Question : Using the Article below," Write a review of the current status of development of antibiotics." There is not Table only from the article below that you should take informations from to write a review of the status of the development of Antibiotics!!
Article: "Bacterial fatty acid metabolism in modern antibiotic discovery."
Abstract:
Bacterial fatty acid synthesis is essential for many pathogens and different from the mammalian counterpart.
These featuresmake bacterial fatty acid synthesis a desirable target for antibiotic discovery. The structural divergence
of the conserved enzymes and the presence of different isozymes catalyzing the same reactions in the
pathwaymake bacterial fatty acid synthesis a narrowspectrumtarget rather than the traditional broad spectrum
target. Furthermore, bacterial fatty acid synthesis inhibitors are single-targeting, rather than multi-targeting like
traditional monotherapeutic, broad-spectrum antibiotics. The single-targeting nature of bacterial fatty acid synthesis
inhibitors makes overcoming fast-developing, target-based resistance a necessary consideration for antibiotic
development. Target-based resistance can be overcome through multi-targeting inhibitors, a cocktail of
single-targeting inhibitors, or by making the single targeting inhibitor sufficiently high affinity through a pathogen
selective approach such that target-based mutants are still susceptible to therapeutic concentrations of drug.
Many of the pathogens requiring new antibiotic treatment options encode for essential bacterial fatty acid
synthesis enzymes. This reviewwill evaluate themost promising targets in bacterial fatty acid metabolismfor antibiotic
therapeutics development and review the potential and challenges in advancing each of these targets to
the clinic and circumventing target-based resistance. This article is part of a Special Issue entitled: Bacterial Lipids
edited by Russell E. Bishop.
Introduction:
Phospholipid synthesis is one of themajor biosynthetic pathways in
all living organisms, and fatty acids form the acyl chains found on all
phospholipids. Fatty acid synthesis is essential inmany bacterial pathogens,
and significant differences exists between the structures of
mammalian type I fatty acid synthesis and bacterial type II fatty acid
synthesis (FASII). The essential nature of bacterial fatty acid synthesis
coupledwith the ability to target bacterial fatty acid synthesis selectively
over themammalian counterpart has made FASII a focus for developing
novel antibiotics. In this review, we will discuss the potential of the
various molecular targets in fatty acid synthesis to be targeted for antibiotic
discovery.Wewill startwith a brief reviewof antibiotic discovery
to understand how history has shaped thinking in the field. Next, we
will discuss the antibiotic discovery targeting bacterial fatty acidmetabolismand
the potential of enzymes in bacterial fatty acidmetabolismas
antibiotic targets. Finally, we will discuss current thinking in antibiotic
discovery and how to develop novel therapeutics against the targets
in bacterial lipid metabolism.
2. A brief history of antibiotic discovery
2.1. Activity based synthetic compound screening â the first step
The history of antibiotic discovery started fromthe crossroads ofmicrobiology
and organic chemistry in the early 1900s. Having recently
shown microorganisms as the cause of infectious diseases, early researchers
screened for compounds that kill the infectious agentwith reduced
toxicity to the human patient (as measured in animal models).
The German Nobel laureate Paul Ehrlich was the first person to formalize
the idea of a hypothetical agent (âmagic bulletâ) that kills the specific
microbe responsible for the diseases without harming the body [1].
Through synthesizing and screening analogues of the organoarsenic
compound Atoxyl which had antisyphilitic activity but also severe
side effects, Ehrlich's team discovered arsphenamine (Salvarsan) and
neoarsphenamine (Neosalvarsan), the first modern chemotherapeutic
agents. The first antibiotic to reach commercial successwas sulfonamide
drugs discovered by scientists in Bayer AG [2]. A red aniline dye was
found to effectively inhibit bacterial infections in mice after screening
hundreds of dyes. The compound was named Prontosil and was
highly effective against Gram-positive streptococci. Further research
found that Prontosil was a prodrug of the active sulfanilamide.
Sulfanilamide was the first broad-spectrum antibiotic, and was
used on the battlefields of World War II for treating wound
infections.
2.2. Activity based natural product screening â the Golden Age
The Golden Age of antibiotic discovery occurred from the activity
screening of natural products. Alexander Fleming was the first to
show that the pure Penicillium mold was able to produce an agent
with antimicrobial activity in 1928. Fleming was able to isolate the active
compound and demonstrated its antimicrobial activity. Penicillin
had greater clinical efficacy than sulfanilamide and reduced toxic side
effects, but Fleming was unable to garner interest for research into the
industrial production of penicillin due to the success of the recently
commercialized sulfanilamide antibiotics. The mass scale production
of penicillin in the United States was a triumph of the allied war
effort during World War II. Penicillin was so successful that it
defined the characteristics of the ideal antibiotic â broad-spectrum,
monotherapeutic, and low toxicity.
