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REVIEW
Next-generation antimicrobials: from chemical biology
to first-in-class drugs
Michelle Lay Teng Ang
1
•Paul Murima
2
•Kevin Pethe
1
Received: 16 April 2015 / Accepted: 29 July 2015
ÓThe Author(s) 2015. This article is published with open access at Springerlink.com
Abstract The global emergence of multi-drug resistant
bacteria invokes an urgent and imperative necessity for the
identification of novel antimicrobials. The general lack of
success in progressing novel chemical entities from target-
based drug screens have prompted calls for radical and
innovative approaches for drug discovery. Recent devel-
opments in chemical biology and target deconvolution
strategies have revived interests in the utilization of whole-
cell phenotypic screens and resulted in several success
stories for the discovery and development novel drug
candidates and target pathways. In this review, we present
and discuss recent chemical biology approaches focusing
on the discovery of novel targets and new lead molecules
for the treatment of human bacterial and protozoan
infections.
Keywords Drug discovery Antimicrobials First-in-
class drugs Screening Lead optimization Chemical
biology
Introduction
Since the dawn of the genomics era, the main focus of drug
discovery has been on targeting cellular processes or
defined enzymes that have a key role in disease patho-
genesis (Hopkins and Groom 2002; Vander Heiden 2011).
The target-centric approach enabled the testing of a specific
biological hypothesis and the rational design of drug can-
didates to modulate it. Although functional genomics has
transformed how antibacterial drug discovery is approa-
ched, very few genomics-driven compounds are currently
in clinical development. Target-based drug discovery is
overly reductionist in concept, reducing the drug–organism
relationship to inordinate drug-target interplay. This dis-
sociation from bacterial systems pharmacology is in con-
trast to the integrated network response to perturbation that
microbes are endowed with.
Prior to the introduction of biochemical screening that
emerged after sequencing of most human pathogens, drug
discovery was driven empirically by a cell-based approach,
abiding by the mantra of ‘‘selectively kill the bacteria first
and understand why later’’. These antibiotics, such as para-
aminosalicylic acid (PAS) and pyrazinamide (PZA), were
introduced to clinical practice with limited knowledge
about the mechanism of action.
Although the field was successful in the discovery and
development of the current antibacterial armamentarium
(Fischbach and Walsh 2009), the limited use of empiric
whole cell screening approach in recent years has con-
tributed in part to the current dearth of new antibacterials
(Sams-Dodd 2005; Fischbach and Walsh 2009; Dick and
Young 2011).
A consequential impediment of empiric cell based
screening is that the target protein remains unknown,
making lead optimization steps more time-consuming and
&Michelle Lay Teng Ang
michelle.ang@ntu.edu.sg
Kevin Pethe
kevin.pethe@ntu.edu.sg
1
Lee Kong Chian School of Medicine and School of
Biological Sciences, Nanyang Technological University, 30
Biopolis Street, #B2-15a, Singapore 138671, Singapore
2
Global Health Institute, Swiss Federal Institute of
Technology in Lausanne (EPFL), 1015 Lausanne,
Switzerland
123
Arch. Pharm. Res.
DOI 10.1007/s12272-015-0645-0
challenging (Plouffe et al. 2008; Swinney 2013). Never-
theless, progress in chemical biology and target deconvo-
lution strategies has revived high-throughput whole-cell
phenotypic screening (Terstappen et al. 2007), whilst
exploiting cutting-edge technologies to accelerate the dis-
covery and development of novel antimicrobials (Andries
et al. 2005; Makarov et al. 2009; Rottmann et al. 2010;
Pethe et al. 2013; Ling et al. 2015). This has culminated in
the discovery of several first-in-class medicines with novel
molecular mechanism of actions (MMOA). Automation
and miniaturization of cellular screening systems enable
the collation of unprecedented amounts of data for a single
small molecule across a diverse collection of cellular
screens, thus providing insights into a compound’s possible
MMOA upon comprehensive evaluation (Plouffe et al.
2008). In fact, a recent analysis suggested phenotypic
screens to be the more successful strategy for the discovery
of first-in-class medicines (Swinney 2013). The rational-
ization for the success of phenotype-based screens was the
unbiased identification of the MMOA.
Given the success of phenotypic screens in delivering new
chemical entities (NCE) inhibiting virgin target space, we
herein review recent chemical biology approaches focusing
on the discovery of novel targets and new lead molecules for
the treatment of human bacterial and protozoan infections.
Chemical biology strategies for protozoan
parasites
Protozoa are a wide group of unicellular organisms that can
be found in multiple ecosystems and are often non-patho-
genic for humans. However, a small subset of protozoa has
evolved as lethal intracellular parasites posing serious global
health challenges. Among the most deadly intracellular
protozoan parasites are those of the genus Plasmodium,
Leishmania and Trypanosoma, which are collectively
responsible for millions of death every year. Due to the
emergence and spread of drug resistance (Dondorp et al.
2009; Tun et al. 2015), treatment options are often limited
with reduced efficacy (Wongsrichanalai and Meshnick
2008). Drug R&D have been largely inexistent for these
three parasites until early 2000’ when initiatives from lead-
ing pharmaceutical industries, largely supported by philan-
thropic and public nonprofit organizations emerged as a new
business model to develop drugs for neglected diseases.
These product development partnerships (PDP) have
brought promising candidates for malaria in a record time.
Plasmodium
There are four species of Plasmodium that cause malaria in
human. Collectively, they are responsible for 584,000
deaths and more than 200 million cases every year. The
most common species are Plasmodium falciparum and
Plasmodium vivax. The discovery and development of
KAE609; an investigational drug to treat malaria is the
most notable example of an antimalarial drug resulting
from the effort of a PDP. The spiroindolone KAE609 was
discovered in a phenotypic screen designed to identify
small-molecules that rapidly clear intracellular P. falci-
parum from human red blood cells (Plouffe et al. 2008).
