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7 Jul 2019

1. One of the first stages of drug design is the identification of a compound (a lead compound) that interacts the target of interest. This active compound is then further modified in the drug development process. Briefly describe how the following can be sources of these active compounds:

Natural products

Chemical (compound) libraries

Existing drugs

2. Describe what is meant by the following two approaches in computer-aided drug design.

Ligand-based drug design

Structure-based drug design

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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 Î²-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 Î²-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 Î³-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 Î²-ketoacyl-ACP reductase FabG catalyzes the reduction of Î²-

ketoacyl-ACP into Î²-hydroxyacyl-ACP [15]. In contrast to enoyl-ACP

reductase, there is only one highly conserved Î²-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 Î³-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 Î²-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 Î¼M vs FabF and 9.2 Î¼M 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 Î²-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 Î²-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 Î²-

lactamases, Î²-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 Î²-lactam and Î²-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 Î¼g/ml minimal inhibitory concentration against

one of the bacterial species, only 1 analogue was able to achieve low

Î¼g/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.

brownhamster745

Question: From the Article Below, write a review of the current status of development of antibiotics." I do not have the figures!"

Article: Antibiotic discovery in the twenty-first century: current trends and future perspectives

New antibiotics are necessary to treat microbial pathogens that are becoming increasingly resistant to available treatment.

Despite the medical need, the number of newly approved drugs continues to decline. We offer an overview of the pipeline for

new antibiotics at different stages, from compounds in clinical development to newly discovered chemical classes. Consistent

with historical data, the majority of antibiotics under clinical development are natural products or derivatives thereof. However,

many of them also represent improved variants of marketed compounds, with the consequent risk of being only partially

effective against the prevailing resistance mechanisms. In the discovery arena, instead, compounds with promising activities

have been obtained from microbial sources and from chemical modification of antibiotic classes other than those in clinical use.

Furthermore, new natural product scaffolds have also been discovered by ingenious screening programs. After providing selected

examples, we offer our view on the future of antibiotic discovery.

The Journal of Antibiotics advance online publication, 16 June 2010; doi:10.1038/ja.2010.62

Keywords: antibiotics; natural products; pipeline

Medical progress in the prevention and treatment of many diseases,

which have resulted in significantly increasing life expectancy, may be

put at risk without the introduction into clinical practice of new

antibiotics effective against multidrug-resistant (MDR) pathogens.

Although most stakeholders agree that new antibiotics could tackle

this unmet medical need, opinions vary on how new antibiotics could

be discovered and brought into the market in a cost-effective manner.

1â3 Two considerations would probably meet with unanimous

consensus: the golden era of antibiotic discovery is gone and it will not

be repeated; and genomics, combinatorial chemistry and highthroughput

screening do not represent the magic bullet to fill the

pipeline with new developmental drug candidates. In this respect, it is

important to underline the contribution that natural products,

especially those of microbial origin, can provide to antibiotic discovery,

as advocated by Demain4,5 on several occasions. The decreasing

number of drugs approved for clinical use, year after year, suggests

that the âailing pharmaceutical industryâ is not yet following the

âprescriptionâ of Demain,6 as spelled out in 2002.

The purpose of this review is to highlight some of todayâs features of

antibiotic discovery in the context of the current medical needs and

the existing pipeline of antibacterial agents in clinical development.

Our main focus will be on chemical classes that, if developed into

drugs, would be new to the clinic. However, these classes would not

necessarily be new to science. For example, a âlook-backâ strategy was

applied to antibiotics discovered during the golden era, which were

then reexamined using contemporary tools in the light of current

medical needs.7 Although some important breakthroughs have also

been made in identifying new promising drug candidates from

synthetic origin, for reason of space, and in the spirit of the important

contributions to the field by Demain, we would limit ourselves

to antibiotics of microbial origin and their derivatives reported

since 2005.

CURRENT ANTIBIOTIC PIPELINE

Infections due to methicillin-resistant Staphylococcus aureus (MRSA),

vancomycin-resistant Enterococcus faecium (VRE) and fluoroquinolone-

resistant Pseudomonas aeruginosa are rapidly increasing in US

hospitals, and even more frightening is the recent occurrence of

panantibiotic-resistant infections, involving Acinetobacter species,

MDR P. aeruginosa and carbapenem-resistant Klebsiella species.8,9

Although antibiotic resistance continues to grow in hospitals and in

the community, involving both Gram-positive and Gram-negative

pathogens, the number of newly approved agents has been decreasing,

with only six new antibiotics approved since 2003.