The success of penicillin stimulated natural product screening
efforts and spawned the âGolden Ageâ of antibiotic discovery from the
1940s to the 1970s [3]. Advances in structural biology and medicinal
chemistry allowed researchers to chemically modify the natural
products to produce semi-synthetic antibiotics with improved clinical
properties. The majority of the broad-spectrum antibiotic classes we
use even today including the β-lactams, tetracycline, and macrolides
were discovered through this process. Unfortunately, resistance quickly
caught up with these newly discovered antibiotics, as the discovery of
new antibiotic classes through natural product screening decreased
over time [4,5]. Additional screens largely rediscovered previously
found chemical entities and failed to find promising new chemical
entities. The decreasing return caused many pharmaceutical companies
to leave antibiotic research.
2.3. Target-based discovery â the draught
Major advances in genetics and molecular biology by the 1990s
allowed the identification of the molecular targets of the antibiotics
[6,7]. The first sequenced bacterial genomes were also released at
this time, with the various proteins encoded by the genomes rapidly
characterized [8]. Several hundred proteins essential for bacterial
growth were identified, and thought to be potential antibiotic targets
[9]. The success of fluoroquinolone, a synthetic antibiotic rationally
designed against DNA topoisomerase suggested that other
essentialmolecular targets could be exploited as novel antibiotic targets
as well [10]. Antibiotic discovery entered a new phase. Rather
than screen for active compounds and determine their molecular
target, hits against essential molecular targets were identified and
chemically modified to become successful antibiotics [11]. However,
the end goal for target-based antibiotic discovery remained the same
- to discover another broad-spectrum, monotherapeutic, and low
toxicity penicillin-like antibiotic. To rationally design such an antibiotic,
researchers startedwith an essentialmolecular target found in a
broad spectrum of bacteria [9]. Furthermore, this target would be
nonexistent or significantly different in humans to decrease the
probability of toxicity. High-throughput screening technology was
used to find the lead compounds against the molecular target, and the
lead compound was modified via medicinal chemistry to penetrate
the bacterial cell membrane, possess the ideal broad-spectrum, and
have drug like pharmacokinetic properties. This compound would
then be tested in mousemodels, and would be brought to clinical trials
in humans if successful.
Several large pharmaceutical companies conducted systemic targetbased
discovery campaigns in the late 1990s. GlaxoSmithKline carried
out the best documented target-based antibiotic discovery campaign
[9]. The goal of the campaign was to discover novel antibacterial inhibitors
with either Gram-positive or broad-spectrum activity. Over 350
conserved gene targetswere identified through comparing the genome
sequences of Haemophilus influenzae, Streptococcus pneumoniae, and
Staphylococcus aureus. These conserved genes were genetically verified
for essentiality, and 127 genes were essential in at least one of the
three bacterial species. Several genes encoding for fatty acid synthesis
were validated as conserved and essential in these screens. A total of
67 high throughput screening campaigns against the SmithKline
Beecham compound collection were conducted against the subset of
essential and conserved targets with known function and amenable assays.
Only 5 leads resulted from the HTS screens, and two of the leads
targeted fatty acid synthesis enzymes FabI and FabH. Nearly a decade
later, none of the leads have passed phase III trials, although some are
still under clinical development. The target-based antibiotic discovery
efforts by the other pharmaceutical companies also did not yield clinical
products despite significant investment [12]. The systematic failure
of target-based antibiotic discovery suggests that some underlying
assumptions about the discovery process must be incomplete or
incorrect.
3. Antibiotic targets in fatty acid metabolism
Fatty acids make up the acyl chains of glycerophospholipids,
the major and essential component of bacterial membrane [13,14].
Bacteria synthesize fatty acids using the type II fatty acid synthesis system,
where fatty acids are synthesized from acetyl-CoA precursors
through rounds of 2 carbon elongations using discrete, monofunctional
enzymes [15] (Fig. 1). In contrast, fatty acids are synthesized by the
multifunctional type I fatty acid synthase in mammals, allowing for
selective targeting of FASII [16]. FASII is a validated target, with two of
the five leads discovered in the GlaxoSmithKline HTS campaign
targeting FASII (FabH and FabI) [9]. Targeting fatty acid synthesis for
novel antibiotic therapies via target-based discovery is also an active
area of research, with the FabI inhibitor AFN-1252 having passed
phase II clinical trials and currently undergoing further clinical development
[17â22]. This section summarizes the antibiotic targets in bacterial
fatty acid metabolism.
