Running title: Renaissance in antibiotic discovery
RENAISSANCE IN ANTIBIOTIC DISCOVERY:
SOME NOVEL APPROACHES FOR FINDING DRUGS TO TREAT BAD BUGS
Bharat Gadakh and *Arthur Van Aerschot
KU Leuven – University of Leuven, Department of Pharmaceutical and Pharmacological
Sciences, Medicinal Chemistry, Rega Institute for Medical Research, 3000 Leuven, Belgium
* Corresponding author: Prof. Dr. Arthur Van Aerschot
Address: KU Leuven – University of Leuven, Medicinal Chemistry, Rega Institute for Medical
Research, Minderbroedersstraat 10, 3000 Leuven, Belgium
Direct Phone (32) 16 372624; Fax: (32) 16 337340;
E-mail: Arthur.VanAerschot@rega.kuleuven.be
Bharat Gadakh
Address: KU Leuven – University of Leuven, Medicinal Chemistry, Rega Institute for Medical
Research, Minderbroedersstraat 10, 3000 Leuven, Belgium
Direct Phone (32) 16 337381; Fax: (32) 16 337340;
E-mail: bharat.gadakh@rega.kuleuven.be
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Abstract
With the alarming resistance to currently used antibiotics, there is a serious worldwide threat to public
health. Therefore, there is an urgent need to search for new antibiotics or new cellular targets which are
essential for survival of the pathogens. However, during the past 50 years, only two new classes of
antibiotics (oxazolidinone and lipopeptides) have reached the clinic. This suggests that the success rate
in discovering new/novel antibiotics using conventional approaches is limited and that we must
reconsider our antibiotic discovery approaches. While many new strategies are being pursued lately,
this review primarily focuses only on a few of these novel/new approaches for antibiotic discovery.
These include structure-based drug design (SBDD), the genomic approach, anti-virulence strategy,
targeting non-multiplying bacteria and the use of bacteriophages. In general, recent advancements in
nuclear magnetic resonance, X-crystallography, and genomic evolution have significant impact on
antibacterial drug research. This review therefore aims to discuss recent strategies in searching new
antibacterial agents making use of these technical novelties, their advantages, disadvantages and
limitations.
Keywords: acylated homoserine lactone, antibiotics, antibiotic discovery, antivirulence strategy,
autoinducers, bacteriophages, genomic approach, non-multiplying bacteria, phage therapy, quorum
sensing, resistance, screening strategies, structure-based drug design.
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1. INTRODUCTION
For more than seven decades, antibiotics are essential components of modern medicine and are
one of the leading causes for increased life expectancy [1, 2]. However, over the last two decades,
development of resistance to antibiotics poses a serious threat to public health worldwide [3, 4].
Nowadays, most of the lethal infections are caused by resistant pathogens like methicillin resistant
Staphylococcus aureus (MRSA), vancomycin resistant Enterococcus (VRE), extended-spectrum lactamase producing enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii.
Looking back at the timeline of antibiotic development from 1935 till present, it can be seen that the
majority of the antibiotics in clinical use have been discovered during the 1940s-1960s and this period
therefore is called the golden period of antibiotic discovery. In the past 50 years only two new classes
of antibiotics (i.e. oxazolidinone and lipopeptides) have reached the clinic. With the emergence of
multidrug resistant pathogens and the withdrawal of pharmaceutical companies from the infectious
disease area, we are in danger that these resistant pathogens may send us back into the pre-antibiotic
era leaving us without any protection against these resistant pathogens. So, we need to ask ourselves
“where did we go wrong?” The factors contributing to this situation are numerous and complex [5-8].
These factors include the shifting research priorities of the pharmaceutical companies, the raised utility
bar by Food and Drug Administration (FDA), limited market due to improved hygiene, regulatory
hurdles, rapid development of resistance, etc. In addition, success in discovering antibiotics using
conventional approaches like high throughput screening (HTS) and combinatorial chemical libraries is
rare [1, 9, 10]. In order to fight these bad bugs (resistant pathogens), new classes of antibiotics must
enter the clinic at regular intervals. Therefore, with alarming bacterial resistance and low productivity
of conventional whole cell-based screening assays, we must consider alternative approaches for
antibiotic discovery.
However, also commercial considerations have driven pharmaceutical companies out of the
antibiotic development area. Most infections can still be treated with current antibiotics at virtually no
cost. With these at hand, companies hardly can expect therefore to get the same price for a new antibiotic
as is now been paid for an antiviral or an anticancer drug. An extravagant example of pricing herein is
seen with the antiviral drug sofosbuvir (SovaldiTM, Gilead Sciences), a once-daily oral nucleotide analog
polymerase inhibitor for the treatment of chronic hepatitis C infection, where a 1000 $ per pill is
charged, resulting in a total 85,000 $ treatment regimen Such examples of exorbitant costs in our
opinion could also provoke a backlash against pharmaceutical industry by both government officials
and private insurance companies [11].
In contrast, in view of the dwelling pipeline and the alarming rise in antimicrobial resistance,
European Commission launched an action plan resulting in the New Drugs for Bad Bugs (ND4BB)
initiative. The latter aims to join forces between public and private partners in order to bring new
antimicrobials closer to patients and to boost research on antibiotics especially against Gram-negative
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bacteria. Likewise, being aware of the lack of adequate reward for companies developing antibiotics,
American government decided to stimulate antibiotic research via altered legislation. Hereto, the GAIN
initiative (Generating Antibiotic Incentives Now), part of the FDA Safety and Innovation Act was
signed into law in July 2012, which for infectious disease products provides a more speedy review and,
if approved, provides companies with five additional years of market exclusivity to recoup their
investments [12]. These initiatives have certainly boosted research on antibiotics lately, both in
academia and in smaller biotech companies.
This review aims to illustrate some recent approaches for discovery of new antibiotics and the
advantages, disadvantages and experimental challenges facing their development. By no means is it
meant to be exhaustive with many new ideas being pursued over the last years. All novel approaches
can be classified into several strategies such as structure-based drug design (SBDD), the (post-)genomic
approach, anti-virulence strategy, the use of bacteriophages, non-multiplying bacteria as a target, noncultivable bacteria as antibiotic sources, antibiotics from marine sources, the search for unexploited and
vital targets, targeting resistance mechanisms (e.g. β-lactamase inhibitors), the RNAi approach,
inhibition of peptidoglycan biosynthesis, methods to enhance uptake of inhibitory products,
antimicrobial peptides, etc. However, in view of length considerations this review focuses on only the
first five approaches mentioned above and the lessons they taught us. All these strategies, however,
showed some successes and failures in their preliminary screening. Other approaches are reviewed
elsewhere, e.g. the RNAi approach [13] or antimicrobial peptides [14, 15] and we refer to these. Many
alternative therapeutic strategies are currently under consideration as well and cannot all be covered
here. Industrial views on the topic can be found amongst others in the opinion papers of David Payne
discussing the (industrial) challenges of antibacterial discovery. Innovation herein can take many forms,
from new physicochemical properties for analogues of an existing class, over modifications to scaffolds
overcoming resistance problems, to agents with new mechanisms of action, development of novel
chemical classes or uncovering of new targets [16, 17]. Another recent review which could be consulted
discusses the different platforms for antibiotic discovery [18]. In our last part, a brief discussion on
bacteriophage therapy is included as it constitutes an often forgotten strategy to combat specific
infections like in patients with serious burn wounds. However, the lack of a legal framework seriously
hampers the use of this latter technique.