In the late 90s, following the global concern regarding the rapid

increase in MRSA, many companies redirected their attention to target

Gram-positive pathogens, particularly MRSA, VRE and penicillinresistant

Streptococcus pneumoniae, as evidenced by the commercial

and clinical success of linezolid and daptomycin, the only antibiotics

belonging to new classes introduced in clinical practice since the early

1960s. However, most antibiotics currently under development for

Gram-positive infections are improved derivatives of existing drugs

(see Table 1). As vancomycin has been increasingly used for the

treatment of a wide range of infections, second-generation glycopep-

tides with improved profile over vancomycin were developed. Among

them, telavancin, a once-a-day derivative of vancomycin, was

approved by the US Food and Drug Administration (FDA) in 2009.

Oritavancin, derived from the vancomycin-related glycopeptide chloroeremomycin,

is highly active against VRE strains and shows a long

plasma half-life. However, in 2008, the FDA did not authorize its

commercialization. The long-acting glycopeptide dalbavancin, a derivative

of the teicoplanin-related glycopeptide A40926, was also not

approved by FDA, because of insufficient clinical evidence of efficacy.

If approved, dalbavancin would be the first antibiotic to be administered

once weekly.10

Resistance to methicillin in S. aureus is mediated by the production

of a penicillin-binding protein with reduced affinity for b-lactams. The

most recent cephalosporins, ceftobiprole and ceftaroline (Table 1),

have been specifically designed to enhance activity against MRSA and,

thanks to their oral availability, are particularly attractive for the

community setting. Ceftobiprole is quickly bactericidal against a wide

range of Gram-positive pathogens, including MRSA and VRE and has

been approved in Canada and Switzerland.11 However, early in 2010,

the FDA did not grant market authorization to ceftobiprole, and later

the European authority issued a negative opinion on this compound.

Ceftaroline, which is active against most Gram-positive pathogens

with the exclusion of enterococci, has completed phase III studies and

may be submitted for FDA approval.12 Both cephalosporins, however,

lose potency against MRSA compared with methicillin-susceptible

S. aureus strains. The injectable carbapenem PZ-601 has shown potent

activity against drug-resistant Gram-positive pathogens, including

MRSA, and is currently undergoing phase II studies.13

After the success of linezolid, many new oxazolidinones are being

developed for Gram-positive infections. Radezolid14 and torezolid15

are currently in phase II trials, whereas RWJ-416457 has completed the

phase I trial. Despite the fact that the use of fluoroquinolones has been

associated with increased incidence of MRSA,16 several new members

of this class are under development: delafloxacin, nemonoxacin,

zabofloxacin and WCK-771 (Table 1) are the most advanced.

The extensive use of fluoroquinolones and other wide-spectrum

antibiotics such as cephalosporins, by affecting the normal gut flora,

has led to the rapid diffusion of Clostridium difficile-associated

diarrhea, particularly in elderly and immunocompromised patients.

Difimicin, currently in phase III, and ramoplanin, with phase II

completed, are microbial products under development for prevention

and treatment of C. difficile-associated diarrhea, acting locally by

decolonizing the gut (Table 1).

Other compounds which have completed phase I clinical trials

include the oral and injectable pleuromutilin BC-3205,17 the FabI

inhibitor AFN-1252 targeting staphylococcal infections18 and the

lipopeptide friulimicin (Table 1).19

The scenario is even more disappointing for compounds targeting

Gram-negative pathogens, in which old drugs have been revamped for

new uses, and none of them has reached phase III yet (Table 1).