3.1. Bacterial fatty acid metabolism is a narrow spectrum antibiotic target
The initiation and elongation steps of FASII are shown in Fig. 1
[13,15]. Some of the validated inhibitors of FASII are shown in
Fig. 2. Two features of bacterial fatty acid synthesis reduce the potential
spectrum of antibiotics targeting fatty acid synthesis [23]. First,
fatty acid synthesis is not essential in certain bacteria, such as the
key pathogen Streptococcus pneumoniae, if they are provided an extracellular
source of fatty acids [24,25]. These bacteria can potentially
bypass the inhibition of endogenous fatty acid synthesis by using
host fatty acids to make their phospholipids,making them refractory
to FASII inhibition. Second, there are different enzyme isoforms
performing the same enzymatic reactions in several key steps of
FASII in different bacteria. For example, there are four structurally
distinct enoyl-ACP reductase enzyme forms [26â29] (Fig. 1). This
feature further reduces the spectrum of potential inhibitors of
bacterial fatty acid synthesis. Inhibitors targeting bacterial fatty
acid metabolism will have at its inception narrower spectrum than
the broad-spectrum, monotherapeutic antibiotics commonly deployed
in clinics today. This factor must be considered when designing
inhibitors of bacterial fatty acid synthesis.
Enzymes catalyzing rate-limiting reactions in key regulatory steps
make for the best chemotherapeutic targets. In contrast, fast turnover
enzymes catalyzing equilibrium reactions make for bad chemotherapeutic
targets because a large amount of inhibition is required to
cause relatively little phenotypic effect. There are three fast turnover enzymes
catalyzing equilibrium reactions â FabD, FabZ, and FabA â which
make for poor drug targets. In contrast, the condensation enzymes
FabF and FabH, enoyl-ACP reductases, and the acetyl-CoA carboxylase
enzyme complex are rate-determining reactions at key regulatory
steps, making them desirable drug targets [30]. A number of natural
products and synthetic inhibitors are known for these enzymes, validating
them as antibacterial targets [31,32].
3.2. Acetyl-CoA carboxylase
The acetyl-CoA carboxylase complex catalyzes the irreversible carboxylation
of acetyl-CoA to make malonyl-CoA using the energy from
hydrolyzing ATP to ADP [33]. This reaction is the first committed, regulated,
and rate-limiting step in fatty acid synthesis, making it a good
chemotherapeutic target. The acetyl-CoA carboxylase complex is composed
of 4 protein subunits catalyzing two half reactions in bacteria. Biotin
carboxylase (AccC) catalyzes the carboxylation of the biotin
prosthetic group attached to biotin-carboxyl carrier protein (AccB) in
the first half reaction. The carboxyltransferase complex (AccA and
AccD) transfers the carboxyl group from the biotin to acetyl-CoA to
form malonyl-CoA. The bacterial acetyl-CoA carboxylase complex is
highly conserved [31,34] and structurally different from the mammalian
acetyl-CoA carboxylase complex which is organized as a single polypeptide
rather than separate protein subunits [35],making it a potential
broad-spectrum antibiotic target.
The effective spectrum of acetyl-CoA carboxylase inhibition is
still being investigated [25]. Several natural products such as
moiramide B and andrimid block the growth of bacteria through
on-target inhibition of fatty acid synthesis in standard laboratory
culture media without fatty acids [36]. Plants also encode for a
bacteria-like acetyl-CoA carboxylase complex, and the plant enzyme
is targeted by commercial herbicides [37]. Extensive effort at designing
ACC inhibitors for antibacterial therapy has been attempted [38,
39], including designing dual-ligand inhibitors that simultaneously
inhibit two subunits of the acetyl-CoA carboxylase complex [40].
Exogenous fatty acids can overcome Andrimid inhibition in both
Staphylococcus aureus and Streptococcus pneumoniae in planktonic
growth, and a Staphylococcus aureus strain encoding an inactive
acetyl-CoA carboxylase can grow in laboratory culturemedia supplemented
with exogenous fatty acids [25]. However, this same strain of
Staphylococcus aureus cannot proliferate in a mouse sepsis model
illustrating the importance of in vivo testing [41]. Acetyl-CoA carboxylase
inhibition is expected to be effective against the model
Gram-negative bacterium Escherichia coli because the essential lipopolysaccharide
synthesis requires β-hydroxyacyl-ACP made from
endogenous fatty acid synthesis [42,43]. Acetyl-CoA carboxylase is
also essential for Pseudomonas aeruginosa [44]. Whether acetyl-CoA
carboxylase is essential for Gram-negative bacteria with nonessential
lipopolysaccharides, such as Neisseria [45,46], remains to be
validated.
3.3. Condensation enzymes
The condensation enzymes, FabH, FabF, and FabB, catalyze a
Claisen condensation using malonyl-ACP as the nucleophile to elongate
the acyl chain by two carbons at a time [30,47]. FabH initiates
fatty acid synthesis by condensing a malonyl-ACP with acetyl-CoA
to acetoacetyl-ACP [15]. Branched-chain acyl-CoA precursors such
as 2-methylbutyryl-CoA are used in place of acetyl-CoA for the synthesis
of branched chain fatty acids [48]. FabF initiates each round
of 2 carbon elongation through the condensation of malonyl-ACP
with acyl-ACP [15]. FabB has similar function as FabF, but is essential
for the elongation of unsaturated fatty acids in bacteria expressing a
FabA [49,50]. FabF and FabB have a Cys-His-His catalytic triad, and
FabH has a Cys-His-Asn triad [51]. Condensing enzymes proceed
via a Ping-Pong mechanism where the active site cysteine attacks
the thioester of the acetyl-CoA to make the acyl-cysteine thioester
intermediate [52â54]. After the release of the CoA, decarboxylation
of the carboxyl group of malonyl-ACP makes an enolate intermediate.
