment based drug discovery; HTS, high-throughput screening.

Figure 5. Specific TCS inhibitors that can be used as a starting point for the design of more potent antibacterial drugs following structure-based drug
design approaches. (A) LED209 inhibits the binding of adrenergic signals to the periplasmic domain of E. coli QseC, preventing its
autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. (B) Thiazolidione derivative was identified
in a structure-based screening for ligands of the CA domain of S. epidermidis WalK. Thiazolidione derivative inhibited S. epidermidis WalK
autophosphorylation (IC50 = 14 μM) and showed antibacterial effect against Gram-positive bacteria with MICs in the range 2−6 μM. Discovery of
thiazolidione derivates as TCSs inhibitors by SBVS demonstrated that this is a viable tool for discovery of HK inhibitors with antibacterial effects.
(C) NSC48630 inhibited the formation of S. enterica PhoP-DNA complex (IC50 = 3.6 μM). NSC4836 was identified as a putative PhoP ligand in a
structure-based screening for ligands of the activated response regulator PhoP.

220 DOI: 10.1021/cb5007135

ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

■ CHALLENGES AND FUTURE PERSPECTIVES FOR

THE DISCOVERY AND DEVELOPMENT OF
There are several ways to improve TCS inhibitor discovery
and drug design. First, solving the structure of model proteins
ANTIBACTERIALS TARGETING TCSS would facilitate SBVS and as a consequence provide proteins
for the library screening approach. These structures should
TCSs are considered attractive drug targets and to date around preferably come from TCS targets in clinically important
a hundred TCS inhibitors belonging to different chemical pathogens in order to facilitate the successful identification of
classes have been described in the literature; most of these specific inhibitors. Also, structural data of HKs or RRs
inhibitors target Gram-positive bacteria (Table 3). cocrystallized with inhibitors are of special interest, as they
So far, two approaches have been used for TCS inhibitor will lead to the discovery of the binding site of the inhibitor, as
discovery: (1) HTS assays with purified model TCSs and well as visualizing the chemical space, which might allow for a
different types of compound libraries and (2) SBVS with further refinement of target binding by novel inhibitors. Indeed,
different TCS structures or structural models. Of special more structure−activity relationship data are needed to reduce
relevance have been the screens for TCS inhibitors (mainly toxicity of TCS inhibitors in eukaryotic cells. All these steps will
WalKR) following classical screening methods with secondary lead to a better understanding of the mechanism of action of
metabolites from soil Streptomyces spp. These screens have led TCS inhibitors. Second, SBVS libraries of compounds should
to the discovery of walkmycin B, singermycin, and contain more diverse structures of potential inhibitors as
waldiomycin. To this date, phenotypic screening of Streptomyces proposed in general for antibacterial drug research.15 In general
secondary metabolites has been the most successful. Addition- terms, a search for novel compounds should not strictly use the
ally, one compound produced by Penicillium XR770 showed Lipinski-rule-of-five as most antibiotics and drugs that are used
inhibition of the NRII (or NtrB) HK from E. coli but had no successfully to treat cancer or central nervous system disorders
activity against whole cell bacteria.65 do not follow the Lipinski rule.15,72 Libraries of compounds
Alternative approaches to target-based screening with should be diversified and specifically prepared to be used in
chemical or natural product libraries are structure based virtual TCS screening, and it may be useful to remove false-positives
screening (SBVS) and rational drug design following SBVS or and analogous compounds from drug discovery libraries.70
fragment-based screening (Figure 4). SBVS has identified a Over the past decade, research aimed at finding novel TCS
range of TCS inhibitors. In SBVS targeted against TCSs, the inhibitors has dwindled. Shifting priorities in the pharmaceut-
most successful screens have found inhibitors that target the ical industry as a consequence of high financial risks and low
conserved catalytic ATP-binding CA domain of HKs.66,67 The return on long-term (10−15 years) investment compared to,
HK dimerization domains have also been proposed as good for instance, anticancer drugs, and the high technical complex-
drug targets for TCSs inhibitors.67 Of particular interest are the ity in antibacterial drug discovery may have been responsible
thiazolidinone derivatives, which were targeted to the CA for the observed decrease in screening for TCS inhibitors. With
domain of WalKR and have antibacterial activity toward the prospect of returning to a preantibiotic era, many public−
S. epidermidis (Figure 5). These hit compounds from SBVS private partnerships have been initiated to aid TCS inhibitor
were further modified with different halogen groups - six of research.73,74
them showed reduced the MICs for S. epidermidis and lower Based on research developments, we propose that TCSs
toxicity in animal kidney cells.68 remain promising antimicrobial drug targets. Around 20
Although the TCSs inhibitors published to date still need to independent research groups from both academia and industry
be improved and better characterized, several of them might be have shown promising preliminary results and proof-of-concept
developed into more potent inhibitors (Figure 5; Table 3) for studies. The role of TCSs in antimicrobial resistance and
instance, hexapeptides.69 It is unfortunate that several inhibitors virulence regulation as well as their essentiality for bacterial
of HK autophosphorylation or phosphotransfer such as RWJ- growth and survival make them attractive topics for future
49815, inhibit TCS through nonspecific protein aggregation.70 collaborative research between academia and industry.
Some benzimidazole TCS inhibitors induce bacterial death
mainly via membrane damage.71 TCS kinase inhibitors may
also have cytotoxic or hemolytic activity in human cells,
■ AUTHOR INFORMATION
Corresponding Author
including RWJ-49815 (mentioned above) and cyclohex- *E-mail: jerry.wells@wur.nl.
enes.70,71 Moreover, initial biochemical screens for TCS Notes
inhibitors may identify a high number of inhibitors acting The authors declare no competing financial interest.