Owing to its high potency in the cellular assay, low toxic
liability and an early proof of efficacy in infected animals
(Rottmann et al. 2010; Yeung et al. 2010), the spiroin-
dolone series was optimized to improve overall properties.
A reverse genetic approach was employed to identify the
Na(?) efflux ATPase PfATP4 as the molecular target of
the spiroindolone series (Rottmann et al. 2010; Spillman
et al. 2013). Having demonstrated a remarkable efficacy in
a phase 2a study at low dose, the optimized clinical can-
didate KAE609 has the potential to revolutionize malaria
treatment (White et al. 2014; Held et al. 2015). The road is
still uncertain before the introduction of KAE609 to clin-
ical practice, but the program is a paradigm of how a PDP
together with international collaborations can revolutionize
the drug discovery process. Besides the discovery of
KAE609, the same phenotypic screen also screen deliver
KAE609, but also the novel class of imidazolopiperazines
(IP) (Meister et al. 2011; Derbyshire et al. 2012), which is
under clinical development (Leong et al. 2014).
A similar forward chemical genetics approach was
conducted by two independent teams to identify hundreds
of novel chemotypes active against drug resistant P. fal-
ciparum (Gamo et al. 2010; Guiguemde et al. 2010). The
release of the chemical structures in the public domain is
yet another example on how open innovation can acceler-
ate drug discovery for neglected diseases. In an elegant
reverse chemical genetics approach targeting 61 proteins
and enzymes of P. falciparum, Crowther et al. identified
the putative target of several promising hits using a thermal
shift assay (Crowther et al. 2009). Even though validation
experiments are yet to be performed to ascertain target
engagement, their preliminary results set a solid foundation
in spearheading translational research. Since the release of
the chemical structures, several lead series have been
evaluated for further evaluation (Sanz et al. 2011; Jimenez-
Diaz et al. 2014; Vaidya et al. 2014). Interestingly, the
promising dihydroisoquinolines compound (?)-SJ733
compound act through PfATP4 to mediate host clearance
of the parasite (Jimenez-Diaz et al. 2014), which is also the
target of mechanism shared with KAE609 (Spillman et al.
2013).
A most challenging subpopulation of Plasmodium to
eradicate is the liver form of the disease. Plasmodium
infect hepatocytes as sporozoites that first undergo a phase
A. L. T. Michelle et al.
123
of maturation before emerging in the blood to cause disease
manifestations. Targeting the early exoerythrocytic form of
the parasite could be prophylactic since it would eradicate
Plasmodium before the onset of the disease. In a technical
tour de force, Meister et al. developed a high-content assay
to quantify replication of P. yeollii inside human hepato-
cytes (Meister et al. 2011). An image-based approach is
particularly well adapted for screening since only 1 % of
the hepatocytes are infected by the parasites. By screening
a collection of more than 4000 commercially available
compounds that have activity against blood stage P. fal-
ciparum, the authors identified several chemicals that kill
exoerythrocytic parasites. The most advanced drug candi-
date GNF179 provided protection against a challenge with
P. berghei sporozoite, demonstrating in vivo activity
against the early-liver stage of the disease. The putative
target for GNF179 is pfcar1, an uncharacterized protein
that is postulated to be involved in protein folding that is
assumed to be essential for the biology of both the liver and
blood stages of Plasmodium infection (Jonikas et al. 2009).
More recently, a team at AstraZeneca utilized another
high-throughput imaging assay to identify 2 more novel
classes of fast-acting antiplasmodial agents; the N-aryl-2-
aminobenzimidazoles, which targets the asexual blood
stages of P. falciparum (Ramachandran et al. 2014), and
the triaminopyrimidines (TAPs), which may potentially be
used for single-dose treatment of malaria when used in
multidrug combination therapy (Hameed et al. 2015).
Exhibiting potent and wide-spectrum antimalarial activity
against multiple life-cycle stages of P. falciparum,
DDD107498 is another optimized lead derived from a 2,
6-disubstituted quinolone-4-carboxamide scaffold previ-
ously identified from a previous phenotypic screen (Bara-
gana et al. 2015).
A key challenge in quest to eradicate malaria is the lack
of potent drugs active against exoerythrocytic Plasmodium
vivax.P. vivax is the most common form of malaria in
South-east Asia. Although less deadly than P. falciparum,
this species can survive in a dormant form as hypnozoites
for extended period of time, leading to recurrent relapse
(Wells et al. 2010). The identification of drug candidates
active against hypnozoites is challenging due to the lack of
predictive ex vivo models amenable to large-scale pheno-
typic screens. (Wells et al. 2010). Recent technical devel-
opments bring about cautious but yet resurgent optimism
for the eventual discovery for an inherent cure for vivax
malaria, through the complete eradication of all forms of
Malaria parasites from the body. Using a low-throughput
assay designed to quantify the number of hypnozoites and
schizont forms of Plasmodium cynomolgi (a model for P.
vivax) inside rhesus hepatocytes, the drug candidate
KAI407 was identified for activity in reducing the forma-
tion of hypnozoites (Zou et al. 2014). In vivo prophylactic
efficacy suggests that KAI407 may indeed represent a
radical cure for vivax malaria. In prospect, the recent
development of transgenic fluorescent P. cynomolgi
(Voorberg-van der Wel et al. 2013), and of platforms that
recapitulate the hepatic stage of P. vivax (Chattopadhyay
et al. 2010; March et al. 2013) will probably be instru-
mental in the coming year to the identification and devel-
opment of drugs targeting P. vivax hypnozoites.