2. CLASSICAL OR CONVENTIONAL APPROACH
Before moving on to a discussion on the new strategies, it is important to briefly look at the
classical approaches of antibiotic discovery. One such approach involves the random screening of
potential inhibitors (obtained from natural product or synthetic chemical libraries) in a whole cell assay,
with growth inhibition or the death of a pathogen as an end point [9, 10]. The active compounds from
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these screening activities are then tested in an in vivo model to determine efficacy and safety. Most of
the clinically used antibiotics have been discovered using this approach. The ensemble of these
antibiotics targets only a few essential cellular pathways such as cell wall biosynthesis, nucleic acid
metabolism, protein synthesis and fatty acid biosynthesis. The conventional screening process gives
valuable information on potency (minimum inhibitory concentration - MIC), the spectrum of activity
and the ability of the compound to enter bacterial cells. However, the drawback of this approach is that
it may eliminate potentially important compounds at the early stages of drug discovery as it is mainly
based on two properties of the compounds: antibacterial activity and the ability to enter the bacterial
cells. Is it however appropriate to demand both afore mentioned properties in a new compound at early
stage? In retrospect, it might be better to get target specificity first and to optimize a lead to address
other factors such as cell permeability. As this conventional strategy targets essential biosynthetic
pathways in multiplying bacteria, these antibacterial agents are inactive or only partially active against
non-multiplying bacteria [19, 20].
Following the ‘golden period’ of antibiotic discovery, subsequent improvements in the infectious
disease area were achieved by chemical modifications of existing antibiotics. This approach is called
the conservative approach which yielded short-term success. For example ampicillin and methicillin
are derived from penicillin with an expanded spectrum and with increased resistance to β-lactamases
respectively. Further, screening of vast chemical libraries was accelerated by high throughput screening
(HTS). However, the hits identified from HTS often fail to reach the clinic due to poor selectivity,
toxicity, inefficient uptake, narrow spectrum of activity etc. Therefore at this point of time, we need to
revise our approaches for antibiotic discovery.
3. NEW/NOVEL APPROACHES FOR DISCOVERY OF ANTIBIOTICS
Although rational design of antibiotics is a valid approach, it has not been practiced much due to
the technical difficulties and limited understanding of the targets. However, recent technical
advancements in nuclear magnetic resonance (NMR), X-ray crystallography, computational tools,
biochemical and genetic tools have contributed significantly in antibacterial drug development. These
new approaches of antibiotic discovery explore either novel cellular targets which are essential for
survival of a pathogen or new antibacterial agents acting by a novel mode of action. As mentioned
before, only the topics SBDD, the (post-)genomic approach, anti-virulence strategy, targeting nonmultiplying bacteria and the use of bacteriophages as biological antibacterial agents will be discussed
in the next sections.
3.1. Structure-Based Drug Design (SBDD)
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Drug discovery and development is a complex and multivariate process driven by economic and
regulatory factors. In 1990s, the application of combinatorial chemistry along with HTS in the
pharmaceutical industry significantly accelerated screening of vast chemical libraries against a potential
target. However, there has not been a proportional increment in the number of new chemical entities
(NCEs) reaching the approval. During the same time many pharmaceutical companies also pursued a
target-based drug discovery approach for antibiotic discovery. This approach was mainly based on a
combined application of the knowledge of the bacterial genome, HTS and SBDD. The technical
advances in X-ray crystallography, NMR and computational tools and the possibility of crystallizing
many vital bacterial targets contributed significantly to the use of SBDD in antibacterial drug discovery.
Numerous literature reports contain a variety of examples where the SBDD approach has been used for
hit identification and lead optimization of known antibiotics [16, 21-23]. Moreover, a better
understanding of the molecular basis of resistance also opens up many opportunities where SBDD
proved to be a powerful tool to fight resistant pathogens. Here, the development of a novel dihydrofolate
reductase (DHFR) inhibitor, iclaprim has been described as an illustrative example where SBDD plays
an important role to address the issue of bacterial resistance. Two very recent examples are added
confirming the power of this matured technique.
3.1.1. SBDD: addressing the issue of resistance
Dihydrofolate reductase (DHFR) is an essential bacterial enzyme which is responsible for
synthesis of tetrahydrofolate from dihydrofolate. It has been used as an antibacterial target over the last
five decades. Trimethoprim (TMP) (compound 1, Fig. 1) is a potent and selective inhibitor of DHFR
(with Ki of 0.9 nM for Staphylococcus aureus DHFR vs Ki of 19 M for human DHFR) and is used in
a synergistic combination with sulfamethoxazole (SMX) (compound 2, Fig. 1). The synergistic
combination of TMP and SMX is marketed by Roche Pharmaceuticals as Bactrim™. After a long-term
clinical use, not surprisingly, the effectiveness of these drugs is reduced due to the emergence of
resistance. The extent of resistance varies among bacterial species and the geographical location. For
example, in 2002, in the USA approximately 20% and in Korea 70% of S. aureus strains were found to
be Bactrim™ resistant.
The crystal structure of DHFR from the resistant stain suggests that resistance to TMP has
occurred via a point mutation in the TMP binding site by substituting phenylanine at position 98 with
tyrosine (F98Y). This mutation results in the loss of a hydrogen bond between the 4-amino moiety of
TMP and the carbonyl oxygen of Leu6 [24-26]. Therefore, researchers at Roche made several attempts
to eliminate the 4-amino group or to replace the 2,4-diaminopyrimidine core with other heterocycles.
However, such replacement resulted in reduced potency as compared to TMP. Finally, combining
detailed structural knowledge and the SBDD approach, they designed modified diaminopyrimidines
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that would compensate for the loss of hydrogen bonding. A few key DHFR inhibitors have been
depicted in Fig. 1 (compounds 3-5). Iclaprim (compound 3, Fig. 1) is the most studied compound in
this series and adds favorable hydrophobic interactions via the iclaprim cyclopropyl group with Ile50
and Leu54 of mutant DHFR [27-29]. Iclaprim has better potency against both the wild type (15 fold)
and the F99Y mutant (100 fold) strains as compared to TMP.
From a properties standpoint, the water solubility of iclaprim is reduced by 20-fold as compared
to TMP. Pharmacokinetic studies indicate that the human oral bioavailability of iclaprim is only 40%
as compared to >90% for TMP (smaller and more water soluble). Recently, iclaprim was evaluated in
a phase III clinical trial for treatment of patients with complicated soft structure skin infections (cSSSI).
The clinical data demonstrated that iclaprim was safe and efficacious. However, additional clinical data
were required by FDA to demonstrate efficacy in order to gain approval [29]. In 2010, Acino Pharma
acquired the rights for this intravenous (i.v.) antibiotic efficacious against MRSA strains resistant to
many antibiotics and beginning of 2014 was taken over by Pharma Strategy partners. The actual fate of
iclaprim is unclear though.
In addition and in brief, a more recent example with defensin mimetics targeting Lipid II
highlights once more the power of SBDD. Lipid II, being an essential precursor in cell wall biosynthesis
and having a high turnover rate has been established before as an ideal molecular target for development
of antibiotics and several peptide antibiotics among which the glycopeptides vancomycin and
teicoplanin have been described to target Lipid II [30]. Following their report on the functional
interaction of the human defensin peptide HNP1 with Lipid II [31], the authors were able to cocrystallize and solve the structure of a HNP-1/Lipid II complex. Following establishment of
pharmacophore models with the required orientation of side chains in space and in silico searching
within libraries of drug-like compounds, the above uncovered detailed interaction allowed to identify
low molecular weight compounds with strong similarity to the defensin HNP1. Several of these showed
strong antibacterial activity and specific killing of S. aureus. The lead compound coined BAS00127538
(Compound 6, Fig. 1) herein most strongly bound to Lipid II, as shown by surface plasmon resonance.
Inhibition of cell wall synthesis was proved to be the primary target, with membrane perturbation as a
secondary mechanism [32]. The compound likewise was shown to strongly inhibit Acinetobacter
baumanii further highlighting that small molecule inhibitors of Lipid II have the potential to be
developed into broad-spectrum therapeutics [33].
A further recent example from Trius Therapeutics used the well-known sulfamate inhibitor
Threonine-AMS as a starting point and came up with a series of potent and bacteria-selective threonyltRNA synthetase (ThrRS) inhibitors (Compound 7, Fig. 1) in which both the sugar and natural
nucleoside base part were substituted for by heterocycles. The study relied on computational modelling
using the structural information of the Thr-AMS/ThrRS complex. Nice antimicrobial activities were
noted for the lead compounds against Haemophilus influenzae, efflux-deficient mutants of Escherichia
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coli and against Burkholderia thailandensis [34]. All of these clearly establish the power of SBDD
provided the necessary high resolution structural information is available.