Ceftazidime is a marketed cephalosporin being developed in combination

with NXL104, a representative of a new class of b-lactamase

inhibitors,20 which renders cephalosporin effective against most

b-lactamase-producing enterobacteria. If approved, this combination

would be the first alternative to piperacillin/tazobactam. NXL104 is

also under investigation in combination with ceftaroline.21 CXA-101 is

a ceftazidime-like compound with improved stability against the

AmpC b-lactamase, but it shows no improvements against MDR

P. aeruginosa,22 unless administered in combination with tazobactam.

The new aminoglycoside ACHN-490, effective against pathogens

resistant to this class, has recently completed phase I.23 The new

monobactam BAL-30072, stable against metalloenzymes, is ready to

start clinical development against difficult-to-treat Gram negatives,

including Pseudomonas and Acinetobacter.24

The increasing spread of MDR Gram-negative pathogens, particularly

P. aeruginosa, Acinetobacter spp. and some Enterobacteriaceae has

renewed the interest toward narrow-spectrum compounds, to avoid

other clinical conditions associated with the use of broad-spectrum

antibiotics. However, because of a long history of success in the

empirical treatment of infections, many hospitals lack rapid and

effective tools for identifying etiological agents. This limitation poses

significant hurdles for the clinical development of narrow-spectrum

compounds.

APPROACHES LEADING TO NEW ANTIBIOTIC CLASSES

It is generally agreed that the best way to overcome the decreasing

efficacy of existing antibacterial agents is to introduce into practice

compounds belonging to classes that are new to the clinic. Microbial

sources can provide a rich reservoir of such compounds, and the

different approaches used usually aim at discovering either a novel

class or an improved variant of a poorly explored class. However,

this must be carried out in a high background of many known

compounds, some of which are encountered in random screening

programs at a relatively high frequency. Thus, the discovery of an

antibacterial agent belonging to a new chemical class or an improved

variant of an existing class is a rare event, and the approaches

described below reflect strategies designed and implemented to

capture this rare event. Appropriate strategies include retrieving

microbial strains from underexplored environments, screening new

microbial taxa, mining microbial genomes and using innovative

assays. These strategies have led to some novel chemical classes, as

illustrated in Figure 1.

As an example of the first strategy, investigation of deep-sea

sediment samples led to the discovery of abyssomicins (Figure 1),

which are polycyclic antibiotics from the new marine actinomycete

taxon Verrucosispora.25 The compounds were discovered using a simple

agar diffusion assay, which involved pursuing antibiotics the action of

which could be reverted upon addition of p-aminobenzoic acid.

Abyssomicins represent a new chemical class, and preliminary studies

indicate that they act as substrate mimics of chorismate. Interestingly,

only abyssomicin C and its atrop stereoisomer show antibiotic activity

against Gram-positive bacteria, including MDR S. aureus.26

An additional example of a new chemical class discovered by

screening new taxa is represented by thuggacins (Figure 1), which

are thiazole-containing macrolides produced by the myxobacteria

Sorangium cellulosum and Chondromyces crocatus.27 These compounds

show activity against Mycobacterium tuberculosis and their target

appears to be the electron transport chain.

Another successful approach has been exploring microbial genomes

for the presence of secondary metabolite pathways. As the corresponding

genes are organized in clusters and bioinformatic tools allow

a reasonable prediction of the pathway product, this bioactivityindependent

approach can directly target structural novelty. On a

pioneering work of this type, scientists at Ecopia Biosciences (now

Thallion Pharmaceuticals, Montreal, QC, Canada) identified ECO-

02301, a linear polyene from Streptomyces aizunensis with antifungal

activity28 and ECO-0501, a glycosidic polyketide from Amycolatopsis

orientalis with activity against Gram-positive pathogens, including

MDR isolates (Figure 1).29 In a similar approach, a novel cyclic

lipopeptide, designated orfamide (Figure 1), was identified from the

Pseudomonas fluorescens genome.30 In this case, the bioinformatic

prediction that the peptide contained four leucine residues suggested

feeding with 15N-Leu, which facilitated compound purification and

characterization. Orfamide shows a moderate antifungal activity

against amphotericin-resistant strains of Candida albicans and may

prove beneficial in agriculture and crop protection.

Another important strategy for discovering new classes of antibiotics

has been the implementation of increased-sensitivity assays in

screening programs. One such approach relied on the antisense

technology. When the level of a desired bacterial target is depleted

by overexpression of the cognate antisense mRNA, the strain becomes

hypersensitive to compounds acting on that target. By using a target

against which few compounds are known to act, the increased

sensitivity of the assay should allow the identification of compounds

routinely missed with growth inhibition assays on the wild-type

strain.31 One assay involved the FabH/FabF enzyme, required for

fatty acid biosynthesis in bacteria. Antimicrobial activities were

detected by agar diffusion in a two-plate assay, in which one plate

was inoculated with S. aureus carrying the antisense construct and the

other plate with an S. aureus control. Different inhibition halos in the

two plates indicated an increased sensitivity of the âantisense strain.â

After screening 4250 000 microbial product extracts, the assay led to

the identification of a new chemical class that includes platensimycin

(Figure 1), produced by Streptomyces platensis, and related compounds.