Other kinetoplastids
Parasites of the genus Trypanosoma and Leishmania are
kinetoplastid protozoan parasites that cause trypanosomi-
asis and leishmaniasis, respectively. These diseases,
prevalent in tropical and subtropical countries, cause sig-
nificant morbidity and mortality. No vaccines are available;
and the current chemotherapies available for these
neglected tropical diseases (NTDs) are limited with mul-
tiple shortcomings including potentially severe side effects,
lengthy drug regimens and variable efficacy.
Chagas disease, caused by Trypanosoma cruzi, is the
major cause of heart failure in Latin America. The only 2
available chemotherapies are benznidazole and nifurtimox,
which have been shown to be largely ineffective and toxic,
commonly causing drug resistance (Filardi and Brener
1987; Viotti et al. 2006). Drug R&D for Chagas disease is
challenging due to the complex infection cycle of T. cruzi
that can persist for many years in infected patients as try-
pomastigotes and amastigotes. The availability of engi-
neered reporter gene expressing-parasites (Bettiol et al.
2009; Canavaci et al. 2010) have triggered the develop-
ment of phenotypic assays suitable for HTS, as well as the
establishment of new in vivo protocols (Canavaci et al.
2010; Rodriguez and Tarleton 2012) that allow faster
evaluation of experimental therapeutic options.
Automated high content microscopy approaches (Engel
et al. 2010; Moon et al. 2014) have been used to identify
new parasitic inhibitors. To mimic the intracellular life
cycle of T. cruzi for drug screening, Engel et al. developed
and validated a flexible cell-based, high-throughput
96-well plate assay that could be used with a variety of
untransfected T. cruzi isolates and host cells (Engel et al.
2010). This allowed the simultaneous measurement of both
efficacies against the intracellular amastigote stage and
host cell toxicity. Validation of the HTS assay enabled the
identification of 55 hits upon screening a library of 909
bioactive compounds. Further drug testing narrowed the
list down to 17 compounds that showed at least 5-fold
selectivity between the inhibition of T. cruzi and host cell
toxicity (Engel et al. 2010). Since these confirmed hits
were selected from a library of clinical drugs, they could
potentially be repurposed for T. cruzi treatment and used
for mode of action and target identification studies. In a
Next-generation antimicrobials: from chemical biology to first-in-class drugs
123
similar vein, a team from the Institut Pasteur Korea cus-
tomized a high content image-based and HTS algorithm for
the quantification of infection ratio and intracellular T.
cruzi amastigote in human cell line in response to drug
treatment (Moon et al. 2014). Based solely on DNA
staining and single-channel images, the algorithm precisely
segments and identifies the nuclei and cytoplasm of
mammalian host cells as well as the intracellular parasites
infecting the cells to produce various statistical parameters
that can be used to assess both drug responses and com-
pound cytotoxicity (Moon et al. 2014).
The Genomics Institute of the Novartis Foundation (GNF)
has also initiated a drug discovery program for Chagas disease
using phenotypic screens that measure the inhibition of pro-
liferation of various kinetoplastid parasites to identify hits
among low molecular mass compounds (Bustamante et al.
2011). To date, GNF has screened around 700,000 small
molecules against the bloodstream form of T. brucei and the
Leishmania donovani axenic amastigotes, which yielded
around 2,000 confirmed hits in total. 44 % of these hits were
also found to be active against intracellular T. cruzi
(IC50 \4lM). Several of these T. cruzi-active compounds
that possess a favorable profile have been selected for medic-
inal chemistry exploration with the goal of identifying lead
scaffolds that could be further optimized. These early efforts
have resulted in the discovery of analogs with sub-nanomolar
potency against T. cruzi (Bustamante et al. 2011). These
screens have also been recently extended to a larger 1.8 million
compound library to identify T. cruzi-active compounds
(Bustamante et al. 2011). It is expected that these screening
efforts by GNF will eventually yield a single pool of anti-T.
cruzi compounds that will be further validated and chemically
optimized to yield preclinical candidates that could address this
crucial gap in drug discovery for Chagas disease.
Leishmaniasis has been ranked among the most
neglected of tropical diseases. As a group of diseases
caused by trypanosomatids from the genus, cutaneous and
mucosal leishmaniases are the predominant pathologies
produced by Leishmania infection. Several drugs are cur-
rently available to treat cutaneous leishmaniasis, but each
has limitations (Cruz et al. 2009). While target-based
screening approaches for anti-leishmanial drug discovery
has yielded little progress for a variety of reasons (Freitas-
Junior et al. 2012), the development and implementation of
HTS phenotypic assays by individual academic groups,
consortia and public–private partnerships have generated
several potential starting points for drug development.
There is a general consensus for an urgent need for more
reliable phenotypic in vitro screening that would mimic as
closely as possible the definitive host environment.
Therefore, genetically modified parasites expressing easily
detectable reporters represent promising tools for pheno-
typic screening (Reguera et al. 2014).
Yet another evident advancement in this field is the
development and validation of a high-content, high-
throughput image-based screening assay targeting the
intracellular amastigote stage of different species of
Leishmania in infected human macrophages without the
need for a reporter gene (Siqueira-Neto et al. 2012). The
in vitro infection protocol was adapted to a 384-well-plate
format, thus enabling acquisition of a large amount of
readouts by automated confocal microscopy. This assay
has enabled the screening of up to 300,000 compounds to
obtain 350 hits (Siqueira-Neto et al. 2012; Reguera et al.
2014). Since the same study also established that only 4 %
of the hits identified by using Leishmania promastigotes
display efficacy against intracellular amastigote forms
(Siqueira-Neto et al. 2012), intracellular amastigote-based
phenotypic screening is reinforced as the most suitable
approach to be developed.