3.1.2. Lessons learned from SBDD
SBDD is a promising strategy for antibiotic discovery and in addition has been used for many
other targets such as the DNA gyrase subunit B (GyrB) [35, 36], LpxC (a bacterial deacetylase essential
for lipopolysaccharide biosynthesis) [37], methionyl-tRNA synthetase (MetRS) [38], -lactamase [39]
and bacterial ribosome [40]. SBDD has proven its value for optimization of the lead structure and for
addressing the issue of resistance. As compared to other strategies, SBDD fairly rapidly leads to
discovery of compounds with antibacterial activities. In addition optimization of hits from HTS has
been relatively effective. Although SBDD can be used to tailor the spectrum, activity of the compounds
against multiple enzyme orthologs are often ignored, not reported or probably not measured. In addition,
the choice of the target plays a vital role in determining the potency and spectrum of the new
antibacterial agents. It is difficult to predict the degree of success at early stage because the lack of in
vivo antibacterial activity despite nice in vitro activity may be due to other factors such as poor bacterial
cell wall penetration. The SBDD strategy often affects drug properties such as hydrophilicity,
lipophilicity and solubility, which in turn influence oral absorption and bioavailability (see also the
issue iclaprim vs TMP).
A comment should be added in the sense that in using SBDD we only discussed here the
targeting of classical resistance elements. In addition, the “intrinsic resistome” as recently defined
comprises all elements that contribute directly or indirectly to bacterial resistance to antibiotics,
independent of horizontal gene transfer. Inhibition of these elements or genes will turn bacteria either
more susceptible or more resistant to antibiotics dependent on the nature of the element, being for
instance a plasmid-encoded -lactamase, an efflux pump or a transcriptional repressor of an efflux
pump. The study of the intrinsic resistome indicates diverse possibilities of modulating antibiotic
efficacy and is a research topic on its own, which has been very nicely covered recently by Martinez et
al. [41].
3.2 Genomic Approach
In 1995, the complete bacterial genomic sequence of H. influenzae was published, which was
considered as the beginning of the ‘genomic era’ [42]. Subsequently, the complete genomic sequence
of Saccharomyces cerevisae (1996) [43] and E. coli K-12 (1997) [44] were published. In recent years,
there has been an explosion in the amount of available genome data which has had a great impact on
antibacterial drug discovery [45, 46]. In fact more than 1000 complete bacterial genome sequences are
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available for comparative analysis in antibiotic drug discovery and many more are currently in progress.
It was assumed that the bacterial genome would reveal a treasure of unknown and therefore unexploited
targets for antimicrobial therapy and would speed up the process of drug discovery. In retrospect, this
approach yielded limited success owing to difficulties in the target selection, the screening procedures
and the compound pools used as source of inhibitors. The process of antibiotic discovery using a
genomic approach is represented schematically in Fig. 2A. As a bioinformatics-driven approach the
target selection is limited to the areas where a full package of information related to a particular gene is
known. But it does provide a framework for comparative analysis of genes and prioritization of new
targets which can be used to investigate genes of unknown function.
Many pharmaceutical companies have embarked on programs to find antibacterial agents using
a genomic approach. Illustrative examples include large screening campaigns of GlaxoSmithKline and
Pfizer [16]. During the years 1995-2001, researchers at GSK identified more than 350 genes of interest
from S. aureus, Streptococcus pneumoniae and H. influenzae by comparative genomic analysis. They
found that out of 350 selected genes, only 127 genes are essential in at least one of these
microorganisms. Of these, 67 were purified and tested by HTS against different libraries ranging from
260,000 to 530,000 compounds which yielded 16 hits, of which five resulted in a lead. Only one of
these five leads (AFN-1252, compound 8, Fig. 3) further progressed to development as a fatty acid
biosynthesis inhibitor. Fig. 2B schematically illustrates the development of AFN-1252 as a lead
following the genomic approach. This compound inhibits the enoyl-acyl carrier protein (ACP) reductase
(Fabl) and reduces the growth of all clinical isolates of S. aureus and S. epidermidis. However, AFN1252 has a narrow spectrum of antibacterial activity as the enoyl-ACP reductase is encoded by distinct
enzyme isoforms in different bacterial species (e.g. Fabl in S. aureus and or FabK in S. pneumoniae
[47] or FabV in P. aeruginosa [48]. The program was out licensed to Affinium. The candidate AFN1252 showed excellent tolerability and efficacy in a phase I clinical trial for oral treatment of S. aureus
infections [49]. More recently, Affimium Pharmaceuticals completed the first oral Phase II clinical
study in 2011 [50, 51]. The FDA in November 2013 designated AFN-1252 as a qualified infectious
disease product (QIDP) affording beneficial regulatory incentives. In February 2014, AFN-1252 was
acquired by Debiopharm™. Similarly, Pfizer reported four leads from their 65 HTS programs; however
none of them progressed to clinical trials. In addition, Cubist pharmaceuticals focused on a family of
enzymes called aminoacyl-tRNA synthetases. They have screened 17 enzymes of this family against
50,000 compounds but without success [52]. Despite of this positive evolution, the question remains
why the genomic strategy was not as successful as anticipated?
3.2.1. Lessons learned from the genomic era regarding the targets
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In the last few years, we have gathered considerable knowledge which explains why target-based
HTS is not as successful as predicted. The reasons are mainly related to the selection of the targets
themselves, the applied screening procedures and the libraries of compounds used as the source of
inhibitors.
The perception that the genomic revolution would reveal a treasure of novel, unexplored broadspectrum targets which would accelerate the process of antibiotic discovery, turned out to be wrong.
Besides the criteria used for target selection like target conservation, evolutionary distance to human
homologs and indispensible nature of the target, a target must fulfill several other and equally important
requisites. The targets need to be validated in every species. For example, while screening quinolinone
derivatives as MetRS inhibitors against 101 S. pneumoniae clinical isolates, researchers at GSK found
that 20% of S. pneumoniae strains are intrinsically resistant due to the presence of a MetRS isoform
[53].
Essentiality of the target must be validated under in vitro as well as in vivo conditions (in the
nutrient rich environment of the human body) and it needs to be ascertained that the bacteria have no
mechanism for circumventing the target reaction by acquiring its reaction product from the host. In
recent studies it has been found that Gram-positive pathogens with low GC content were resistant to
inhibitors of fatty acid biosynthesis type II in vitro, when medium is supplemented with exogenous fatty
acids [54]. Examples of fatty acid inhibitors include AFN-1252 (GSK/Affinium) (compound 8, Fig. 3)
[51], plantensimycin (compound 9, Fig. 3) [55], platencin (Merck) [56] and pyrroline diones (Bayer)
[57]. In addition, deletion of essential fatty acid biosynthesis genes does not diminish the virulence of
Streptococcus agalactiae in mice which further questioned the essentiality of the target under in vivo
infection conditions [54]. Moreover, the new antibiotics should have a low tendency to provoke
development of bacterial resistance due to a single point mutation.
In summary, validation of the essential nature of the novel target for all species under the in vitro
conditions as well as in the host (during the infection process) is mandatory. Although the techniques
of in vivo target validation have been described, they are only used intermittently. Moreover, in the
absence of a model target inhibitor, validation of the essentiality of the target becomes methodologically
challenging which is often the case with novel or unexplored targets.
3.2.2. Lessons learned from the screening strategies
The target selection and validation are only one part of the problem. The screening strategies
themselves also play an important role in the success of this approach. When the compounds are tested
against an isolated (cell-free) target, penetration through the bacterial cell wall is not an issue. As a
result, the majority of the hits obtained turn out not to be able to penetrate the cell wall and thus will
not exert antibacterial effect [16]. A way to overcome this problem is the design of whole-cell assays
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that allow measurement of the target function in intact bacterial cells. For example, an assay involves
differential sensitivity of target-depleted strains where the target can be silenced (by siRNA) or down
regulated (by reduced promoter expression). In both cases, an inhibitor will exhibit differential
antibacterial activity against mutant versus wild type strains. However, this technique leads to doubling
of the screening efforts as it involves two detection points (i.e. for mutant and wild type strain) for each
time point and inhibitor concentration.