Nucleophilic attack from the enolate group on the acyl-cysteine
thioester elongates the acyl group by two carbons.
The condensing enzymes have been an active area of antibacterial
research. FabH was genetically essential in the GlaxoSmithKline antibacterial
screening efforts and FabH inhibitors have demonstrated
in vivo activity [9]. A variety of natural product and synthetic inhibitors
also target the condensing enzymes [13,32]. Due to the similar enzymatic
reactions catalyzed by the condensing enzymes, inhibitors against
the condensing enzymes have the potential to be dual targeting. Several
natural product inhibitors have been demonstrated to target both
classes of condensing enzymes [55,55â57], including cerulenin which
is a covalent inhibitor of FabB and FabF [58,59].
3.4. Reductases
There are two reduction reactions in each elongation cycle of fatty
acid synthesis [15]. Enoyl-ACP reductase catalyzes the reduction of
trans-2-enoyl-ACP into acyl-ACP. Because the dehydration step preceding
enoyl-ACP reductase in the elongation cycle is an equilibrium
reaction, enoyl-ACP reductase is one of the rate-determining steps in
fatty acid synthesis [60,61]. There are four characterized isoforms of
enoyl-ACP reductase. The FabI enoyl-ACP reductase isoform was first
discovered in E. coli and a target of the biocide triclosan [26,62,63].
The FabL isoform is distantly related to FabI and found in addition to
FabI in Bacillus species [27]. The FabK isoform is a flavoprotein with no
structural relationship to FabI [28]. FabK is the primary enoyl-ACP reductase
in Streptococcus species and a significant number of other
firmicutes [64]. The FabV isoform is the least characterized isoform
and is found predominately in γ-Proteobacteria clades such as Vibrio,
Yersinia, and Pseudomonas species [29,65]. The FabV isoform may have
originated from Pseudomonas fluorescens encoding for the synthesis of
natural product inhibitors of FabI [66].
FabI is a validated antibiotic target [63,67], and FabI inhibitors are
currently being evaluated in clinical trials [17â22]. Although FabI is
not the most widely distributed enoyl-ACP reductase isoform, FabI is
the essential enoyl-ACP reductase in a number of key pathogens
including Staphylococcus, Neisseria, Enterococcus, Acinetobacter, and Enterobacter
species [25,68,69]. The biocide triclosan targets the FabI in
Staphylococcus aureus and E. coli [63,67] in addition to acting as a nonspecific
biocide [28,70]. The anti-tuberculosis drug isoniazid also targets
InhA in mycolic acid synthesis, which is the homologue of FabI in
Mycobacterium tuberculosis [71]. Although concerns have been raised
that exogenous fatty acids could bypass FabI inhibition [24], experiments
in Staphylococcus and Neisseria have conclusively shown that
exogenous fatty acids cannot bypass FabI inhibition in these organisms
[25,68]. Because FabI is not broadly distributed in bacteria,most FabI antibiotic
discovery has focused on Staphylococcus.
The β-ketoacyl-ACP reductase FabG catalyzes the reduction of β-
ketoacyl-ACP into β-hydroxyacyl-ACP [15]. In contrast to enoyl-ACP
reductase, there is only one highly conserved β-ketoacyl-ACP reductase
isoform that has been characterized, suggesting that inhibitors
targeting this enzyme would have broad-spectrum activity. However,
FabG does not appear to have a rate-controlling role in fatty acid
synthesis, making it less desirable as a target. While several natural
product FabG inhibitors have been reported [72,73], there has been
very little follow through research to validate the selectivity of
these inhibitors or develop the compounds further. FabG remains
to be validated as a suitable target for drug discovery.