■
through nonspecific inhibitory mechanisms,71 yielding a large
number of compounds that cannot be further developed into ACKNOWLEDGMENTS
drugs.
To develop inhibitors with higher specificity for selected All authors gratefully acknowledge the financial support of EU
FP7 Marie Curie ITN grant no. 238490. A.E.B. and N.V. were
TCS such as WalKR we need more molecular structures of the
Marie Curie Fellows. A.E.B. thanks J. Blaazer for fruitful
HK domains from relevant pathogens.66 Already some chemical
discussions on the manuscript.

■
scaffolds that interact with conserved motifs of the CA domain
of HKs have been proposed.66 For SBVS, it is also important to REFERENCES
include protein flexibility data and protein interactions with
water molecules. SBVS has the advantage of screening much (1) Infectious Diseases Society of America. (2004) Bad Bugs, No
Drugs: As Antibiotic R&D Stagnates, a Public Health Crisis Brews; IDSA,
higher numbers of chemical compounds in shorter time than Alexandria, VA.
can be achieved by conventional methods. However, SBVS also (2) Lewis, K. (2013) Platforms for antibiotic discovery. Nat. Rev.
results in many false positive hits, necessitating whole-cell Drug Discovery 12, 371−387.
phenotypic screening methods and biochemical assays to (3) Silver, L. L. (2011) Challenges of antibacterial discovery. Clin.
validate the mechanism of action of hit compounds. Microbiol. Rev. 24, 71−109.