3.2.3. Lessons learned from the compound pool
Our inadequate knowledge about the bacterial physiology and suboptimal screening strategies
does not explain the lack of success in almost all antibacterial high-throughput target-based screening
campaigns. The problem also resides within the compound libraries. It is now widely accepted that the
Lipinski’s ‘rule of five’ is irrelevant for antibiotics when coming from natural sources [58-60].
Moreover, the necessity to penetrate through the bacterial cell wall leads to physicochemical properties
which are significantly different from that of other drugs [60]. In general, it has been observed that
antibiotics are more hydrophilic and high molecular weight compounds. However, the dilemma is that
most of the compound libraries from big pharma companies follow the Lipinski’s ‘rule of five’ (average
physiochemical properties needed for all therapeutic area). As a result only a small subset of compounds
is screened for antibacterial activity. Therefore, it can be concluded that the antibacterial screening
approach should not be limited to compounds which follow Lipinski’s ‘rule of five’, rather it should
consider the more natural product-like physicochemical properties [16].
3.2.4. The valuable contribution of different “omics” strategies
Overall, the genomic era should be recognized for its valuable contributions in our never ending
fight against microbes. While indeed the genomic approach did not deliver what was originally
anticipated in terms of new targets and/or new lead compounds, the contribution of genome mining,
proteomics, comparative genomics and expression profiling and analogous techniques cannot be
underestimated and has helped us gain a better understanding of antimicrobial action and of resistance
mechanisms. Genomic differences between antibiotic-susceptible versus resistant strains can help to
understand resistance mechanisms at the molecular level, which in turn helps to find additional targets
to combat resistance. This could lead to the development of new antimicrobial compounds or co-drugs
which enhance each other’s activities. This in turn will avoid or postpone the development of resistance
[61, 62]. Genome profiling also taught us that antibiotics can have a differential effect on bacterial cells
with in contrast low concentrations having a stimulating and differentiating effect. Herein, some
antibiotics used at sub-inhibitory concentration even induce biofilm formation. This bacterial signaling
mediated by antibiotics is not discussed here but has been nicely covered by Romero et al. [63].
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3.3. Targeting Virulence Factors: to Kill or Not to Kill?
With the emergence of multidrug resistance, it is not sufficient to target only essential enzymes
in the biosynthetic pathways, rather one should consider the virulence factors as a target for antiinfectives [64, 65]. The idea behind this strategy is not to kill but rather to hinder the pathogen so as to
cause harm to the host. This is a ‘kinder and gentler’ approach in antibiotic discovery [65]. This
approach is debatable because the virulence factors are important for causing harm to a host but not
essential for the survival of pathogens [66]. Moreover, the progress in the field has been hampered by
the difficulties in development of an in vitro assay to screen inhibitors. On the contrary, virulence factors
are attractive targets for a number of reasons. Firstly, being outside the infected cell, targeting virulence
factors would circumvent the problem of bacterial cell penetration. Secondly, these targets are absent
in the host which is an added advantage. Thirdly, there is a possibility that compounds that target a
‘non-essential’ process are less prone to develop resistance [67]. Lastly, virulence specific therapeutics
would have minimal impact on human gut flora. Although there are several potential advantages in
targeting virulence factors, questions about the utility of such inhibitors as prophylactic agents, solo
therapeutics or in combination with current antibiotics are always argued [65, 68, 69]. With the current
improvements in the screening methodologies using genetically modified strains, there is hope for new
and effective anti-virulence drugs [70]. In fact, the potential of anti-virulence agents as effective
antibacterial agents is demonstrated by the positive results for the monoclonal antibodies against
Clostridium difficile toxin A [71] and E. coli Shiga-like toxin 2 [72], although this overview does not
intend to include passive immunotherapy. However, targeting virulence factors is more challenging due
to our limited knowledge about the evolution of virulence factors and their roles in the bacterial
physiology [73]. Here below, quorum sensing (QS) and the two components signal transduction (TCST)
system have been described as a possible target.
In general, virulence agents are responsible for the symptoms of an infection, allowing bacteria
to adapt to the host environment. Therefore, one can also aim to modulate the production of these agents
instead of directly inhibiting their action. Hence, blockade of virulence gene expression can be
envisaged and has been shown to significantly attenuate infections by disarming bacteria. This gene
regulation strategy for virulence factors has been adequately documented by Williams and colleagues
and will not be discussed further [74, 75].
3.3.1. Quorum sensing: an unexplored therapeutic strategy
Before discussing about QS inhibitors as potential antibacterial agents, a brief overview of QS is
necessary. QS is a fundamental process of bacterial cell-cell communication by producing and
responding to small diffusible molecules that act as signals. These small molecules are called
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autoinducers (AIs) [76, 77]. AIs are produced in response to the changing population density to
coordinate the expression of virulence factors in both Gram-positive and Gram-negative bacteria. There
are three types of AIs that have been identified so far: N-acylated homoserine lactone (acyl-HSL),
autoinducing peptide (AIP) and the boronate ester autoinducer-2 compounds (AI-2). In Gram-negative
bacteria, acyl-HSL acts as an autoinducer whereas small peptides (AIP) play the same role in Grampositive bacteria [78, 79]. AI-2 is an interspecies autoinducer. Representative structures for all three
types of AIs are depicted in Fig. 4 (compound 10-12).
The LuxI/LuxR bioluminescence system of Vibrio fischeri is the most intensely studied QS
system and will be discussed here as representative example. As shown in Fig. 5 [80], bacteria produce
diffusible AIs (e.g. acyl-HSL) via the inducer synthase (LuxI-type protein). As cell population density
increases, the concentration of AI inside and outside the cell increases. At critical AI concentration, an
autoinducer receptor protein (LuxR) binds to the AI. The AI-LuxR complex most often homodimerizes
and binds to adjacent QS promoter (luxICDABE, termed as ‘lux boxes’), which then activates a variety
of group behavior such as expression of virulence factors, swarming and biofilm formation, besides
many other activities [67]. As QS plays a vital role in establishing infections by most of the pathogens,
interception of QS represents an ideal strategy to disarm the pathogen. The QS system can be targeted
at three points: suppressing the acyl-HSL production (LuxI), inactivating the signal molecule (AI) and
blocking the receptor (LuxR). Inhibition at any one of these points is expected to lead to a bacterial
communication breakdown.
3.3.2. QS inhibitors
3.3.2.1. Targeting the synthase (I)
Acyl-HSL synthases are considered as ideal antimicrobial targets as they are absent in eukaryotes
and they are essential for QS. Inhibition of synthase protein appears a straightforward approach, as cellcell communication is impossible without AI. However, as these crystal structures of LuxI from
different species were reported more than a decade ago [81, 82] there are only very few reported studies
that specifically target LuxI [83]. Moreover, measuring the enzymatic activity is also cumbersome.
Lately, a few synthase inhibitors have been identified from HTS [84]. In Burkholderia glumae wild
type (wt) N-octanoyl-homoserine lactone (C8-HSL) is synthesized by TofI (acyl-HSL synthase from B.
glumae). In 2011, Rhee et al. screened a library of 55 compounds for inhibition of QS-mediated
production of toxoflavin and C8-HSL and identified two hits along with a few minor hits. The natural
AI (C8-HSL, 13) and the hits J8-C8 (14) and E9C-3oxoC6 (15) are depicted in Fig. 6. Although both
compounds inhibited the synthesis of C8-HSL and also inhibit QS-dependent ahpF expression, E9C3oxoC6 was shown to be more potent than J8-C8. Detailed mechanistic studies suggest that the
13
compound J8-C8 inhibits TofI (C8-HSL synthesis) whereas E9C-3oxoC6 competitively inhibits
binding of C8-HSL to the toxoflavin receptor (TofR).