3.5. Acyltransferases
Acyl-ACP of the appropriate length becomes substrate for the
acyltransferases and the synthesis of phosphatidic acid, the precursor
to all phospholipids in bacteria [48]. The structures of the enzymes involved
in bacterial phospholipid synthesis are poorly defined because
they are typically membrane bound proteins. Phosphatidic acid is
made by two successive acylations of glycerol-3-phosphate using acyl-
ACP. The PlsC enzyme involved in the second acylation step is conserved
in both bacteria andmammals and make a poor antibiotic target. Three
enzymes, PlsB, PlsE, and PlsY, are involved in the first acylation step. The
PlsX/PlsY acyltransferase system is the most widely distributed in
bacteria [74]. All characterized bacteria use the PlsX/PlsY acyltransferase
system except for γ-Proteobacteria which uses PlsB and Chlamydia
which uses PlsE [75]. PlsB and PlsE share the same HX4D catalytic
motif and substrate as PlsC and may not be expected to allow for
selective antibacterial targeting [76]. Incontrast,PlsYbelongs to-another
protein family and uses acyl-phosphate made by PlsX for the acylation
reaction [77]. Analogues of acyl-phosphate show that the on-target inhibition
of PlsY inhibits bacterial growth [78,79]. However, current
acyl-phosphate analogues are too hydrophobic to be âdrug likeâ, and
the lack of PlsY structure hampers improved compound design [78,
79]. Because PlsX catalyzes the reversible conversion between acyl-
ACP and acyl-phosphate [80,81], acyl-phosphate analogues could be
dual targeting inhibitors of PlsX and PlsY.
4. Antibiotic discovery in bacterial fatty acid metabolism
4.1. Resistance in antibiotic discovery
In addition to targeting an essential molecular target, resistance
must also be considered in antibiotic discovery. Themechanismof resistance
against an inhibitor determines the time frame that the inhibitor
is therapeutically relevant. If high level resistance to the inhibitor is
able to develop during the course of treatment, then the inhibitor stands
a significant chance of failure during therapeutic treatment and will
likely fail clinical trials. Resistance development to the inhibitor must
be considered during target selection, inhibitor design, and clinical development.
There are fourmechanisms for bacterial resistance to antibiotics
[82]. First, bacteria canmodify the cellwall/membrane to decrease
the permeability of the antibiotic to render the intracellular concentration
of the antibiotic ineffective. Second, bacteria can increase the efflux
of the antibiotic to again decrease the intracellular concentration of the
antibiotic. Third, bacteria can modify the antibiotic to render it ineffective
such as hydrolyzing the β-lactamring or methylation of aminoglycosides.
Finally, bacteria can modify the molecular target of the
antibiotic to decrease the affinity of the antibiotic to the molecular
target.
These four resistance mechanisms arise and spread by lateral gene
transfer and spontaneousmutation. Themode that the resistancemechanismoccurs
affects the rate of resistance development [82]. Of the four
resistance mechanisms, modification of cell wall, increase in efflux, and
molecular target modification occur through spontaneous mutations.
Spontaneous mutation arises from errors in DNA replication leading to
changes in gene function or the expression level of the gene function.
Missense mutations occur at approximately one in 109 bacterial cells,
approximating the error rate in DNA replication [83]. Environmental
stresses can further accelerate themutation rate. This feature of bacterial
physiology means that a strain encoding for a mutant gene product
resistant to the antibiotic might already be present at low populations
in nature, and could quickly develop if it is not already present. While
resistance through modification of cell wall or increased efflux lead to
low level of antibiotic resistance, single amino acid changes via a missense
mutation in the drug binding pocket of the molecular target can
increase resistance against the antibiotic by several hundred fold. This
makes the modification of the molecular target resistance mechanism
a key consideration during antibiotic discovery because thismechanism
gives rise to fast-developing, high level resistance. One of the key reasons
for the failure of target based antibiotic discovery (which targets
a single molecular target) is due to high level resistance quickly developing
via modification of themolecular target [84]. Therefore, overcoming
resistance development, particularly missense mutations in the
gene target, must be considered in the early phases of antibiotic
discovery.
In contrast, chemicalmodification of antibiotics causes high level resistance
by spreading through lateral gene transfer. Spontaneousmutations
to an existing gene that give it the ability to modify a xenobiotic
are possible in theory, but evolving such new gene functions requires
long periods of selection in practice [85]. Therefore, resistance through
chemical modification of antibiotics develops slowly at first, but spreads
with frightening speed once a certain threshold has been reached. The
development of this mode of resistance is largely unpredictable, and
the appropriate focus is to slowthe spread of this resistance mechanism
through proper use of the antibiotic [82].
4.2. Multi-targeting monotherapeutic antibiotics â FabH/FabF/FabB
inhibitors
The well-reasoned and provocative âmulti-targetâ hypothesis posits
that all broad-spectrum, monotherapeutic antibiotics in clinical use
today target multiple gene targets, and stipulates that the multitargeting
feature of these antibiotics limit the fast development of
high level resistance through modifying the molecular target [84]. Antibiotics
targeting two or more gene targets require simultaneous resistance
causing missense mutations in all the gene targets to cause high
level resistance. The probability of developing a resistantmissense mutation
in a gene target is approximately one in 109 bacteria [83,86]
meaning that a resistant bacteria can be found in an approximately
1ml OD600=1 culture. However, the probability of developing two resistantmissensemutations
simultaneously is approximately one in 1018
bacteria,meaning a 1,000,000 l OD600=1 culture would be required to
find the resistancemutant. Themultiplicative probability of developing
resistant missense mutations means that targeting two or more essential
targets simultaneously is an effectiveway of slowing the emergence
of resistance throughmodifying themolecular target. A summary of the
design principles of a broad-spectrum antibiotic is summarized in
Table 1. Resistance against these antibiotics occurs via a combination
of decreasing cell permeability against the antibiotic, efflux of the
antibiotic, modification of the antibiotic, and stepwise accumulation of
multiple target-based resistance mutations [84,87].