221 DOI: 10.1021/cb5007135

ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

(4) Tralau-Stewart, C. (2014) U.K. academic drug discovery. Nat. (26) Laub, M. T., and Goulian, M. (2007) Specificity in two-
Rev. Drug Discovery, 15−16. component signal transduction pathways. Annu. Rev. Genet. 41, 121−
(5) Bragginton, E. C., and Piddock, L. J. (2014) U.K. and European 145.
Union public and charitable funding from 2008 to 2013 for (27) Howell, A., Dubrac, S., Andersen, K. K., Noone, D., Fert, J.,
bacteriology and antibiotic research in the U.K.: An observational Msadek, T., and Devine, K. (2003) Genes controlled by the essential
study. Lancet Infect. Dis. 14, 857−868. YycG/YycF two-component system of Bacillus subtilis revealed
(6) Gwynn, M. N., Portnoy, A., Rittenhouse, S. F., and Payne, D. J. through a novel hybrid regulator approach. Mol. Microbiol. 49,
(2010) Challenges of antibacterial discovery revisted. Ann. N.Y. Acad. 1639−1655.
Sci. 123, 5−19. (28) Dubrac, S., and Msadek, T. (2004) Identification of genes
(7) O’Shea, R., and Moser, H. E. (2008) Physicochemical properties controlled by the essential YycG/YycF two-component system of
of antibacterial compounds: Implications for drug discovery. J. Med. Staphylococcus aureus. J. Bacteriol. 186, 1175−1181.
Chem. 51, 2871−2878. (29) Dubrac, S., Boneca, I. G., Poupel, O., and Msadek, T. (2007)
(8) Singh, S., and Barret, J. F. (2006) Empirical antibacterial drug New insights into the WalK/WalR (YycG/YycF) essential signal
discoveryFoundation in natural products. Biochem. Pharmacol. 71, transduction pathway reveal a major role in controlling cell wall
1006−1015. metabolism and biofilm formation in Staphylococcus aureus. J. Bacteriol.
(9) Anne, J., Maldonado, B., Impe, J. v., Mellaert, L. v., and Bernaerts, 189, 8257−8269.
K. (2012) Recombinant protein production and streptomycetes. J. (30) Bisicchia, P., Bui, N. K., Aldridge, C., Vollmer, W., and Devine,
Bacteriol. 158, 159−167. K. M. (2011) Acquisition of VanB-type vancomycin resistance by
(10) Simmons, K. J., Chopra, I., and Fishwisk, C. W. G. (2010) Bacillus subtilis: The impact on gene expression, cell wall composition
Structure-based discovery of antibacterial drugs. Nat. Rev. Microbiol. 8, and morphology. Mol. Microbiol. 81, 157−178.
501−510. (31) Jansen, A., Türck, M., Szekat, C., Nagel, M., Clever, I., and
(11) Scott, D. E., Coyne, A. G., Hudson, S. A., and Abell, C. (2015) Bierbaum, G. (2007) Role of insertion elements and yycFG in the
Fragment-based approaches in drug discovery and chemical biology. development of decreased susceptibility to vancomycin in Staph-
Biochemistry 51, 4990−5003. ylococcus aureus. Int. J. Med. Microbiol. 297, 205−215.
(12) Chalker, A., and Lunsford, R. D. (2002) Rational identification (32) Depardieu, F., Podglajen, I., Leclercq, R., Collatz, E., and
of new antibacterial drug targets that are essential for viability using a Courvalin, P. (2007) Modes and modulations of antibiotic resistance
genomics-based approach. Pharmacol. & Ther. 95, 1−20. gene expression. Clin. Microbiol. Rev. 20, 79−114.
(13) Dutta, R., and I, M. (2000) GHKL, an emergent ATPase/kinase (33) Dieppois, G., Ducret, V., Caille, O., and Perron, K. (2012) The
superfamily. Trends Biochem. Sci. 25, 24−28. transcriptional regulator CzcR modulates antibiotic resistance and
(14) Tanaka, T., S, S., Tomomori, C., Ishima, R., Liu, D., Tong, K. I., quorum sensing in Pseudomonas aeruginosa. PloS One 7, No. e38148.
Park, H., Dutta, R., Qin, L., Swindells, M. B., Yamazaki, T., Ono, A. M., (34) Srinivasan, V., Venkataramaiah, M., Mondal, A., Vaidyanathan,
Kainosho, M., Inouye, M., and Ikura, M. (1998) NMR structure of the V., Govil, T., and Rajamohan, G. (2012) Functional characterization of
a novel outer membrane porin KpnO, regulated by PhoBR two-
histidine kinase domain of the E. coli osmosensor EnvZ. Nature 396,
component system in Klebsiella pneumoniae NTUH-K2044. PloS One
88−92.
7, No. e41505.