Platensimycin shows antibacterial activity against Grampositive

pathogens, including MDR strains, and was also effective in

an experimental model of infection.32

In another increased-sensitivity assay, a high-throughput screening

program was implemented to identify inhibitors of a cell-free translational

system affecting steps other than elongation. The assay made

use of a model âuniversalâ mRNA that could be translated with similar

efficiency by cell-free extracts from bacterial, yeast or mammalian cells.

The rationale behind the approach was to use a sensitive assay and to

discard frequently encountered compounds using a polyU-based assay.

This program led to the identification of GE81112 (Figure 1), a novel

tetrapeptide produced by a Streptomyces sp., which targets specifically

the 30S ribosomal subunit by interfering with fMet-tRNA binding to

the P-site.33 The compound was highly effective against a few Grampositive

and Gram-negative strains, if grown in minimal or chemically

defined medium, suggesting active uptake by the cells.34

The above examples illustrate how different approaches can lead to

novel antibiotic classes. Usually, when unexploited microbial diversity

is accessed, there is no need for specific, high-sensitivity assays.

Whichever the approach chosen, there is no guarantee of success.

The reader is referred to a recent review for suggestions on how to

increase the probabilities of success.35

IMPROVED VARIANTS FROM MICROBIAL SOURCES

New variants of known classes can be found by screening microbial

strains, by varying cultivation procedures or by manipulating the

biosynthetic pathway. There is an increasing amount of literature

related to pathway manipulation and this trend is likely to continue as

methodological advancements result in increased success rates. In

some cases, the desired variant might not be a more active compound,

but a molecule carrying functional groups suitable for further chemical

modifications. As the antibiotics in clinical use belong to a few

classes, which have been extensively explored by screening and

chemical modification, there is probably little space for finding

improved variants within those classes. We provide selected examples

of microbial strains producing improved variants of chemical classes

not yet in clinical use.

Lantibiotics, which are ribosomally synthesized peptides that

undergo posttranslational modifications to yield the active structures

containing the typical thioether-linked (methyl)lanthionines, are produced

mostly from strains belonging to the Firmicutes and, to a lesser

extent, to the Actinobacteria. Their antimicrobial activity is limited to

Gram-positive bacteria. The prototype molecule is nisin, discovered in

the 1920s and used as a food preservative for440 years.36 Lantibiotics

with antibacterial activity are divided into two classes according to

their biogenesis: lanthionine formation in class I compounds requires

two separate enzymes, a dehydratase and a cyclase, whereas a single

enzyme carries both activities for class II lantibiotics. Until recently,

the occurrence of class I compounds was limited to the Firmicutes (see

below). Although compounds from both classes exert their antimicrobial

activity by binding to Lipid II, they do so by binding to

different portions of this key peptidoglycan intermediate.