An exciting approach that is midway between in vitro
cell infections and experimental infections in mice consists
of the use of ex vivo explants of Leishmania-infected
organs. Target-infected organs are harvested from fluores-
cent or bioluminescent Leishmania-infected rodents for
development of ex vivo explant culture. These splenic or
lymph node ex vivo infected explants are advantageous
over in vitro systems by including the whole cellular
population involved in the host-parasite interaction: mac-
rophages, CD3 ?and CD4 ?T cells, B lymphocytes and
granulocytes; which could affect the therapeutic effect of
the tested compound. Since a single infected spleen can
yield up to four 96-well plates, the use of these ex vivo
explants will also drastically reduce the number of animals
used for screening, whilst still enabling medium-through-
put screening capabilities. Using this approach, 4,035
compounds were screened at a single-dose concentration
against luciferase-transfected L. donovani-infected ex vivo
hamster spleens, revealing more than 200 active hits
(Osorio et al. 2011). More recently, the same group has
also validated a lymph node ex vivo explant model using
the same bioluminescent reporter in a L. major strain
(Peniche et al. 2014).
Furthermore, lead compounds can be scaled up to
in vivo preclinical trials using rodent models of infection
monitoring parasite loads by means of advanced bioimag-
ing devices. The use of quick and reproducible fluorescent
and bioluminescent readouts would greatly reduce the
number of animals used for these trials and allow for an
earlier stage detection of the infective process as compared
with classical methods (Reguera et al. 2014). A total of
near half million compounds have been screened for vis-
ceral leishmaniasis treatment through a series of cutting-
edge technologies combined with optimized assays (Fre-
itas-Junior et al. 2012). However, since more systemic
approaches to develop new chemical entities for
A. L. T. Michelle et al.
123
leishmaniasis have started only recently, the late discovery
process is still in its infancy.
Human African trypanosomiasis (HAT), also known as
sleeping sickness, is yet another example of a vector-
transmitted disease caused by 2 T. brucei subspecies
namely, T. b. gambiense and T. b. rhodesiense. Untreated
HAT leads to a fatal outcome, causing significant mor-
bidity and mortality. With no vaccine available, current
chemotherapy against HAT relies on four drugs that pos-
sess several limitations and occasional severe side-effects.
Whole-cell assays in HTS format for T. brucei are rela-
tively new, with the development of luciferase and resa-
zurin-based cell viability assays a 384-well format
(Mackey et al. 2006; Sykes and Avery 2009a,b). HTS
resazurin-based assays have been recently used to screen
87,296 compounds, resulting in 6 hits from 5 new chemical
classes with activity confirmed against the causative spe-
cies of HAT (Sykes et al. 2012). Being intensively used in
antimalarial drug discovery as well (Smilkstein et al. 2004;
Johnson et al. 2007; Izumiyama et al. 2009; Vossen et al.
2010), SYBR Green whole-cell assays, which are an
indirect assessment of cell number based on quantitative
detection of nuclei acids, have also been found to be
applicable to T. brucei. To aid in drug discovery efforts for
HAT, both assays were semi-automated to screen a library
of 4,000 putative kinase inhibitors (Faria et al. 2015). The
compounds with the most potent anti-trypanosomal activity
could be grouped into 13 structural clusters. Several of the
identified compounds had IC
50
\1lM coupled with high
selectivity toward the parasite, thus providing promising
starting points for lead optimization.
The limitation for the development of novel lead candi-
dates for kinetoplastid parasites remains in target identifi-
cation. Early identification of the candidate target and
demonstration of target engagement would indeed acce lerate
drug development by using enzymatic assays or biophysical
methods to rationally optimize drug candidates. While
efforts can be further expanded for drug discovery for these
NTDs, it is anticipated that novel drug candidates are on the
horizon for the treatment of trypanosomiasis and leishma-
niasis. On the model of P. falciparum, selection of escape
mutants followed by whole-genome sequencing is probably
the most straightforward approach to identify the target, or at
least mechanisms of resistance.
Chemical biology strategies for pathogenic
bacteria
The emergence of multi-drug resistant (MDR) bacteria
poses enormous public health issues. Despite recurrent call
for actions, efforts and investments from the public and
private sector do not match the threat posed by MDR
germs, especially by emerging gram-negative bacteria,
often referred to as superbugs. Among the most deadly
bacteria are Mycobacterium tuberculosis, the etiological
agent of human tuberculosis, and those known as the
‘‘ESKAPE’’ pathogens Enterococcus faecium,Staphylo-
coccus aureus,Klebsiella pneumoniae,Acinetobacter
baumanii,Pseudomonas aeruginosa, and Enterobacter
species that cause the lion’s share of hospital-acquired
infections (Rice 2008; Boucher et al. 2009; Rice 2010).
The global R&D pipeline remains extremely thin for
broad-spectrum antibiotics, especially for the gram-nega-
tive ESKAPE bugs (Boucher et al. 2013). Innovation in
chemical biology and target deconvolution strategies rep-
resents the most promising approach to discover novel
antimicrobials, as exemplified by recent success stories for
tuberculosis.
Mycobacterium tuberculosis
Tuberculosis (TB) remains a major global health challenge,
with nearly 1.5 million TB-related deaths in 2013 alone
(WHO 2013). Underlying the endemic is the emerging
epidemic of multi-drug resistant (MDR-TB) and exten-
sively-drug resistant (XDR-TB) TB strains. With dwin-
dling treatment options for MDR and XDR-TB, one of the
pertinent key issues faced by the TB research community is
the daunting challenge of discovering and developing new
anti-TB drugs. Phenotypic screens have been used exten-
sively in the last decade to discover innovative drug can-
didates, contributing to replenish the drug pipeline.