3.3.2.2. The acyl-HSL inactivation
Degradation or inactivation of the signal molecule would be a complementary strategy to combat
the QS dependent virulence factor expression. Indeed, several organisms including prokaryotes and
eukaryotes are known to degrade the acyl-HSL signal in order to block QS of invading pathogens. There
are several ways by which degradation of acyl-HSL can take place such as increase of pH at the infection
site (in certain plants species) provoking hydrolysis of the lactone, secretion of oxidized halogenated
compounds which can react with acyl-HSL leading to inactive derivatives, secretion of degrading
enzymes such as lactonase or acylase, use of acyl-HSL as carbon and nitrogen source. Although acylHSL inactivation according to current views does not represent a viable strategy in human and animals,
it can have significant value for agricultural application. For example, transgenic plants producing acylHSL lactonase have shown to be resistant to infections caused by several Gram-negative plant
pathogens [85].
3.3.2.3. Targeting the receptor (R)
The third target in the QS system is the receptor (LuxR type) protein. As compared to the
synthase, it is a widely studied medicinal chemistry target with significant genetic, biochemical and
structural data being available. In 1986, Eberhard et al. [86] evaluated ~20 acyl-HSL analogues for their
agonistic and antagonistic activity against V. fischeri B-61 (a strain that produces more light with
addition of exogenous 3-oxo-acyl-SHL). The authors concluded that the -lactone ring is a prerequisite
for LuxR agonistic activity whereas replacement of the -lactone ring with the thiolactone or lactam
yielded compounds with inhibitory or no activity. Secondly, the optimal chain length for agonistic
activity was six carbons and deviating from this length resulted in inhibitory activity, with a nine carbon
long side chain being the optimum for antagonistic activity [87]. Moreover, 3-oxo groups were claimed
to be important for potency. The most active compounds of the series are shown in Fig. 7 (structures
16-18). A decade later, in 1996, Greenberg et al. [87] evaluated the activity of acyl-HSL analogues in
the E. coli reporter strain VJS533 pHV200I, which harbors LuxR and V. fischeri luminescence gene
cluster with luxI inactivated. This study revealed the same results as reported by Eberhard et al. [86].
More recently, De Keersmaecker et al. [88] reported different strong activators of a LuxR homologue
from Salmonella enterica and of V. fischeri.
In 2002, inspired by halogenated furanone modulators of QS, Reverchon et al. [89] reported a
series of acyl-HSL analogues having various substitutions on the 3-and 4-position of -lactone ring and
14
studied their activities against LuxR. The authors discovered that substitution on the 4-position resulted
in decreased agonistic activity whereas substitution at the 3-position is well tolerated. In fact, the
compounds 19 and 20 (Fig 7) act as antagonists at high concentration. Moreover, R stereochemistry at
the 3-position (compound 21, Fig. 7) is important for agonistic activity while inversion of
stereochemistry resulted in a 1000-fold reduced activity.
The authors concluded that branched alkyl or cycloalkyl acyl-HLSs are potent agonists, with
notably cyclohexyl acyl-HSL being as potent as the natural ligand [89]. Further, replacement of the
acyl-HSL amide with sulfonyl or urea moieties resulted in compounds that were potent LuxR inhibitors
[90, 91] (see Fig. 7 compounds 22 and 23). In 2008, Blackwell et al. [78] revealed several modulators
of LuxR ranging from agonist to antagonist. They found that phenylacetanoyl homoserine lactones
(PHLs) with an electron withdrawing group at the para-position of the phenyl ring were the most potent
inhibitors of LuxR (compound 24, Fig. 7). Lately, Wang et al. [92-94] reported a series of AI-2 quorum
sensing inhibitors through structure-based virtual screening. Representative examples of these sulfone
and adenosine derivatives are depicted in Fig. 8 (compound 25-30). A recent and extensive review on
acyl-homoserine lactone quorum sensing by Greenberg et al. [95] is worthwhile consulting for this
specific topic.
3.3.3. Challenges in the QS targeting strategy
There are many hurdles while considering QS as a therapeutic target. Development and
standardization of the assay method is one of the largest hurdles in QS targeting. It has been found that
not all compounds display similar activity versus different strains. This lack of compatibility can be
overcome by using an in vitro assay with purified protein. However, the progress has been hampered
due to difficult purification and manipulation of these proteins in the absence of a natural ligand.
Researchers have used invertebrate or plant species as a surrogate model to study bacterial pathogenesis
[96-98]. While these surrogate models cannot be used to address pharmacokinetic issues, they can be
used for rapid screening of large compound libraries [99, 100]. Moreover, it has been shown that QS
mutant strains of P. aeruginosa displayed reduced virulence as compared to wild-type P. aeruginosa,
but none of the mutations resulted in avirulent strains [67, 85, 101]. This suggests that besides QS, other
factors also play a key role in infection [102]. Finally, QS is a new and rapidly expanding field and we
have limited knowledge about the mode of action of the QS inhibitors.
3.3.4. Two-component signal transduction systems (TCSTS)
The bacterial two-component signal transduction system is responsible for sensing the
extracellular environment and for responding accordingly so that the bacteria can survive. The essence
15
of TCSTS lies in the recognition and interpretation of external signals and conversion of those signals
into transcription activation or repression of specific genes. It is one of the important systems in which
information is processed. The factors regulated by TCSTS include host invasion, drug resistance,
motility, phosphate uptake, osmoregulation, nitrogen fixation and other functions. Bacterial TCSTS
typically comprise of a membrane-bound histidine kinase (HK, sensor kinase) and a cytoplasmic
regulator [103]. In response to external signals, the HK catalyzes ATP-dependent autophosporylation
on a histidine residue. The phosphoryl group is subsequently transferred to an aspartate residue on a
cognate response regulator. Phosphorylation of the response regulator activates the DNA-binding
properties of the C-terminal domain, resulting in expression modulation of the virulence factors (Fig.
9) [104].
A number of features of TCSTS make them a potential target for antimicrobial therapy. At First,
significant homology exists among the HK and response regulator proteins, in particular around the
active sites, so that TCSTS inhibitors can yield broad-spectrum antibiotics [105, 106]. Secondly,
TCSTS is responsible for expression of virulence factors which are essential for survival of pathogens
inside the host [107]. Furthermore the Ser/Thr kinase signaling pathways in eukaryotes are different
from HK-mediated signal transduction in prokaryotes. This difference can be used for selective
targeting of bacterial TCSTS [104].
3.3.5. Inhibitors of TCSTS
Understanding how TCSTS works, provides several potential points of intervention like signal
sensing, autophosphorylation of the sensor kinase, phosphotransfer to the regulator, or
dephosphorylation of the regulator. Biochemical assays used to screen potential inhibitors are based on
the autophosphorylation reaction of HK and the subsequent phosphoryl transfer to the response
regulator with ATP as the phosphate donor [108]. In recent years, some TCSTS inhibitors have been
discovered by pharmaceutical industry through screening of large chemical libraries and a structure
activity relationship (SAR) program of the resulting lead compounds. One representative structure of
each chemical class found, is depicted in Fig. 10 (compounds 31-39). Among them, the compound
RWJ-49815 (compound 31) and its derivatives are claimed to exhibit bactericidal activity against
several Gram-positive bacteria. Their activity spectrum includes MRSA, VRE and penicillin resistant
S. pneumoniae [104]. Most of the hits obtained via screening of large compound libraries often suffer
from poor selectivity and specificity. However, with recent advancements in the structure determination
of several proteins of TCSTS, the stage is now set for rational structure-based drug design. Finally, in
a recent review Gordon et al. [74] discussed the medicinal chemistry perspective of attenuating
virulence gene regulation for the possible treatment of staphylococcal infections.