As lantibiotics bind Lipid II at a site different from that affected by

vancomycin and related glycopeptides, they are active against MDR

Gram-positive pathogens and have attracted attention as potential

drug candidates. The compound NVB302, a derivative of deoxyactagardine

B (Figure 2a) produced by a strain of Actinoplanes liguriae, is

currently a developmental candidate for the treatment of C. difficileassociated

diarrhea.37 Independently, a screening program, designed to

detect cell-wall-inhibiting compounds turned out to be very effective

in identifying lantibiotics from actinomycetes.38 It consisted of identifying

extracts active against S. aureus but inactive against isogenic

L-forms, discarding extracts the activity of which was abolished by

b-lactamases or by excess N-caproyl-D-alanyl-D-alanine. Among the

new lantibiotics identified, the most active compound was NAI-107

(Figure 2a), produced by Microbispora sp.39 This compound represents

the first example of a class I lantibiotic produced by actinomycetes. It

is currently a developmental candidate for the treatment of nosocomial

infections by Gram-positive pathogens.40 The same screening

program led to the identification of additional class I lantibiotics from

actinomycetes. Among them, the compound 97518 (Figure 2a),

structurally related to NAI-107,41 afforded improved derivatives by

chemical modification.42 Another interesting advancement in the

lantibiotic field has been the discovery of two-component lantibiotics

produced by members of the class Bacilli. The best characterized

compound is haloduracin43,44 (Figure 2a), whereas lichenicidin has

been proposed from genomic studies but has not yet confirmed by

structural elucidation.45 Although their antimicrobial activities have

not been described in detail, recent work suggests similar activities for

haloduracin and nisin.44

Thiazolylpeptides are highly modified, ribosomally synthesized

peptides that inhibit bacterial protein synthesis by affecting either

one of two targets: elongation factor Tu, as for GE2270 and related

compounds; or the loops defined by 23S rRNA and the L11 protein,

exemplified by thiostrepton. Most thiazolylpeptides show potent

activity against Gram-positive pathogens, yet their poor solubility

has limited clinical progress, and only a derivative of GE2270 has

entered clinical trials for the topical treatment of acne.46 Novel

members of this class have been described (Figure 2b): thiomuracins47

belong to the subgroup targeting EF-Tu, with an antibacterial profile

similar to GE2270; thiazomycin48 and philipimycin,49 which target the

50S subunit, show high activity against Gram-positive strains, and a

similar profile to thiostrepton.

For ribosomally synthesized peptides, such as lantibiotics and

thiopeptides, new representatives can be generated by site-directed

mutagenesis of the corresponding structural genes. Libraries of new

molecules have been obtained, many of which, as in the examples of

actagardine50 and thiocillin,51 retained antibiotic activities comparable

with those of the parent molecule.

CHEMICAL DERIVATIVES

Many papers have been published in the past 5 years reporting

chemical programs aimed at overcoming the prevailing resistance

mechanisms and/or to improve the drug profile of known microbial

products. Novel approaches included the use of new tools, such as

click chemistry and total synthesis. For the classical approach of semisynthesis,

we will limit the examples to selected compounds not yet in

clinical use.

Click chemistry is a new synthetic approach that can accelerate drug

discovery by using a few practical and reliable reactions. A âclickâ

reaction must be of wide scope, giving consistently high yields with

various starting materials; it must be easy to perform, insensitive to

oxygen or water and use only readily available reagents; finally,

reaction work-up and product isolation must be simple, without

chromatographic purification.52 As an example, this approach was

used to produce new lipophilic teicoplanin and ristocetin aglycons

with improved activity against Gram-positive bacteria, including

VRE.53 For aminoglycosides, which usually require multiple protectionâ

deprotection steps to selectively manipulate the desired amino

and hydroxyl groups, click chemistry allowed the transformation

of neomycin B into several novel building blocks that were used for

the specific modification of the ring systems, thus generating new

neomycin analogs the biological activity of which is currently under

investigation.54

For some low-molecular-weight compounds, total synthesis has

become available and will be useful to design preliminary SAR for new

classes of antibiotics (such as platensimycin) or to access new

derivatives for already known classes (such as tetracyclines). Indeed,

the novel scaffold and intriguing biological property of platensimycin

captured the interest of several research groups, which reported

different elegant total syntheses.55 In addition, medicinal chemistry

studies have been conducted, and the design, synthesis and biological

evaluation of several platensimycin analogs incorporating varying

degrees of molecular complexity have been reported.56â58 Preliminary

data indicate that certain modifications of the intricate cage region can

be made without detrimental effects on potency, whereas even small

modifications of the benzoic acid region result in a drastic loss of

activity (Figure 1). Another remarkable chemical improvement in the

synthesis of natural product analogs was a short and enantioselective

synthetic route to a diverse range of 6-deoxytetracycline antibiotics

(Figure 3a). This new approach targeted not a single compound but a

group of structures with the D ring as a site of structural variability.

A late-stage, diastereoselective C-ring construction was used to couple

structurally varied D-ring precursors with an AB precursor containing

much of the essential functionality for binding to the bacterial

ribosome. Results of antibacterial assays and preliminary data

obtained from a murine septicemia model show that many of the

novel tetracyclines synthesized have potent antibiotic activities. This

synthetic platform gives access to a broad range of tetracyclines that

would be inaccessible by semi-synthesis and provides a powerful

engine for the discovery of new tetracyclines.59,60

Even on larger molecules, semi-synthetic and synthetic chemistry

has been successfully applied to study and optimize lead compounds.