Historically, most anti-tuberculosis drugs were identi-
fied by screening candidate drugs against Mycobacterium
tuberculosis (Mtb) replicating in culture broth medium.
The exception is pyrazinamide, a sterilizing first-line anti-
TB drug that was discovered by a bold phenotypic screen
in infected animals (Malone et al. 1952). Mode of action
was usually not understood at the time of introduction to
clinical practice. The sequencing of the Mtb genome in
1998 (Cole et al. 1998) led to the emergence of target based
drug discovery and prompted the field to reconsider the
utility of phenotypic screens. The difficulty in finding tar-
gets inhibited by the compounds with whole cell activity
was central to the departure from the phenotypic screening
paradigm. Nevertheless, the landscape changed rapidly.
Improved technologies for de-orphaning phenotypic
screening hits has prompted renewed interest in pursuing
whole cell phenotypic screens for novel, constructive
sources of lead molecules/series.
The success of chemical biology for TB drug discovery
is best epitomized by the discovery of the diarylquinoline
bedaquiline (Sirturo
Ò
) (Andries et al. 2005). Bedaquiline is
the first drug approved for the treatment of tuberculosis in
the last 40 years. The drug was identified from a corporate
Next-generation antimicrobials: from chemical biology to first-in-class drugs
123
collection screening campaign against Mycobacterium
smegmatis, a surrogate environmental, non-pathogenic
mycobacteria (Andries et al. 2005). Limited chemical
optimization of a diarylquinolone series led to bedaquiline,
a compound with exceptional potency against Mtb in vitro
and in animal models (Andries et al. 2005). Whole-genome
sequencing of spontaneous mutants resistant to bedaquiline
revealed the F
0
F
1
ATP synthetase as the drug target.
Despite its evolutionary conservation, bedaquiline is highly
specific for the mycobacterial complex. The inhibition of
ATP synthase leads to ATP depletion and pH homeostasis
imbalance, triggering bacterial death of both replicating
and non-replicating mycobacteria (Deckers-Hebestreit and
Altendorf 1996; Rao et al. 2001). Bedaquiline recently
underwent accelerated approval by the Food and Drug
Administration (FDA) for the treatment of MDR and XDR
TB (Cohen 2013).
Delamanid is another drug available for the treatment
MDR-TB that was granted conditional approval by the
European Medicine Agency late in 2013 (Barry 2015). Full
safety and efficacy is currently being evaluated in Phase III
clinical trials. Delamanid was optimized from a series of
highly potent nitro-imidazole (Stover et al. 2000) that
inhibits mycobacterial growth by targeting multiple
essential processes, including mycolic acid synthesis and
respiration (Stover et al. 2000; Matsumoto et al. 2006;
Singh et al. 2008). PA-824, another clinical phase nitro-
imidazole drug candidate discovered by whole-cell
screening, showed the promise to shorten the time of
MDR-TB therapy when given in combination with moxi-
floxacin, pyrazinamide and/or bedaquiline (Diacon et al.
2012; Dawson et al. 2015; Tasneen et al. 2015).
Using a similar approach, ppromising drug candidates
with a novel mode of action were recently reported. Of
particular interest is a series of indolcarboxamine that
inhibits mycobacteria growth by interfering with the
function of MmpL3, a transporter of trehalose monomy-
colate that is essential for mycobacterial cell wall biosyn-
thesis (Rao et al. 2013). The optimized indolcarboxamine
drug candidate NITD-304 and NITD-349 have excellent
in vivo efficacy, low toxicity and favorable pharmacoki-
netic properties in multiple species, which are properties
that support clinical development of the series for the
treatment of MDR-TB. The same team recently reported on
the discovery of yet another promising class of mycolic
acid inhibitors, the 4-hydroxy-2-pyridones (Manjunatha
et al. 2015). This novel chemical series inhibits InhA, an
essential enzyme involved in mycolic acid synthesis
(Takayama et al. 1975), which was also recently estab-
lished as the target of the natural compound pyridomycin
(Hartkoorn et al. 2012,2014). GSK has also released a set
of 177 potent non-cytotoxic hits active against Mtb using
comparable screening methodologies with the aim of
fuelling open-source early-stage drug discovery activities
(Ballell et al. 2013). The set of compounds represent a
valuable resource for the TB community to initiate lead
optimization and/or target identification programmes.
By virtue of being a facultative intracellular bacterium
that becomes partially phenotypically drug resistant to
multiple antibacterial when hiding inside macrophages, an
attractive approach to circumvent the limitations of in vitro
culture broth media is to screen for compounds that kill
Mtb replicating inside macrophages (Kumar et al. 2010).
Indeed, the nutrients and metabolites that Mtb use to
replicate and survive in eukaryotic cells is largely predic-
tive of the in vivo situation, making it an attractive plat-
form to discover novel classes of anti-TB drugs. Several
teams have reported on the development of systems to
screen for drugs or siRNA that interfere with the survival
of Mtb inside macrophages (Christophe et al. 2010; Sun-
daramurthy et al. 2013).
Using automated confocal fluorescent microscopy to
monitor the intracellular growth of GFP-expressing Mtb
H37Rv, a team from the Institut Pasteur Korea developed a
rapid phenotypic assay to screen large chemical libraries in
384-well format (Christophe et al. 2009). This phenotypic
HTS assay was the first successful demonstration for the
feasibility of large scale screens against intracellular
mycobacteria. A series of dinitrobenzamide derivatives
(DNB) targeting the decaprenyl-phosphoribose 20epimer-
ase DprE1/DprE2, thus blocking arabinogalactan synthesis,
was discovered on this platform (Christophe et al. 2009).
Interestingly, an independent study validated further the
decaprenyl-phosphoribose 20epimerase as an attractive
drug target (Makarov et al. 2009).