A monotherapeutic antibiotic under this model must inhibit molecular
target(s) encoded for by 2 or more genes in the bacterial genome.
This means that the active site and function of these molecular targets
must be sufficiently similar to be inhibited by a single pharmacophore.
Thesemolecular target(s)must also be highly conserved in awide spectrum
of bacteria if broad-spectrum activity is desired. Despite having
nearly a dozen different classes of antibiotics, all broad-spectrum
monotherapeutic antibiotics target 3 molecular target classes - penicillin
binding proteins, ribosomes, and topoisomerases [84]. Each of
these targets represents a family of essential and related protein functions
encoded for by multiple genes. These three targets are also highly
conserved in a wide range of bacteria. With the genomes of many
bacterial pathogens sequenced and the protein functions of model
organisms like E. coli largely characterized, an educated guess can be
ventured onwhether there aremoremolecular target(s) that are highly
conserved, widely distributed, and encoded for 2 or more times in the
bacterial genome. There appear to be few enzyme classes that fulfill all
three criteria.
There are three dual targeting inhibitors of bacterial fatty acid
metabolism. The natural product inhibitor platencin is a dual inhibitor
of the condensing enzymes FabH and FabF in Staphylococcus aureus
[55,88]. Platencin has relatively balanced activity against the two enzymes
(IC50 of 4.6 μM vs FabF and 9.2 μM vs FabH). Unfortunatelyplatencin has poor pharmacokinetic properties and a continuousinfusion
is required to effectively treat Staphylococcus aureus in the
mousemodel [88]. Little progress has beenmade inmodifying platencin
to have better pharmacokinetic properties due to the complex chemical
nature of platencin that makes generating chemical analogue libraries
difficult [89].
The natural product inhibitor thiolactomycin is a dual inhibitor
of FabF and FabB in bacteria encoding both enzymes such as E. coli
[56,57,90â92]. Staphylococcus aureus only encodes for FabF, so
thiolactomycin is only a single-target inhibitor of FabF in Staphylococcus
aureus. Thiolactomycin is a structural mimic of malonyl-ACP
and binds to the acyl-enzyme (Pong form) of the condensing enzyme
[53,93]. Thiolactomycin has an antibacterial spectrum against both
Gram-positive and Gram-negative bacteria, low cytotoxicity, effectiveness
in a mouse model, and favorable pharmacokinetic properties
[94,95]. However, despite considerable effort to structurally
improve thiolactomycin [96â98], successful therapeutic products
have not emerged from this lead. Optimizing thiolactomycin remains
an active area of continuing research [99].
The natural product inhibitor cerulenin is a covalent dual inhibitor of
FabF and FabB [58,59]. Like thiolactomycin, cerulenin is only singletargeting
in bacteria with only FabF. The catalytic cysteine in the condensing
enzymes attacks the epoxide group in cerulenin to forma covalent
intermediate that inhibits the enzyme reaction [100]. The covalent
natural of cerulenin inhibitionmeans that selectivity is for the active site
catalytic cysteine, and the inhibitor could tolerate some differences in
the rest of the active site architecture (similar to β-lactam antibiotics).
Unfortunately, cerulenin reacts with eukaryotic fatty acid synthase
and sterol synthesis enzymes using similar enzymatic mechanisms as
the condensing enzymes [101â103], so it lacks bacterial selectivity.
The covalentmode of inhibition and the lack of bacterial selectivity suggest
that designing a bacterial selective cerulenin derivative will be
challenging and little progress has been made to improve its antibacterial
properties.
4.3. Pathogen-selective antibiotic discovery â FabI
Three companies continue to be involved in developing FabI inhibitors
for antibiotic therapy.Mutabilis [104], CrystalGenomics [105â107],
and Affinium [17,20,21] are all focused on developing FabI inhibitors
with Staphylococcus selective activity rather than broad-spectrum
activity. These inhibitors are the first reported single-target, pathogenselective
inhibitors to enter into clinical evaluation. CG400549 from
CrystalGenomics has successfully passed phase 1 trials and recently
completely phase 2a trials (the results are currently under review).
AFN-1252 from Affinium has successfully completed phase 2 trials and
is undergoing further clinical development by Debiopharm [19]. The
extensive research surrounding AFN-1252 provides an informative
case study in the potential benefits and challenges of designing singletarget
pathogen-selective inhibitors, Fig. 3.