(15) Payne, D. J., Gwynn, M. N., Holmes, D. J., and Pompliano, D. L.
(35) Gossage, L., and Eisen, T. (2010) Targeting multiple kinase
(2007) Drugs for bad bugs: Confronting the challenges of antibacterial
pathways: A change in paradigm. Clin. Cancer Res. 16, 1973−1978.
discovery. Nat. Rev. Drug Discovery 6, 29−40.
(36) Hopkins, A. L. (2008) Network pharmacology: The next
(16) Velikova, N., Bem, A. E., van Baarlen, P., Wells, J. M., and
paradigm in drug discovery. Nat. Chem. Biol. 4, 682−690.
Marina, A. (2013) WalK, the path towards new antibacterials with low (37) Pokrovskaya, V., and Baasov, T. (2010) Dual-acting hybrid
potential for resistance development. ACS Med. Chem. Lett. 4, 891− antibiotics: A promising strategy to combat bacterial resistance. Expert
894. Opin. Drug Discovery 5, 883−902.
(17) Thomason, P., and Kay, R. (2000) Eukaryotic signal (38) Guarnieri, M. T., Zhang, L., Shen, J., and Zhao, R. (2008) The
transduction via histidine-aspartate phosphorelay. J. Cell Sci. 113, Hsp90 inhibitor radicicol interacts with the ATP-binding pocket of
3141−3150. bacterial sensor kinase PhoQ. J. Mol. Biol. 379, 82−93.
(18) Casino, P., Rubio, V., and Marina, A. (2010) The mechanism of (39) Casino, P., Rubio, V., and Marina, A. (2009) Structural insight
signal transduction by two-component systems. Curr. Opin. Struct. Biol. into partner specificity and phosphoryl transfer in two-component
20, 763−771. signal transduction. Cell 139, 325−336.
(19) Kenney, L. J. (2010) How important is the phosphatase activity (40) World Health Organization (2014) Antimicrobial Resistance:
of sensor kinases? Curr. Opin. Microbiol. 13, 168−176. Global Report on Surveillance, pp 1−232, WHO Press, Geneva.
(20) Gao, R., and Stock, A. M. (2010) Molecular strategies for (41) Healy, V. L., Lessard, I. A., Roper, D. I., Knox, J. R., and Walsh,
phosphorylation-mediated regulation of response regulator activity. C. T. (2000) Vancomycin resistance in enterococci: Reprogramming
Curr. Opin. Microbiol. 13, 160−167. of the D-Ala-D-Ala ligases in bacterial peptidoglycan biosynthesis.
(21) Yamada, S., and Shiro, Y. (2008) Structural basis of the signal Chem. Biol. 7, R109−R119.
transduction in the two-component system. Adv. Exp. Med. Biol. 631, (42) Pootoolal, J., Neu, J., and Wright, G. D. (2002) Glycopeptide
22−39. antibiotic resistance. Annu. Rev. Pharmacol. Toxicol. 42, 381−408.
(22) Djordjevic, S., Goudreau, P. N., Xu, Q., Stock, A. M., and West, (43) Hong, H.-J., Hutchings, M. I., Buttner, J. M. (2008) Vancomycin
A. H. (1998) Structural basis for methylesterase CheB regulation by a resistance VanS/VanR two-component systems. In Bacterial Signal
phosphorylation-activated domain. Proc. Natl. Acad. Sci. U.S.A. 95, Transduction: Networks and Drug Targets (Utsumi, R., Ed.) Springer
1381−1386. Science+Buisness Media, New York.
(23) Lee, S. Y., De La Torre, A., Yan, D., Kustu, S., Nixon, B. T., and (44) Macielag, M. J., Demers, J. P., Fraga-Spano, S. A., Hlasta, D. J.,
Wemmer, D. E. (2003) Regulation of the transcriptional activator Johnson, S. G., Kanojia, R. M., Russell, R. K., Sui, Z., Weidner-Wells,
NtrC1: Structural studies of the regulatory and AAA+ ATPase M. A., Werblood, H., Foleno, B. D., Goldschmidt, R. M., Loeloff, M. J.,
domains. Genes Dev. 17, 2552−2563. Webb, G. C., and Barrett, J. F. (1998) Substituted salicylanilides as
(24) Stock, A., Robinson, V. L., and Goudrea, P. N. (2000) Two- inhibitors of two-component regulatory systems in bacteria. J. Med.
component signal transduction. Annu. Rev. Biochem. 69, 183−215. Chem. 41, 2939−2945.
(25) Parashar, V., Mirouze, N., Dubnau, D. A., and Neiditch, M. B. (45) Delaune, A., Dubrac, S., Blanchet, C., Poupel, O., Mader, U.,
(2011) Structural basis of response regulator dephosphorylation by Hiron, A., Leduc, A., Fitting, C., Nicolas, P., Cavaillon, J. M., Adib-
Rap phosphatases. Plos Biol. 9, No. e1000589. Conquy, M., and Msadek, T. (2012) The WalKR system controls