The lipoglycodepsipeptide ramoplanin (Figure 3b) is 2â10 times more

active than vancomycin against Gram-positive bacteria and maintains

full activity against VRE and all MRSA strains. However, its systemic

use has been prevented by its low tolerability at the injection site,

apparently related to the length of the fatty acid side chain.

To overcome this problem, the fatty acid side chain was selectively

removed and replaced with different carboxylic acids. Many derivatives

showed an antimicrobial activity similar to that of the precursor,

and a significantly improved local tolerability.61 The recently

described, fully synthetic lactam analog of ramoplanin showed the

same biological activity as the natural product. Moreover, a set of

alanine analogs, obtained by total synthesis, has provided insights into

the importance of individual amino-acid residues on ramoplanin

activity. The MICs of each alanine-containing analog parallels its

ability to bind Lipid II. Apart from positions 5, 6 and 9, which can

tolerate alanine substitutions, MICs increased 415-fold upon alanine

replacement, with dramatic effects observed for positions 4, 8, 10 and

12. The new data thus confirm the importance of the ornithine

residues at positions 4 and 10, with the latter directly involved in

target binding, most likely by ion pairing with the diphosphate of

Lipid II.62,63

The mannopeptimycins, which were originally isolated in the late

1950s from Streptomyces hygroscopicus, have been recently revived

because of their promising activity against clinically important Grampositive

pathogens, including S. pneumoniae, MRSA and VRE. They

also bind to Lipid II, but in a manner different from ramoplanin,

mersacidin and vancomycin. Multiple approaches have been used to

optimize the mannopeptimycin activity profile, including selective

chemical derivatization, precursor-directed biosynthesis and pathway

engineering. The SAR data have shown that substitution of a hydrophobic

ester group on the N-linked mannose or serine moieties

suppressed antibacterial activity, whereas hydrophobic acylation on

either of the two O-mannoses, particularly the terminal mannose,

significantly enhanced activity. AC98-6446 (Figure 3b) represents an

optimized lead obtained by adamantyl ketalization of a cyclohexyl

analog prepared by cyclohexylalanine-directed biosynthesis. AC98-

6446 showed superior antimicrobial potency and properties, both

in vitro and in vivo.7,64

Laspartomycin is active against VRE and MRSA strains. Recently,

enzymatic cleavage of its lipophilic moiety allowed the synthesis of

various acylated derivatives (Figure 3b), even if none was more potent

than the parent antibiotic.65 The cyclic heptapeptide GE23077 is a

potent and selective inhibitor of bacterial RNA polymerase

that, probably because of its hydrophilicity, is unable to cross

bacterial membranes. New derivatives obtained by modifying different

moieties were reported. Although many of them retained activity

on the enzyme, none showed a significant antibacterial activity

apart from marginal inhibition of Moraxella catarrhalis growth

(Figure 3b).66

FUTURE PERSPECTIVES

This brief and nonexhaustive excursus on the present and future

pipeline of antibacterial agents for treating human diseases provides

opportunities for additional considerations. The first is that, of the

antibiotics under clinical development (Table 1), 67% are natural

products themselves, or natural product-derived compounds, a percentage

perfectly in line with that found with exisiting drugs.67

The second consideration is that the major players in antibacterial

development are small companies, which are not deterred by the small

market size for these drugs. However, it should be noted that a

significant number of the compounds listed in Table 1 were not

discovered by small companies, but actually represent projects abandoned

by large pharmaceuticals companies. Thus, it remains to be

seen whether small biotechs will dedicate sufficient resources and be

successful in discovering and developing novel antibacterial agents.

In this relatively grim scenario, microbial products continue to

provide new chemical classes or unexpectedly active variants of

chemical classes already known to science. New technologies can

now provide access to unexplored microbial diversity or to hypersensitive

assays to detect bioactive compounds. Furthermore, the information

derived from rapidly accessing the genome of many microbial

strains can provide new routes to natural product discovery, as well as

making more effective traditional, bioassay-based screening efforts.

In our opinion, no single technology will represent the magic bullet

for antibiotic discovery, but only the painstaking integration of a

multidiscplinary team with profound knowledge of microbiology,

chemistry and bioinformatics will ultimately lead to new antibacterial

agents of medical relevance and commercial success.

mauvemammoth698

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