Pathogen-selective inhibitors could potentially overcome targetbased
resistance by being so potent that therapeutic doses of antibiotics
are still curative against the missense resistant mutant. Rather
than compromising affinity to target a broad spectrum of bacteria,
pathogen-selective inhibitors can be designed to have extremely high
affinity and activity against the particular pathogen. AFN-1252 has a
4 nM apparent Ki against the Staphylococcus FabI and 4 ng/ml minimal
inhibitory concentration against Staphylococcus aureus in planktonic
growth [18,20,108]. The only mutations causing high level resistance
to AFN-1252 are missense mutations to two residues (M99T and
Y147H) in FabI. The M99T mutation causes a 64 fold increase in AFN-
1252 resistance (MIC of 250 ng/ml) withminimal change in growth fitness.
The Y147Hmutation causes a 128 fold increase (MIC of 500 ng/ml)
in AFN-1252 resistance along with severe growth defects [25,108].
Mutants with higher levels of resistance were not found by selecting
the single missense mutants, while a mutant enzyme with both the
M99T and Y147H mutations is catalytically inactive [108]. This result
suggests that there is a ceiling in short term resistance development
(at least in Staphylococcus aureus), and that a sufficiently potent
inhibitor can overcome target-based resistance. The key question for
AFN-1252 is whether the drug can be dosed at the levels required to
eradicate the M99T and Y147H mutants.
Pathogen-selective inhibitors also have the potential to minimize
the collateral damage to the microbiome. Recent advancesin
microbiome research show that broad-spectrum antibiotics damage
the beneficial microbiomeof the human body, leading to a variety of adverse
short and long termconsequences [109,110]. In particular, broadspectrumantibiotics
are known to cause antibiotic associated secondary
infections. These diseases arise from broad-spectrum antibiotic treatment
wiping out the beneficial host microbiome and allowing drug
resistant pathogens that can't normally compete with the microflora
to proliferate [111,112]. The effect of AFN-1252 on the mouse gut
microbiome was examined as a case study [64]. AFN-1252 caused no
significant change in gut bacterial abundance and minimal disturbance
in bacterial composition in contrast to broad-spectrum antibiotics that
reduced the gut bacterial abundance several thousand fold along with
severe disturbance in bacterial composition. Similar results are expected
in humans, but this conclusion requires verification. One of the
major demands for newantibiotics comes frompatientswithweakened
immune systems [112,113]. A pathogen-selective approach would
benefit these patients by minimizing the disturbance to the beneficial
microbiome.
A summary of the design principles of a pathogen-selective antibiotic
are summarized in Table 1. This pathogen-selective paradigm faces
two major hurdles. First, how resistance will develop against an inhibitor
must be taken into account during the course of development. The
inhibitor must be designed to be high-affinity while minimizinginteractionswith the side chains of non-conserved residues that can undergo
mutations that decrease the affinity of the inhibitor [114]. Resistance
against the inhibitor must be evaluated early in the discovery
phase, and minimizing the impact of resistance must be an additional
criterion that guides the structure optimization process. The inhibitor
must also be tested against the resistant mutant in animal models to
verify that the inhibitor is still effective against the missense resistant
mutants. Second, the specific nature of a pathogen-selective inhibitor
requires accurate pathogen diagnostic technology. Because the antibioticmust
bematchedwith the correct pathogen, rapid and accurate identification
of the pathogen responsible for the disease must precede
antibiotic prescription. One major challenge in pathogen detection is
determining which bacterial species is responsible for diseases because
many diseases causing pathogens are also permanent residents of the
human microflora [115]. Fortunately, rapid pathogen detection is an
area of major research and advances [116].
4.4. Combinations of single-target antibiotics
The success of the β-lactam antibiotics has made combinations of
antibiotics a less favorable choice due to the difficulty in dosingmultiple
compounds. However, combinational antibiotic therapy has long been
the standard for treating Mycobacterium tuberculosis. Because Mycobacterium
tuberculosis encodes for and produces extended spectrum β-
lactamases, β-lactamdrugs are largely ineffective [117]. Mycobacterium
tuberculosis also encodes only a single rRNA gene so ribosomeinhibitors
must be used in combination therapy with other antibiotics to prevent
fast-developing, target-based resistance [118]. Fluoroquinolones are effective
against tuberculosis, but has been largely held in reserve for
treatment of drug resistant tuberculosis. Instead, the front line treatment
against tuberculosis is a combination of isoniazid, rifampin, ethambutol,
and pyrazinamide. Each of these drugs target a single gene
target and experiences high rates of resistance development when
used individually [119], but the drug cocktail is effective at preventing
resistant mutants. The large scale use of this effective drug cocktail
against M. tuberculosis suggests that the combination of single antibiotics
can be an effective therapeutic strategy to fight target-based
resistance. Another example of this strategy is the sulfamethoxazoletrimethoprim
combination [120]. Sulfamethoxazole is a sulfanilamide
analogue that inhibits dihydropteroate synthetase in tetrahydrofolic
acid synthesis. Trimethoprim inhibits dihydrofolate reductase, another
enzyme in tetrahydrofolic acid synthesis. Target-based resistance arises
quickly for sulfamethoxazole or trimethoprim treatment alone, which
limits the clinical efficiency of these drugs. However, combining these
two drugs for treatment decreases the occurrence of spontaneous
target-based mutations.