222 DOI: 10.1021/cb5007135

ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

major staphylococcal virulence genes and is involved in triggering the and Mycobacterium tuberculosis dormancy. Infect. Immun. 77, 3258−
host inflammatory response. Infect. Immun. 80, 3438−3453. 3263.
(46) Howden, B. P., McEvoy, C. R., Allen, D. L., Chua, K., Gao, W., (61) Leistikov, R., Morton, R. A., Bartek, I. L., Frimpong, I., Wagner,
Harrison, P. F., Bell, J., Coombs, G., Bennett-Wood, V., Porter, J. L., K., and Voskuil, M. I. (2010) The Mycobacterium tuberculosis DosR
Robins-Browne, R., Davies, J. K., Seemann, T., and Stinear, T. P. regulon assists in metabolic homeostasis and enables rapid recovery
(2011) Evolution of multidrug resistance during Staphylococcus aureus from nonrespiring dormancy. J. Bacteriol. 192, 1662−1667.
infection involves mutation of the essential two component regulator (62) Chauhan, S., and Tyagi, J. S. (2008) Cooperative binding of
WalKR. PLoS Pathog. 7, e1002359. phosphorylated DevR to upstream sites is necessary and sufficient for
(47) Mohedano, M. L., Overweg, K., de la Fuente, A., Reuter, M., activation of the Rv3134c-devRS operon in Mycobacterium tuberculosis:
Altabe, S., Mulholland, F., de Mendoza, D., Lopez, P., and Wells, J. M. Implication in the induction of DevR target genes. J. Bacteriol. 190,
(2005) Evidence that the essential response regulator YycF in 4301−4312.
Streptococcus pneumoniae modulates expression of fatty acid biosyn- (63) Gupta, R., Thakur, T. S., Desiraju, G. R., and Tyagi, J. S. (2009)
thesis genes and alters membrane composition. J. Bacteriol. 187, 2357− Structure-based design of DevR inhibitor active against nonreplicating
2367. Mycobacterium tuberculosis. J. Med. Chem. 52, 6324−6334.
(48) Cafiso, V., Bertuccio, T., Spina, D., Purrello, S., Campanile, F., (64) Mai, D., Jones, J., Rodgers, J. W., Hartman, J. L., Kutsch, O., and
Di Pietro, S., Purrello, M., and Stefani, S. (2012) Modulating activity of Steyn, A. J. (2011) A screen to identify small molecule inhibitors of
vancomycin and daptomycin on the expression of autolysis cell-wall protein−protein interactions in mycobacteria. Assay Drug Dev. Technol.
turnover and membrane charge genes in hVISA and VISA strains. PloS 9, 299−310.
One 7, No. e29573. (65) Brewer, S. (2000) Biodiversity: New Leads for the Pharmaceutical
(49) Howden, B. P., Peleg, A. Y., and Stinear, T. P. (2013) The and Agrochemical Industries (Wrigley, S. K., Hayes, M. A., Thomas, R.,
evolution of vancomycin intermediate Staphylococcus aureus (VISA) Crystal, E. J. T., Nicholson, N., Ed.), pp 59−65, The Royal Society of
and heterogenous-VISA. Infect., Genet. Evol., 575−582. Chemistry, Cambridge.
(50) McEvoy, C. R. (2013) Decreased vancomycin susceptibility in (66) Francis, S., Wilke, K. E., Brown, D. E., and Carlson, E. E. (2013)
Staphylococcus aureus caused by IS256 tempering of WalKR expression. Mechanistic insight into inhibition of two-component system
Antimicrob. Agents Chemother. 57, 3240−3249. signaling. MedChemComm 4, 269−277.
(51) Mwangi, M., Wu, S. W., Zhou, Y., Sieradzki, K., de Lencastre, H., (67) Igarashi, M., Watanabe, T., Hashida, T., Umekita, M., Hatano,
Richardson, P., Bruce, D., Rubin, E., Myers, E., Siggia, E. D., and M., Yanagida, Y., Kino, H., Kimura, T., Kinoshita, N., Inoue, K., Sawa,
Tomasz, A. (2007) Tracking the in vivo evolution of multidrug R., Nishimura, Y., Utsumi, R., and Nomoto, A. (2013) Waldiomycin, a
resistance in Staphylococcus aureus by whole-genome sequencing. Proc. novel WalK-histidine kinase inhibitor from Streptomyces sp. MK844-
Natl. Acad. Sci. U.S.A. 104, 9451−9456. mF10. J. Antibiot. 66, 459−464.
(52) Qin, Z., Zhang, J., Xu, B., Chen, L., Wu, Y., Yang, X., Shen, X., (68) Liu, H., Zhao, D., Chang, J., Yan, L., Zhao, F., Wu, Y., Xu, T.,
Molin, S., Danchin, A., Jiang, H., and Qu, D. (2006) Structure-based Gong, T., Chen, L., He, N., Wu, Y., Han, S., and Qu, D. (2014)
discovery of inhibitors of the YycG histidine kinase: New chemical Efficacy of novel antibacterial compounds targeting histidine kinase
leads to combat Staphylococcus epidermidis infections. BMC Microbiol. YycG protein. Appl. Microbiol. Biotechnol. 98, 6003−6013.