16
3.4. Targeting Non-Multiplying Bacteria
In clinical infections, bacteria exist in two different states which are described as multiplying
(logarithmic phase) and non-multiplying (stationary, dormant or latent phase) [20, 109]. Most of the
multiplying bacteria are killed by antibiotic therapy whereas non-multiplying or slowly multiplying
bacteria can survive with repeated doses of antibiotics. Although, non-multiplying bacteria do not cause
disease, they act as a pool for multiplying bacteria and are responsible for recurrence of infection. These
non-multiplying bacteria are difficult to eradicate by antibiotics resulting in prolonged therapy,
emergence of resistance and reduced patient compliance (e.g. tuberculosis (TB) and leprosy) (Fig. 11)
[20]. A possible solution to this problem, which at present is mostly related to mycobacterial infections,
could be to target multiplying and non-multiplying bacteria simultaneously. This strategy in theory
should shorten the duration of therapy, slow down or avoid emergence of resistance, decrease the cost
of therapy and increase the patient compliance.
3.4.1. Existing antibiotics and new developments against non-multiplying bacteria
There are few marketed antibiotics known to kill non-multiplying bacteria more effectively than
others and these are found among the inhibitors of mycobacterial infections. Examples of these
antibiotics include pyrazinamide (PZA, 40, Fig. 12), moxifloxacin (41, Fig. 12) and rifampicin (42, Fig.
12). The antitubercular activity of pyrazinamide is pH dependent. Recent studies have shown that
pyrazinamide enters Mycobacterium tuberculosis by passive diffusion. It is then metabolized by
nicotinamidase or pyrazinamidase to pyrazinoic acid (POA) which is then excreted by a weak efflux
pump. Under acidic conditions, protonated POA (HPOA) is reabsorbed and accumulates in the cell (due
to the inefficient efflux pump) leading to increased acidity and cell death. Despite of excellent in vivo
activity, PZA is inactive against M. tuberculosis under in vitro or culture conditions close to neutral pH.
Furthermore, it has no bactericidal activity against rapidly growing bacilli in patients during the first
two days of treatment [110, 111]. Addition of moxifloxacin and gatifloxacin to the regimen of
rifampicin, pyrazinamide and isoniazid increases the death of a non-multiplying culture of M.
tuberculosis [112]. These results motivate researchers to search for new antibiotics targeted to nonmultiplying bacteria and aimed at shortening treatment, increased patient compliance and cure rates.
In the last eight years, five drug candidates have entered clinical trials for the treatment of
genetically resistant strains of M. tuberculosis. Chemical structures of these drug candidates are given
in Fig. 13. These drug candidates include PA-824 (compound 43, Fig. 13) (Pathogenesis/Chiron and
GATB), [113] diarylquinolone R207910/TMC 207 (compound 44, Fig. 13) (Tibotec/Johnson and
Johnson) [114], nitroimidazole OPC-67683 (compound 45, Fig. 13) (Otsuka Pharmaceuticals) [115],
diamine SQ109 (compound 46, Fig. 13) (Sequella Inc and NIH) [116] and Pyrole LL 3858 (compound
47, Fig. 13) (Lupin Ltd) [117].
17
PA-824 (compound 43, Fig. 13) is a nitroimidazole derivative with a complex mode of action
which is active against both multiplying and non-multiplying bacteria (M. tuberculosis). Under aerobic
condition, it acts by inhibiting the cell wall biosynthesis (i.e., the mycolic acid part) through an unclear
mechanism whereas under anaerobic condition, it acts as an oxide nitric (NO) donor that causes
respiratory poisoning through formation of nitric oxide. Recently it has been tested in a Phase II clinical
trial for treatment of resistant TB and was shown to be safe, well tolerated and effective at doses of 100200 mg/day [118]. Bedaquiline (TMC 207, compound 44, Fig. 13) belongs to the diarylamine class and
has a unique mode of action inhibiting the ATP-synthase. It is currently in a phase III trial for treatment
of patients with pulmonary multidrug resistance (MDR)-TB. The compound OPC 67683 (compound
45, Fig. 13) likewise is under phase III clinical evaluation for treatment of MDR-TB. It acts by inhibiting
the synthesis of methoxy and keto-mycolic acid biosynthesis. SQ 109 (compound 46, Fig. 13) was
identified through random screening of large chemical libraries based on a 1,2-diethylamine scaffold.
Phase I and Phase II data suggest it to be safe and well tolerated. Although the pyrrole LL 3858
(compound 47, Fig. 13) showed good efficacy in a mouse model (12.5 mg/Kg, for 12 weeks), its mode
of action is unclear.
Extensive research likewise has been ongoing for the membrane-active agents XF-70 (compound
48, Fig. 13) and XF-73 (compound 49 Fig. 13) [119], which showed nice bactericidal activity against
slow-growing and non-dividing cultures of S. aureus, including against biofilms. With HT61, a small
quinolone-derived compound (undisclosed structure) has been heralded recently being strongly active
against non-multiplying bacteria as well as against mupirocin resistant MRSA [120]. It effectively kills
these non-multiplying cells very rapidly by permeabilization of the cell membrane and destruction of
the cell wall. Synergistic activities were noticed upon combination with either neomycin, gentamicin or
chlorhexidine [121, 122].
3.4.2. New molecular targets in non-multiplying bacteria
In spite of the complex signaling pathway regarding virulence gene expression, and the multitude
of effects that these virulence factors can have, some research groups have tried to identify specific
genes responsible for antibiotic tolerance in non-multiplying bacteria. Uncovering such a gene could
make it possible to develop an inhibitor interfering with its gene product thus rendering non-multiplying
bacteria susceptible to antibiotics. E. coli has been studied extensively in search for such target in nonmultiplier bacteria. These studies revealed that bacteria express the sigma factor RpoS (RNA
polymerase sigma S, main regulator of stationary phase genes) when reaching the stationary phase. This
factor is responsible for decreased drug sensitivity in an acrAB mutant (inactive efflux pump) [123]
which implies that RpoS might be involved in multidrug resistance. Deletion of rpoS in P. aeruginosa
results in a mutant which is much more sensitive to antibiotics than the wild-type, confirming this
hypothesis [124]. The phosphate regulon (PhoU) is also an interesting target and believed to be involved
18
in tolerance to antibiotics and stress. Deletion of phoU in E. coli results in increased susceptibility of
the mutant as compared to wild-type, in both multiplying and non-multiplying bacteria. PhoU is
proposed to regulate cell mobility, nutrient transport and phosphate and energy metabolism, functions
which are all linked to antibiotic tolerance. Another potential target is the penicillin-binding protein 1b
(PBP1b) [125], or the intergenic region between aldB (aldehyde dehydrogenase B) and yiaW (inner
membrane protein) [126], which both also are linked to antibiotic tolerance [127].
3.4.3 Challenges in targeting non-multiplying bacteria
Although, theoretically, targeting non-multiplying bacteria seems a promising and attractive
strategy, there are several important issues which need to be addressed. Above mentioned antibiotics
are relatively ineffective at killing non-multiplying bacteria. In fact, none of these drugs (either
marketed or in clinical trials) have been screened using whole non-multiplying bacteria or molecular
targets that are essential for bacteria in their dormant state. This approach is less viable due to poor
penetration of compounds into the bacterial cell and there might be a limited number of targets due to
low or no metabolic activity [123, 128]. Secondly, each microorganism has many non-multiplying
forms. For example, persisters contribute only 1% of the non-multiplying population, and were found
to have a different expression profile than log phase and non-multiplying bacteria (with “persisters”
referring to genetically drug susceptible quiescent (non-growing or slow growing) organisms that
survive exposure to a given antibiotic or drug and have the capacity to revive (regrow or resuscitate and
grow) under highly specific conditions [129]). Here the question which needs to be addressed is which
form of non-multiplying bacteria should be targeted [130]? It is still not clear which forms of in vitro
non-multipliers are equivalent to those found in the host during clinical infection [131]. Finally,
standard tests need to be developed for comparing antibiotic potency [132].