The benefit of the macrophage platform was further rati-
fied with the discovery of the optimized imidazopyridine
amide (IPA) drug candidate Q203 (Pethe et al. 2013). Q203
is the lead drug candidate that was selected after an evalua-
tion of 477 derivatives (Kang et al. 2014). The initial dis-
covery of the anti-TB activity of the IPA series was reported
in 2011 (Moraski et al. 2011). Q203 displayed potent inhi-
bitory growth against MDR and XDR Mtb clinical isolates
in vitro by inhibiting the function of the respiratory chain, a
mechanism shared with the drug bedaquiline. Genetic and
biochemical evidences pointed to the mycobacterial cyto-
chrome bc-1 as the molecular target of the IPA series
(Abrahams et al. 2012; Pethe et al. 2013). Q203 combines
favorable properties including bactericidal activity in the
mouse model of tuberculosis at low dose and favorable
pharmacokinetic with safety profiles, making this compound
a promising drug candidate for tuberculosis. Intriguingly,
scientists at EPFL have identified the existing drug lanso-
prazole as an antituberculous prodrug that also targets
cytochrome bc-1 in a host cell-based high throughput screen
as well, albeit using lung fibroblasts (Rybniker et al. 2015).
A. L. T. Michelle et al.
123
Recently, another large-scale compound screen in
infected macrophages revealed several interesting com-
pound series that represses the intracellular Mtb growth by
inhibiting mycobacterial cholesterol metabolism (Van-
derVen et al. 2015). Since cholesterol is a carbon source
used by Mtb in macrophages and in vivo (Rohde et al.
2012) but not in vitro, these interesting compounds series
could not have been identified by screening in classical
culture broth media, illustrating the benefit of using rele-
vant models for bacterial phenotypic screen.
The added benefit of screening for anti-TB drugs in
eukaryotic cells opens the possibility of identifying anti-
virulence drugs (Rybniker et al. 2014) or host-targeted
drugs that stimulate cellular pathway critical for the
maintenance of the infection (Sundaramurthy et al. 2013;
Stanley et al. 2014).
Finally, the development of a rapid screening platform
for anti-TB agents in whole animals represents a very
attractive approach. Two systems were recently devel-
oped using Mycobacterium marinum as a surrogate for
Mtb. Being a natural fish pathogen, the infection process
in zebrafish larvae recapitulates many aspects of tuber-
culosis pathogenesis in humans (Swaim et al. 2006). The
zebrafish larvae platform was developed in 96 well plates
and relies on automated fluorometric in situ measurement
of drug efficacy and toxicity. Since the entire procedure
is performed in 96 well-plates, numerous drugs can be
evaluated in parallel. The assay was validated using
known direct- and host-acting drugs (Takaki et al. 2012,
2013). More recently a comparable screening approach
was optimized in amoeba (Kicka et al. 2014). The assay
was developed in the amoeba Acanthamoeba castellanii
infected with M. marinum and validated with several
known anti-TB drugs. In principle, the system is suitable
to identify host-targeted drug candidate and compounds
interfering with virulence, which represent an advantage
compared to classical screening approaches. The perfor-
mance and benefits of such platforms remain to be
determined in a large-scale compound screening
campaign.
Broad spectrum antibacterials
Gram-positive bacteria
Antibiotic resistance has become a major problem in the
treatment of Gram-positive bacterial infections, with some
of the most important gram-positive resistant organisms
including penicillin-resistant Streptococcus pneumonia and
methicillin-resistant Staphylococcus aureus (MRSA)
(Klevens et al. 2007; Arias and Murray 2009). Lately, a
number of contemporary and prominent approaches have
produced promising data in the quest for broad-spectrum
bactericidal antibiotics that could improve the clinical
outcomes for the treatment of Gram-positive bacterial
infections.
The first notable study involved a cell-based screen of
1280 bioactive compounds from the Library of Pharma-
cologically Active Compounds (LOPAC) library against a
constitutively-expressing luciferase Mtb strain under
nutrient-deprivation conditions in order to seek inhibitors
with activity against replicating and nonreplicating Mtb
(Harbut et al. 2015). This screen culminated in the unex-
pected identification of auranofin, an orally bioavailable
FDA-approved anti-rheumatic drug, as possessing potent
bactericidal activities against a number of other clinically
important Gram-positive bacterial species. Auranofin
exerts its effects through a unique mechanism involving the
inhibition of the bacterial thioredoxin reductase, a protein
essential in many Gram-positive bacteria for maintenance
of the thiol-redox balance and protection against reactive
oxygen species (Scharf et al. 1998; Uziel et al. 2004).
These findings not only suggest auranofin as a viable
candidate worth repurposing for antibacterial therapy; but
its associated universal mode of action also highlight the
prospects of targeting the thioredoxin-mediated redox
cascade of Gram-positive pathogens for the development
of novel broad-spectrum antibacterials.
Another significant breakthrough is the discovery of
novel antibiotics identified from soil bacteria recalcitrant to
grow under laboratory conditions (Ling et al. 2015). The
development of several meticulous methods to grow
uncultured organisms via in situ cultivation (Kaeberlein
et al. 2002; Nichols et al. 2010) or by using specific growth
factors (D’Onofrio et al. 2010) hold great promise to
identify novel antibiotics from natural sources. The mul-
tichannel iChip (Nichols et al. 2010) device was designed
to isolate and grow uncultured bacteria in diffusion
chambers in situ, enabling the identification of numerous
novel soil bacteria. A large scale screen of secreted sec-
ondary metabolites from 10,000 bacterial isolates against S.
aureus led to the discovery of the novel antibiotic teix-
obactin. Teixobactin is an unusual depsipeptide that dis-
plays astounding activity against Gram-positive pathogens,
including experimentally proven in vivo efficacies against
drug-resistant pathogens in a number of animal models of
infection (Ling et al. 2015). Due to its unusual mode of
action involving multiple targets and binding to a highly
conserved motif of lipid II and lipid III to inhibit pepti-
doglycan synthesis, teixobactin has been construed as a
promising therapeutic candidate with favorable pharma-
cokinetic parameters (Ling et al. 2015). Primarily, the
results of this pioneer study suggest that additional natural
compounds with similarly low susceptibility to resistance
are present in nature and may potentially be discovered
following a similar approach.