6, 96. (69) Roychoudhury, S., Blondelle, S. E., Collins, S. M., Davis, M. C.,
(53) Okada, A., I, M., Okajima, T., Kinoshita, N., Umekita, M., Sawa, McKeever, H. D., Houghten, R. A., and Parker, C. N. (2000) Use of
R., Inoue, K., Watanabe, T., Doi, A., Martin, A., Quinn, J., Nishimura, combinatorial library screening to identify inhibitors of a bacterial two-
Y., and Utsumi, R. (2010) Walkmycin B targets WalK (YycG), a component signal transduction kinase. Mol. Diversity 4, 173−182.
histidine kinase essential for bacterial cell growth. J. Antibiot. 63, 89− (70) Stephenson, K., Yamaguchi, Y., and Hoch, J. A. (2000) The
94. mechanism of action of inhibitors of bacterial two-component signal
(54) Eguchi, Y., Kubo, N., Matsunaga, H., Igarashi, M., and Utsumi, transduction systems. J. Biol. Chem. 275, 38900−38904.
R. (2011) Development of an antivirulence drug against Streptococcus (71) Hilliard, J. J., Goldschmidt, R. M., Licata, L., Baum, E. Z., and
mutans: Repression of biofilm formation, acid tolerance, and Bush, K. (1999) Multiple mechanisms of action for inhibitors of
competence by a histidine kinase inhibitor, walkmycin C. Antimicrob. histidine protein kinases from bacterial two-component systems.
Agents Chemother. 55, 1475−1484. Antimicrob. Agents Chemother. 43, 1693−1699.
(55) Watanabe, T., Igarashi, M., Okajima, T., Ishii, E., Kino, H., (72) Kell, D. B. (2013) Finding novel pharmaceuticals in the systems
Hatano, M., Sawa, R., Umekita, M., Kimura, T., Okamoto, S., Eguchi, biology era using multiple effective drug targets, phenotypic screening
Y., Akamatsu, Y., and Utsumi, R. (2012) Isolation and characterization and knowledge of transporters: Where drug discovery went wrong and
of signermycin B, an antibiotic that targets the dimerization domain of how to fix it. FEBS J. 280, 5957−5980.
histidine kinase WalK. Antimicrob. Agents Chemother. 56, 3657−3663. (73) Projan, S., and Steven, J. (2003) Why is Big Pharma getting out
(56) Clarke, M. B., Hughes, D. T., Zhu, C., Boedeker, E. C., and of antibacterial drug discovery? Curr. Opin. Microbiol. 6, 427−430.
Sperandio, V. (2006) The QseC sensor kinase: A bacterial adrenergic (74) So, A. D., Gupta, N., Brahmachari, S. K., Chopra, I., Munos, B.,
receptor. Proc. Natl. Acad. Sci. U.S.A. 103, 10420−10425. Nathan, C., Outterson, K., Paccaud, J. P., Payne, D. J., Peeling, R. W.,
(57) Rasko, D. A., Moreira, C. G., Li de, R., Reading, N. C., Ritchie, J. Spigelman, M., and Weigelt, J. (2011) Towards new business models
M., Waldor, M. K., Williams, N., Taussig, R., Wei, S., Roth, M., for R&D for novel antibiotics. Drug Resist. Updates 14, 88−94.
Hughes, D. T., Huntley, J. F., Fina, M. W., Falck, J. R., and Sperandio, (75) Hiron, A., Valle, F. M., Debarbouille, M., and Msadek, T. (2011)
V. (2008) Targeting QseC signaling and virulence for antibiotic Bacitracin and nisin resistance in Staphylococcus aureus: A novel
development. Science 321, 1078−1080. pathway involving the BraS/BraR two-component system (SA2417/
(58) Raman, K., Yeturu, K., and Chandra, N. (2008) targetTB: A SA2418) and both the BraD/BraE and VraD/VraE ABC transporters.
target identification pipeline for Mycobacterium tuberculosis through an Mol. Microbiol. 81, 602−622.
interactome, reactome, and genome-scale structural analysis. BMC Syst. (76) Fridman, M., W, G., Muzamal, U., Hunter, H., Siu, K. W., and
Biol. 2, 1−21. Golemi-Kotra, D. (2013) Two unique phosphorylation-driven signal-
(59) Park, H., Guinn, K. M., Harrell, M. I., Liao, R., Voskuil, M. I., ing pathways crosstalk in Staphylococcus aureus to modulate the cell-
Tompa, M., Schoolnik, G. K., and Sherman, D. R. (2003) Rv3133c/ wall charge: Stk1/Stp1 meets GraSR. Biochemistry 52, 7995−7986.
dosR is a transcription factor that mediates the hypoxic response of (77) Srinivasan, V., Mondal, A., Venkataramaiah, M., Chauhan, N. K.,
Mycobacterium tuberculosis. Mol. Microbiol. 48, 833−843. and Rajamohan, G. (2013) Role of oxyRKP, a novel LysR-family
(60) Honaker, R., Leistikow, R. L., Bartek, I. L., and Voskuil, M. I. transcriptional regulator, in antimicrobial resistance and virulence in
(2009) Unique roles of DosT and DosS in DosR regulon induction Klebsiella pneumoniae. Microbiology, 1301−1314.