More recently, however, a new milestone was reached by the research group of Den et al. [133,
134], who succeeded in reducing the persistence during the growth of both E. coli and P. aeruginosa
and to restore the antibiotic susceptibility of persister cells using the quorum sensing inhibitor (Z)-4bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8, compound 50, Fig. 13). Persister control
was noted for both planktonic and biofilm cells for both bacteria. While this compound showed potent
activity in persister control, it could not be determined whether this was through genuine QS inhibition
or whether the effect is mediated by another target. The latter seems plausible as a similar effect of
reverted antibiotic tolerance was noted for an E. coli luxS mutant.
3.5. Bacteriophage Therapy: Old Dogma New Tricks?
In the early part of 20th century, Frederick Twort and Felix d’Herlle discovered bacteriophage
independently [135]. Bacteriophages or simply phages are viruses with an ability to infect and kill
bacteria, which forms the basis for using phages as therapeutic agents [136, 137]. Immediately
19
following their discovery, bacteriophages have been used clinically during the pre-antibiotic era (19201930s) [138]. The use of bacteriophages as biological antibacterials has persisted globally. This includes
former Soviet Republic of Georgia, Poland, France, Russia, United States, etc. Use of phages as
therapeutic agent offers several advantages. Firstly, phages are bacterium specific, attacking specific
bacteria without affecting normal microflora. Secondly, phages replicate naturally and exponentially
and are therefore effective in treating infections. Finally, they are easy to prepare and to use and show
no adverse effect when used in a cocktail (multi-component phage preparation).
3.5.1. Limitation of phage therapy
The use of bacteriophages as antibacterial agents was abandoned due to discovery of broadspectrum antibiotics coupled with other factors like poor understanding of phage biology, shortage of
data from clinical trials and lack of documentation at that time. Despite of their advantages, there are
several key issues regarding the use of phages as antibacterial agents. Firstly, phages are pathogen
specific, therefore displaying a narrow spectrum of activity which limits their use as therapeutics in
absence of a confirmed diagnosis of a specific pathogen. Secondly, in order to use a phage preparation
effectively, it is necessary to have a set of well characterized phages available for a broad-range of
bacterial pathogens [139]. Thirdly, there are serious difficulties in standardizing protocols for
preparation and quality control. Further, when used systemically, phages may induce an immunogenic
response or neutralizing antibodies. Finally, there is the potential of triggering a toxic shock due to
massive bacterial lysis [140, 141].
Despite of these disadvantages, phage therapy was part of the standard healthcare system in the
Soviet Union even during the 1960s and 1970s. In former Soviet Union, phage preparations were used
for therapy, prophylaxis and diagnosis of many bacterial infections [142]. In 1990, Abdul Hassan et
al.[143] documented the use of phage therapy for the treatment of 30 cases of burn wound associated
antibiotic-resistant P. aeruginosa sepsis. In 18 of 30 cases there was good (12 cases, 60-70% recovery)
or even excellent (6 cases with >90% recovery) improvement in skin graft take [143]. However, the
authors recommended phage therapy should be restricted to antibiotic-resistant strains.
In 2005, Jikia et al. [144] reported the use of bacteriophage to treat 90Sr radiation burns suffered
by three lumberjacks from Georgia. Subsequently, the burns became infected with MRSA. After
unsuccessful treatment with antibiotics for one month, phage therapy was attempted using
PhageBioDerm along with ciprofloxacin. Following 2 days of treatment, purulent drainage decreased
to almost none and testing failed to detect MRSA on the seventh day. As no negative controls were
employed, this study does not provide proof-of-concept for phage therapy. Similarly positive outputs
of phage therapy have been reported by Lazareva et al. [145]. In 2007, Fralick et al. [146] studied the
effect of a phage cocktail in treatment of P. aeruginosa infection in a mouse burn wound model. In this
20
study, a number of 2x102 to 3x103 rifampicin-resistant P. aeruginosa (POA1Rif) were injected at the
burn site. A phage cocktail inoculum (~3x108/100 μL) was administered by intraperitoneal (i.p.),
intramuscular (i.m.) or subcutaneous (s.c.) route, respectively. The authors observed that not all treated
mice did survive and that the route of phage administration played an important role in the treatment
efficacy with the i.p. route providing maximum protection (87%) [146].
Herein, “lysis from without” or a non-productive phage infection kills bacteria as of its high
multiplicity of virion adsorption [138]. This is an important mechanism of killing bacteria via high
dosing regimens, but is not the strategy which should be aimed for in view of a possible immunogenic
response or toxic shock. Moreover, in view of concerns for an immune response even with a lower
inoculum followed by the normal “lysis from within” induced by phage proteins, Paul et al. [147] in
contrast developed lysis-deficient phages by disruption of the endolysin gene. These still kill the target
cells but are incapable of host cell lysis, but such recombinant phage still proved lethal to methicillinresistant S. aureus [147]. A recent review paper also discusses the biocontrol of bacterial infections
using phage therapy from a physiological pharmacology standpoint [148].
Moreover, even today, scientists of the military hospital in Brussels and of KU Leuven are joining
force to evaluate and use phages for the treatment of severe burns [149]. Recently, use of phage therapy
to treat burn wound was evaluated in a small phase I study at the Burn Wound Centre of the Queen
Astrid Military Hospital, Brussels, Belgium. A small group of 9 patients were treated with the BFC-1
phage cocktail containing a mixture of 3 lytic phages: Myovirus, Podovirus against P. aeruginosa and
Myovirus against S. aureus. In the study, a large burn section was exposed to a single spray application
of a phage cocktail while a distant portion of the wound was taken as control. The complete results of
this study are yet to be published. However, a clear legal framework for bacteriophage therapy is largely
lacking and this forms the bottleneck to establish this methodology as a valid treatment option [150,
151].
4. CONCLUSIONS
With the emergence of multidrug resistance, there is an urgent need for new antibacterial
agents. Chemical modifications of existing antibiotics yielded short term success with rapid
development of cross resistance among the same class of antibiotics. Moreover, the success in
discovering antibiotics through a conventional approach is limited (only two new classes of antibiotics
entered the market in last 50 years). The SBDD approach has been successfully used for the design of
a DHFR inhibitor (iclaprim) where the molecular basis of resistance has been utilized for antibiotic
design. Phase III clinical trials demonstrate that iclaprim is well tolerated and effective in treating
complicated soft structure skin infection which clearly shows that SBDD is a powerful tool for antibiotic
discovery. Moreover, SBDD also successfully has been used for the discovery and optimization of
21
ligands for the targets such DNA gyrase subunit B, LpxC, MetRS, etc. Targeting of Lipid II by the
HNP1 defensin small molecule analogues recently uncovered (BAS00127538), seems a valuable
strategy for development of broad-spectrum therapeutics as glycopeptides like vancomycin already
established the essentiality of Lipid II. Likewise, the heterocyclic sulfonamide analogues as inhibitors
of ThrRS and studied by Trius Therapeutics hold potential in view of their high activity and bacterial
selectivity. While they permeate cellular membranes, the antibacterial activity remains mediocre.
Moreover, they also prove efficient substrates for efflux pumps and further optimization of these
compounds is needed to improve their potency.
Although the genomic revolution provided us with a treasure of targets known to be essential
for the bacterial survival, it is not clear yet how many of them are druggable targets. The genomic
revolution is just beginning and it therefore is too early to comment on the success of the genomic
approach. Moreover, validation of the novel target, screening strategies and compound pools also
contribute to failures with the genomic approach in terms of establishing new targets or new leads.
However, comparative expression profiling has thoroughly increased our understanding of resistance
mechanisms and of biofilm formation, which will assist us in developing improved antibiotics.
Anti-virulence strategy is often debatable due to the non-essential nature of the virulence
factors. Targeting virulence factors, either directly or via modulation of their gene expression, seems a
promising strategy as demonstrated by positive results of clinical trials for monoclonal antibodies
against C. difficile toxin A and E. coli Shiga like toxin 2. Moreover, with current improvements in the
screening methodologies, there is hope for a new and effective anti-virulence drug. However, further
research efforts are needed to get insight into the mechanism by which the expression of a virulencerelated gene is activated or inhibited and the development of small molecule inhibitors for modulation
of virulence gene expression has a long way to go. The same holds for the different strategies to inhibit
quorum sensing: some nice activities have been obtained in the test tube, but many challenges remain
before quorum sensing will be established as a viable target for clinically related infections.