Next-generation antimicrobials: from chemical biology to first-in-class drugs
123
Table 1 Novel chemical classes identified through chemical biology screens
Disease Chemical class Lead compound(s) Chemical
structure
Identified
target
MMOA Key reference
Malaria Spiroindolones KAE609 PfATP4 Disrupts intracellular sodium homeostasis Rottmann et al. (2010)
Dihydroisoquinolines SJ733 PfATP4 Disrupts intracellular sodium homeostasis Jimenez-Diaz et al. (2014)
Imidazolopiperazines GNF179 Pfcar1 Unclear Meister et al. (2011)
Imidazopyrazines KAI407 PI(4)K Alters the intracellular distribution of
phosphatidylinositol-4-phosphate
Zou et al. (2014)
N-aryl-2-aminobenz
-imidazoles
Compound 12 in
Ramachandran et al. (2014)
NA Unclear Ramachandran et al. (2014)
Triaminopyrimidines Compound 12 in
Hameed et al. (2015)
Unclear Unclear Hameed et al. (2015)
Tuberculosis Diarylquinolones Bedaquiline F
0
F
1
ATP
synthetase
ATP depletion and pH homeostasis imbalance Andries et al. (2005)
Nitro-imidazoles Delamanid PA-824 Unclear Inhibits mycobacterial growth by targeting multiple
essential cellular processes, including protein,
mycolic acid synthesis and respiration
Stover et al. (2000)
Indolcarboxamines NITD-304 MmpL3 Inhibits mycobacterial cell wall synthesis Rao et al. (2013)
NITD-349
A. L. T. Michelle et al.
123
Table 1 continued
Disease Chemical class Lead compound(s) Chemical
structure
Identified
target
MMOA Key reference
4-hydroxy-2-pyridones NITD-916 InhA Inhibits mycolic acid synthesis Manjunatha et al. (2015)
Dinitrobenzamides NA NA DprE1/DprE2 Blocks arabinogalactan synthesis Christophe et al. (2009)
Imidazopyridine amides Q203 bc-1 complex Inhibits ATP synthesis Pethe et al. (2013)
Pyridomycins Pyridomycin InhA Blocks both the NADH cofactor– and lipid
substrate–binding pockets of InhA
Hartkoorn et al. (2012)
Others Depsipeptide Teixobactin Multiple targets Inhibits peptidoglycan synthesis Ling et al. (2015)
Pyridopyrimidines NA NA biotin carboxylase (BC)
complex
Inhibits fatty acid synthesis Miller et al. 2009
Pyridinemidazoles NA NA LolCDE complex Inhibits lipoprotein trafficking from the inner to
the outer membrane
McLeod et al. (2015)
NA Not available
Next-generation antimicrobials: from chemical biology to first-in-class drugs
123
Gram-negative bacteria
Gram-negative bacteria are best defined by their ability to
acquire and transfer drug resistance genes to other bacteria
(Hall and Collis 1998); along with their characteristic cell
envelope which comprise of a unique outer membrane of
lipoproteins, b-barrel proteins, lipopolysaccharides and
phospholipids, and an inner membrane composed of a
phospholipids bilayer. Compounding the problem of
antimicrobial-drug resistance is the immediate threat of a
reduction in the discovery and development of new
antibiotics for the treatment of Gram-negative bacteria
(Boucher et al. 2009). Accordingly, there is an urgent need
for atypical, originative and alternative modes of drug
discovery veering away from the conventional target-based
approach for drug screening.
One such notable approach was the utilization of the
Pfizer compound library consisting of *1.6 million com-
pounds in an unbiased whole-bacterial cell screening for
growth inhibition of a membrane-compromised, efflux
pump-deficient strain of E. coli (tol C, imp) (Miller et al.
2009). This radical methodology focused on targeting an
antibacterial space resembling those of eukaryotic targets,
thus facilitating the identification of a series of antibacterial
pyridopyrimidines derived from a protein kinase inhibitor
pharmacophore. These compounds were later found to
possess a previously undescribed MMOA for antibacterial
activity by targeting the ATP-binding site of biotin car-
boxylase (BC), which catalyzes the first enzymatic step of
fatty acid biosynthesis (Cronan and Waldrop 2002).
Remarkably, these BC inhibitors also exhibited outstanding
potency against clinical isolates of fastidious Gram-nega-
tive pathogens including H. influenza and M. catarrhalis;
causative agent of many respiratory tract infections.
Despite the structural similarities of the BC active site to
those of eukaryotic protein kinases, inhibitor binding to the
BC ATP-binding site was found to be disparate from the
protein kinase-binding mode with the inhibitors, displaying
selectivity for bacterial BC. The implications behind the
identification of antibacterials derived from a protein
kinase inhibitor pharmacophore suggest that the huge array
of eukaryotic inhibitors present in these pharmaceutical
libraries could be mined for their activity against struc-
turally related bacterial targets such as protein kinases
involved in cell–cell signaling lipopolysaccharide sugar
kinases involved in Gram-negative cell wall formation
(Miller et al. 2009).