223 DOI: 10.1021/cb5007135

ACS Chem. Biol. 2015, 10, 213−224
ACS Chemical Biology Reviews

(78) Srinivasan, V., Venkataramaiah, M., Mondal, A., Vaidyanathan, (93) Kanoija, R. M., Murray, W., Bernstein, J., Fernandez, J., Foleno,
V., Govil, T., and Rajamohan, G. (2012) Role of the two component B. D., Krause, H., Lawrence, L., Webb, G., and Barret, J. F. (1999) 6-
signal transduction system CpxAR in conferring cefepime and Oxa isosteres of anacardic acids as potent inhibitors of bacterial
chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044. histidine protein kinase (HPK)-mediated two-component regulatory
PloS One 7, 1−15. systems. Bioorg. Med. Chem. Lett. 9, 2947−2952.
(79) Marchand, I. (2004) Expression of the RND-type efflux pump (94) Hlasta, D. J., Demers, J. P., Foleno, B. D., Fraga-Spano, S. A.,
AdeABC in Acinetobacter baumannii is regulated by the AdeRS two- Guan, J., Hilliard, J. J., Macielag, M. J., Ohemeng, K. A., Sheppard, C.
component system. Antimicrob. Agents Chemother. 48, 3298−3304. M., Sui, Z., Webb, G. C., Weidner-Wells, M. A., Werblood, H., and
(80) Wei Q, T. S., Dötsch, A., Häussler, S., Müsken, M., Wright, V. J., Barrett, J. F. (1998) Novel inhibitors of bacterial two-component
Cámara, M., Williams, P., Haenen, S., Boerjan, B., Bogaerts, A., systems with Gram-positive antibacterial activity: Pharmacophore
Vierstraete, E., Verleyen, P., Schoofs, L., Willaert, R., De Groote, V. N., identification based on the screening hit closantel. Bioorg. Med. Chem.
Michiels, J., Vercammen, K., Crabbé, A., and Cornelis, P. (2011) Lett. 8, 1923−1928.
Phenotypic and genome-wide analysis of an antibiotic-resistant small (95) Gilmour, R., Foster, J. E., Sheng, Q., McClain, J. R., Riley, A.,
colony variant (SCV) of Pseudomonas aeruginosa. PloS One 6 (12), Sun, P. M., Ng, W. L., Yan, D., Nicas, T. I., Henry, K., and Winkler, M.
No. e29276. E. (2005) New class of competitive inhibitor of bacterial histidine
(81) Nguyen HT, W. K., Cartabuke, R. H., Ogwang, S., and Nguyen, kinases. J. Bacteriol. 187, 8196−8200.
L. (2010) A lipoprotein modulates activity of the MtrAB two- (96) Foster, J., Sheng, Q., McClain, J. R., Bures, M., Nicas, T. I.,
component system to provide intrinsic multidrug resistance, Henry, K., Winkler, M. E., and Gilmour, R. (2004) Kinetic and
cytokinetic control, and cell wall homeostasis in Mycobacterium. mechanistic analyses of new classes of inhibitors of two-component
Mol. Microbiol. 76, 348−364. signal transduction systems using a coupled assay containing HpkA-
(82) Comenge, Y., Quintiliani, R., Jr, Li, L., Dubost, L., Brouard, J. P., DrrA from Thermotoga maritima. Microbiology 150, 885−896.
Hugonnet, J. E., and Arthur, M. (2003) The CroRS two-component (97) Tang, Y. T., Gao, R., Havranek, J. J., Groisman, E. A., Stock, A.
regulatory system is required for intrinsic β-lactam resistance in M., and Marshall, G. R. (2012) Inhibition of bacterial virulence: Drug-
Enterococcus faecalis. J. Bacteriol. 185, 184−192. like molecules targeting the Salmonella enterica PhoP response
(83) Guerrero P, C. B., Á lvarez, R., Salinas, H., Morales, E. H., regulator. Chem. Biol. Drug Des. 79, 1007−1017.
Calderón, I. L., Saavedra, C. P., and Gil, F. (2013) Salmonella enterica (98) Qin, Z., Zhang, J., Xu, B., Chen, L., Wu, Y., Yang, X., Shen, X.,
serovar Typhimurium BaeSR two-component system positively Molin, S., Danchin, A., Jiang, H., and Qu, D. (2006) Structure-based
regulates sodA in response to ciprofloxacin. Microbiology 159, 2049− discovery of inhibitors of the YycG histidine kinase: New chemical
2057. leads to combat Staphylococcus epidermidis infections. BMC Microbiol.
(84) Baranova, N. a., and N, H. (2002) The baeSR two-component 6, 96.