Given the difficulties of finding new monotherapeutic, broadspectrum
antibiotics, combination therapy is gaining more interest.
Combining β-lactam and β-lactamase inhibitors is a significant area of
recent antibiotic development [121]. Given the rather limited target options
in developing broad-spectrum, monotherapeutic antibiotics,
combination therapy of single-target inhibitors with orthogonal gene
targets should be considered as an alternative. The rate-determining
steps in bacterial fatty acid synthesis are validated targets for these
single-target inhibitors. Combining two inhibitors targeting two different
gene targets provides the benefit ofminimizing resistance via single
missense mutations. Furthermore, if resistance is the key reason why
single-target, broad-spectrum antibiotics failed to reach the clinic,
then combining these failed single-target antibiotics should provide an
effective therapy. This hypothesis could be tested directly using the
low toxicity, FASII inhibitors that have already been discovered [13,
32]. The challenge tomulti-drug therapy is avoiding adverse drug interactions
and matching the dosage of the drug components so both drugs
are at active concentrations over the course of treatment to avoid stepwise
resistance. Stepwise resistance against one component of the cocktail
at a time when the other components are low due to improper
antibiotic use is a major concern for the development of resistance in
Mycobacterium tuberculosis [122].
A variety of single-target FASII inhibitors have been described in
literature [13,32]. These include both natural product inhibitors and
synthetic inhibitors. The two best characterized molecular targets are
FabI and FabH, which have been subject to numerous discovery campaigns
by a variety of investigators. One major recurring result from
these campaigns is the difficulty in getting both spectrum and potency
[9,21,99,104â107,123]. For example, a recent FabH discovery campaign
reported a series of 22 thiolactomycin analogues and tested these analogues
againstMycobacteriumtuberculosis, Francisella tularensis, Yersinia
pestis, and Staphylococcus aureus [99]. While several analogues were
able to achieve low μg/ml minimal inhibitory concentration against
one of the bacterial species, only 1 analogue was able to achieve low
μg/ml minimal inhibitory concentration against two bacterial species
and none against 3 or more species. These results show that FASII enzymes
are sufficiently structurally divergent that even broad-spectrum
single-targeting is exceptionally difficult. Outside of cell wall synthesis,
ribosome, and DNA synthesis, there simply do not appear to be
more highly conserved molecular targets in bacteria. A cocktail of narrow
spectrum antibiotics should be considered in future antibiotic
applications.
5. Summary
The Centers for Disease Control released the âAntibiotic
resistance in the United Statesâ report in 2013 [124]. The report
identified Clostridium difficile, Neisseria gonorrhoeae, Enterobacter,
Acinetobacter, Campylobacter, Psuedomonas aeruginosa, Salmonella,
Shigella, Staphylococcus, Enterococcus, and Streptococcus as major
pathogen threats in need of new antibiotic treatments. Bacterial
fatty acid metabolism has been validated as essential in most of
these pathogens. Of these pathogens, only Streptococcus has the ability
to bypass FASII inhibition through using exogenous fatty acids
[23â25]. FASII has been demonstrated to be essential in Staphylococcus,
Enterocococcus, Neisseria, and E. coli [23,25,68,69], so FabI, FabH, FabF,
and acetyl-CoA carboxylase inhibitors are predicted to be effective
against these pathogens. Bioinformatic predications suggest that the organization
of fatty acid, phospholipid, and lipopolysaccharide synthesis
in Enterobacter, Acinetobacter, Campylobacter, Salmonella and Shigella
are similar to E. coli, so inhibition of the enzymes in FASII would be expected
to work in these pathogens as well. Additionally, dual FabF and
FabB targeting would be possible for the Gram-negative pathogens in
the above list because these Gram-negative bacteria synthesize unsaturated
fatty acids using a FabB [59,125]. Psuedomonas aeruginosa encodes
for multiple enoyl-ACP reductase and FabH isoforms [66,126], but is
predicted via bioinformatics or validated experimentally to encode
only a single, essential copy of acetyl-CoA carboxylase [44], FabF, and
FabB. Finally, Clostridium difficile is an opportunistic pathogen that
proliferates in the gut when the normal microflora is eliminated by
broad-spectrum antibiotic treatment [112,127,128]. The narrow
spectrum of a FabI inhibitor designed using a pathogen-selective
paradigm preserves the gut microflora [64] and prevents Clostridium
difficile colitis from occurring in the first place. The various application
of FASII inhibition holds great potential for the future development of
antibiotics against the most urgent pathogens.