In a similar manner, the AstraZeneca compound col-
lection (*1.2 million compounds) was screened in
384-well plate format against a permeabilized E. coli strain
(W3110 DwaaP) (McLeod et al. 2015). The shortened
lipopolysaccharide chain in this modified strain increased
membrane permeability to small molecules and warranted
the discovery of novel pyridinemidazole compounds that
inhibited lipoprotein trafficking from the inner to the outer
membrane. Subsequent studies via resistance mutation
mapping and biochemical transport assays further demon-
strated the inhibition of function of the lipoprotein-releas-
ing system transmembrane proteins (LolCDE complex) via
these compounds. Being the first reported inhibitors of the
LolCDE complex, this novel compound class not only
displayed a unique and specific mechanism of inhibition
for Gram-negative bacteria, but the identification of their
associated novel drug target suggest further exploitation of
the outer membrane lipoprotein transport pathway as a
target for antimicrobial therapy.
Although phenotype-based screens have expanded the
repertoire of new potential drug candidates, one of the
biggest impediments is unraveling the relationships
between the phenotype(s) caused by biologically active
small molecules and their respective mechanisms (Burdine
and Kodadek 2004). Moreover, due to the poor predictive
value of conventional in vitro culture conditions used to
analyze drug candidates (Garber 1960; Brown et al. 2008;
Brinster et al. 2009; Pethe et al. 2010), it is necessary to
innovate alternative means to characterize the MMOA of
biologically active molecules. A more unconventional
approach has been established to identify inhibitors of
E. coli grown under nutrient limitation. Employing meta-
bolomics, the authors demonstrated the promise of
metabolite suppression profiling as a novel method for
assigning a possible mode of action for metabolic inhibi-
tors that are frequently identified in phenotypic-based
screens (Zlitni et al. 2013). This inventive strategy led to
the discovery of novel inhibitors of glycine and biotin
biosynthetic pathways in E. coli and outlined a novel
platform for the identification of small-molecule inhibitors
of essential metabolic pathways that would be potentially
applicable and easily adapted for most bacteria.
Nonreplicating and metabolically quiescent bacteria are
causal for latent infections and relapses following ‘‘steril-
izing’’ chemotherapy (Manina et al. 2015). Actively
shifting the characteristic metabolic flux of drug-tolerant
non-growing but metabolically active (NGMA) microbes
toward an actively growing state offers a fresh prospect to
control such populations. The notion of metabolic modu-
lation to enhance the efficacy of current antibiotics pro-
vides key insights into the Achilles’ heel of persistent cells
(Murima et al. 2014). This was recently demonstrated by
Kim et al. with the unprecedented approach of screening a
compound library against persistent E. coli in combination
with ampicillin in a bid to identify specific killers of bac-
terial persisters (Kim et al. 2011). A small polycyclic
molecule C10 was shown to sensitize bacterial persisters to
ampicillin killing. Although the detailed mode of action for
C10 remains undeciphered, such rudimentary yet
A. L. T. Michelle et al.
123
elucidative studies capitalizing on the use of single-
chemical supplementation could be applied on a universal
basis to other bacterial systems to discover inhibitors tar-
geting NGMAs (Wakamoto et al. 2013). Other potential
metabolic strategies for eliminating persister cells include
collapsing the proton motive force (Rao et al. 2008; Allison
et al. 2011; Farha et al. 2013; Peng et al. 2015), dissipating
intracellular nutrient storage in mycobacteria (Baek et al.
2011), or inducing reactive oxygen species potency of
bactericidal antibiotics (Kohanski et al. 2007).
Future prospects for chemical biology screens
To support the discovery of novel antimicrobials, it is
heartening to observe how public and private initiatives
have collaborated, thereupon allowing researchers to gain
access to vast collections of bioactive compounds for drug
screens. This avenue has provided further motivation for
more researchers to concert towards the identification of
new leads, expanding efforts in the drug discovery sector.
Several of the chemical biology approaches discussed in
this review have proven to be quintessential for detecting
small-molecules with optimal selective indexes for speci-
fied pathogens that usually warrant favorable results in a
preclinical model of the disease; avoiding the nuances
encountered in target-based screenings associated with
poor permeability or degradation into inactive metabolites
(Reguera et al. 2014).
Indeed, chemical biology screens have proven to be
effective for the discovery of antimicrobials, as evidenced by
the discovery of a number of potential and validated first-in-
class drugs associated with novel drug targets/pathways
emerging from these screens. In terms of drug discovery for
infectious diseases, chemical biology screens appear to have
gained the most traction and advancements in the fields of
malaria and Mtb likely due to the motivation behind relevant
interest groups, but have also proven to be applicable to other
human pathogens as well (Table 1). Furthermore, many of
the concepts behind chemical biology approaches discussed
in this review have the potential to be adapted to other human
pathogens with some prudent remodeling. Due to the limited
chemical diversity in screening libraries (Payne et al. 2007),
it is also plausible to widen the scope of these screens to
include metabolism curated libraries and natural products,
which have shown potential in pilot studies (Miller et al.
2009; Ling et al. 2015). Additionally, chemical biology
screens have advanced by quantum leaps in terms of tech-
nology, having progressed from simplistic in vitro whole cell
screens to the incorporation of reporter genes for biolumi-
nescent, fluorescent or enzymatic measurements via HTS
platforms, and more recently, to bioimaging readouts in
ex vivo explants. Ultimately, the development of large-scale
in vivo chemical biology screening platform, with cutting
edge imaging and target deconvolution strategies, certainly
has the potential to accelerate even further antimicrobial
drug discovery.
Acknowledgments This work was supported by a start-up grant
from the Lee Kong Chian School of Medicine, Nanyang Techno-
logical University.
Compliance with ethical standards
Conflict of Interest The authors declare no conflict of interest with
any person or any organization.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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