regulatory system activates transcription of the yegMNOB (99) Sui, Z., Guan, J., Hlasta, D. J., Macielag, M. J., Foleno, B. D.,
(mdtABCD) transporter gene cluster in Escherichia coli and increases Goldschmidt, R. M., Loeloff, M. J., Webb, G. C., and Barret, J. F.
its resistance to novobiocin and deoxycholate. J. Bacteriol. 184, 4168− (1998) Studies of diaryltiazoles against bacterial two-componen
4176. regulatory systems and their antibacterial activities. Bioorg. Med.
(85) Nagakubo, S., Nishino, K., Hirata, T., and Yamaguchi, A. (2002) Chem. Lett. 8, 1929−1934.
The putative response regulator BaeR stimulates multidrug resistance (100) Klitgaard, J. K., Skov, M. N., Kallipolitis, B. H., and Kolmos, H.
of Escherichia coli via a novel multidrug exporter system, MdtABC. J. J. (2008) Reversal of methicillin resistance in Staphylococcus aureus by
Bacteriol. 184, 4161−4167. thioridazine. J. Antimicrob. Chemother. 62, 1215−1221.
(86) Deng, Z., Shan, Y., Pan, Q., Gao, X., and Yan, A. (2013) (101) Bonde, M., Hojland, D. H., Kolmos, H. J., Kallipolitis, B. H.,
Anaerobic expression of the gadE-mdtEF multidrug efflux operon is and Klitgaard, J. K. (2011) Thioridazine affects transcription of genes
primarily regulated by the two-component system ArcBA through involved in cell wall biosynthesis in methicillin-resistant Staphylococcus
antagonizing the H-NS mediated repression. Front. Microbiol. 4, 194. aureus. FEMS Microbiol. Lett. 318, 168−176.
(87) Hirakawa, H., Nishino, K., Hirata, T., and Yamaguchi, A. (2003) (102) Kunze, B., Reck, M., Dotsch, A., Lemme, A., Schummer, D.,
Comprehensive studies of drug resistance mediated by overexpression Irschik, H., Steinmetz, H., and Wagner-Dobler, I. (2010) Damage of
of response regulators of two-component signal transduction systems Streptococcus mutans biofilms by carolacton, a secondary metabolite
in Escherichia coli. J. Bacteriol. 185, 1851−1856. from the myxobacterium Sorangium cellulosum. BMC Microbiol. 10,
(88) Li, X., Zhang, L., and Poole, K. (2002) SmeC, an outer 199.
membrane multidrug efflux protein of Stenotrophomonas maltophilia. (103) Reck, M., Rutz, K., Kunze, B., Tomasch, J., Surapaneni, S. K.,
Antimicrob. Agents Chemother. 46, 333−343. Schulz, S., and Wagner-Dobler, I. (2011) The biofilm inhibitor
(89) Roychoudhury, S., Zielinski, N. A., Ninfa, A. J., Allen, N. E., carolacton disturbs membrane integrity and cell division of
Jungheim, L. N., Nicas, T. I., and Chakrabarty, A. M. (1993) Inhibitors Streptococcus mutans through the serine/threonine protein kinase
of two-component signal transduction systems: Inhibiton of alginate PknB. J. Bacteriol. 193, 5692−5706.
gene activation in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U.S.A.
90, 965−969.
(90) Ulijasz, A. T., and W, B. (1999) Dissecting the VanSR signal
transduction pathway with specific inhibitors. J. Bacteriol. 181, 627−
631.
(91) Weidner-Wells, M. A., Ohemeng, K. A., Nguyen, V. N., Fraga-
Spano, S., Macielag, M. J., Werblood, H. M., Foleno, B. D., Webb, G.
C., Barret, J. F., and Hlasta, D. J. (2001) Amidino benzimidazole
inhibitors of bacterial two-component systems. Bioorg. Med. Chem.
Lett. 11, 1545−1548.
(92) Barret, J. F., Goldschmidt, R. M., Lawrence, L. E., Foleno, B.,
Chen, R., Demers, J. P., Johnson, S., Kanojia, R., Fernandez, J.,
Bernstein, J., Licata, L., Donetz, A., Huang, S., Hlasta, D. J., Macielag,
M. J., Ohemeng, K., Frechette, R., Frosco, M. B., Klaubert, D. H.,
Whiteleey, J. M., Wang, L., and Hoch, J. A. (1998) Antibacterial agents
that inhibit two-component signal transduction systems. Proc. Natl.
Acad. Sci. U.S.A. 95, 5317−5322.

224 DOI: 10.1021/cb5007135

ACS Chem. Biol. 2015, 10, 213−224