Theoretically, targeting multiplying and non-multiplying bacteria simultaneously can shorten the
duration of therapy, decrease the chances of development of resistance and also reduce the cost for
therapy and increase the patient compliance. However, further clinical relevance of an in vitro inhibitor
of a non-multiplier is still lacking. Recently, five drug candidates have shown effectiveness for treating
MDR-TB and are at various stages of clinical trials. While persistence is vital problem in antibacterial
therapy, recent efforts succeeded in restoring the sensitivity to antibiotics using a quorum sensing
inhibitor. If the latter finding can be confirmed this might prove to be a new milestone in the continuous
fight against bacteria.
Bacteriophages, a revisited treatment modality, offer several advantages which make them
attractive therapeutic agents. The positive outcome of many previous studies suggests that phage
22
therapy, if properly applied, can play a vital role in controlling infectious diseases which remain
otherwise untreated. Herein, genetically modified bacteriophages like the lysis-deficient phages
recently developed maybe could remediate for the missing legal framework for therapeutic use of
phages.
Although none of the discussed approaches (either old or new) has potential to discover new
resistance-breaking antibiotics solely, we must consider applying two or more approaches to improve
the antibiotic discovery process at every stage (e.g. the genomic approach can be used for validation of
the target, HTS for rapid screening of large chemical libraries, SBDD for optimization of a lead, etc).
Besides the discovery of new antibiotics, efforts should also be directed to increase the hygiene, to
reduce misuse or abuse of antibiotics and self-medication. Moreover, a combination of drug regimens
likewise is very effective to reduce the chances of resistance development.
Finally, we wish to come back briefly on our economic statements put forward in the
introduction. As the financial reward for developing new antibacterials was inadequate, American and
European government agencies have wisely implemented additional incentives among which improved
IP protection to drive innovation. Whether this will be sufficient remains to be seen, but the overall cost
for developing new therapeutic classes is skyrocketing. Most researchers aim for and hope to find broad
spectrum antibiotics also for economic reasons, allowing more patients to be treated. Wider use at the
same time however increases the chances for resistance development, especially if only a single cellular
target is under attack. Combination therapies like with HIV and HCV antivirals maybe could become
standard when treating serious infections, but at which economic cost? Narrow spectrum antibiotics
therefore should be reserved for treating deadly infections in an intensive care unit as a last resort
therapy and to avoid resistance generation. Obviously, sufficient remuneration to compensate for the
limited therapeutic use should be implemented. However, society can no longer carry the exuberant
costs as charged for some antivirals, with a multitude of highly specialized treatments coming up for
fighting different cancers, viruses, bacteria and rare diseases. Finding the right balance between
adequate incentives and remuneration versus the overall cost for drug development will need a lot of
consideration in the near future.
CONFLICT OF INTEREST
Both authors declare no conflicts of interest are involved.
ACKNOWLEDGEMENTS
Bharat Gadakh thanks Erasmus Mundus External Cooperation Window lot 13 (EMECW13)
and KU Leuven (GOA/10/13) for financial support. Antibiotic research is made possible by a grant
23
from the FWO (G.0778.14N; Flemish Government) and from the Research Fund KU Leuven
(OT/14/105). We also thank all four reviewers and the guest editor for their constructive criticisms
which considerably improved this review.
ABREVIATIONS
AIP = Autoinducing peptide
AIs = Autoinducers
ATP = Adenosine triphosphate
cSSSI = Complicated soft structure skin infection
DHFR = Dihydrofolate reductase
HK = Histidine Kinase
HSL = Homoserine lactone
HTS = High throughput screening
MDR-TB = Multidrug resistant tuberculosis
MIC = Minimum inhibitory concentration
MRSA = Methicillin resistant Staphylococcus aureus
NCEs = New chemical entities
NMR = Nuclear Magnetic Resonance
PHL = Phenylacetanoyl homoserine lactone
POA = Pyrazinoic acid
PZA = Pyrazinamide
QIDP = Qualified infectious disease product
QS = Quorum sensing
SAR = Structure activity relationship
SBDD = Structure-based drug design
siRNA = Small interfering RNA
SMX = Sulfamethoxazole
TB = Tuberculosis
24
TCSTS = Two components signal transduction system
TMP = Trimethoprim
VRE = Vancomycin Resistant Enterococcus
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FIGURE LEGENDS:
Fig. (1). Chemical structure of trimethoprim (1), sulfamethoxazole (2) and a few key inhibitors obtained
during SBDD (3-5).
Fig. (2). panel A: Flow chart for the process of antibiotic discovery using a genomic approach; panel
B: An illustrative example of large screening campaign of GSK.
Fig. (3). Representative chemical structures of fatty acid biosynthesis inhibitors.
Fig. (4). Representative chemical structures of autoinducer acyl-HSL (10), AIP-1 (11) and AI-2 (12).
Fig. (5). Vibrio fischeri LuxI/R quorum sensing circuit (adapted from reference 80).
Fig. (6). Chemical structures of natural autoinducer (C8-HSL, 13) and TofI inhibitors.
Fig. (7). Representative structures of acyl-HSL antagonists tested against LuxR.
Fig. (8). Some examples of AI-2 mediated QS (LuxPQ, the receptor protein) inhibitors.
Fig. (9). Basic two-component signal transduction system (TCSTS) (adapted from reference 104).
32
Fig. (10). Representative structures of different chemical classes as TCSTS inhibitors.
Fig. (11). Conventional treatment of infections: multiplying bacteria die when treated with antibiotics
(left panel) while little or no effect is noticed on non-multiplying bacteria (right panel). Non-multiplying
bacteria can serve as a reservoir for multiplying bacteria and are responsible for recurrence of the
infection and emergence of resistance (adapted from reference 20).
Fig. (12). Existing antibiotics effective in killing non-multiplying bacteria.
Fig. (13). New drug candidates in clinical trials which target non-multiplying bacteria.
FIGURES
33
Fig. (1). Chemical structure of trimethoprim (1), sulfamethoxazole (2) and a few key inhibitors obtained
during SBDD (3-5).
34
Fig. (2). panel A: Flow chart for the process of antibiotic discovery using a genomic approach; panel
B: An illustrative example of large screening campaign of GSK.
35
Fig. (3). Representative chemical structures of fatty acid biosynthesis inhibitors.
Fig. (4). Representative chemical structures of autoinducer acyl-HSL (10), AIP-1 (11) and AI-2 (12).
Fig. (5). Vibrio fischeri LuxI/R quorum sensing circuit (adapted from reference 80).
36
O
O
O
N
H
N
O
O
O
N
N
H
OH
C8-HSL (13)
J8-C8 (14)
E9C-3oxoC6 (15 )
Fig. (6). Chemical structures of natural autoinducer (C8-HSL, 13) and TofI inhibitors.
Fig. (7). Representative structures of acyl-HSL antagonists tested against LuxR.
37
Fig. (8). Some examples of AI-2 mediated QS (LuxPQ, the receptor protein) inhibitors.
38
Fig. (9). Basic two-component signal transduction system (TCSTS) (adapted from reference 104).
39
Fig. (10). Representative structures of different chemical classes as TCSTS inhibitors.
40
Fig. (11). Conventional treatment of infections: multiplying bacteria die when treated with antibiotics
(left panel) while little or no effect is noticed on non-multiplying bacteria (right panel). Non-multiplying
bacteria can serve as a reservoir for multiplying bacteria and are responsible for recurrence of the
infection and emergence of resistance (adapted from reference 20).
Fig. (12). Existing antibiotics effective in killing non-multiplying bacteria.
41
Fig. (13). New drug candidates in clinical trials which target non-multiplying bacteria.
42