Question: From the Article Below, writea review of the current status of development of antibiotics.\" I donot have the figures!\"

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

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

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

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

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

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

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

have been obtained from microbial sources and from chemicalmodification of antibiotic classes other than those in clinicaluse.

Furthermore, new natural product scaffolds have also beendiscovered by ingenious screening programs. After providingselected

examples, we offer our view on the future of antibioticdiscovery.

The Journal of Antibiotics advance online publication, 16 June2010; doi:10.1038/ja.2010.62

Keywords: antibiotics; natural products; pipeline

Medical progress in the prevention and treatment of manydiseases,

which have resulted in significantly increasing life expectancy,may be

put at risk without the introduction into clinical practice ofnew

antibiotics effective against multidrug-resistant (MDR)pathogens.

Although most stakeholders agree that new antibiotics couldtackle

this unmet medical need, opinions vary on how new antibioticscould

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

1–3 Two considerations would probably meet with unanimous

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

be repeated; and genomics, combinatorial chemistry andhighthroughput

screening do not represent the magic bullet to fill the

pipeline with new developmental drug candidates. In thisrespect, it is

important to underline the contribution that naturalproducts,

especially those of microbial origin, can provide to antibioticdiscovery,

as advocated by Demain4,5 on several occasions. Thedecreasing

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

that the ‘ailing pharmaceutical industry’ is not yet followingthe

‘prescription’ of Demain,6 as spelled out in 2002.

The purpose of this review is to highlight some of today’sfeatures of

antibiotic discovery in the context of the current medical needsand

the existing pipeline of antibacterial agents in clinicaldevelopment.

Our main focus will be on chemical classes that, if developedinto

drugs, would be new to the clinic. However, these classes wouldnot

necessarily be new to science. For example, a ‘look-back’strategy was

applied to antibiotics discovered during the golden era, whichwere

then reexamined using contemporary tools in the light ofcurrent

medical needs.7 Although some important breakthroughs havealso

been made in identifying new promising drug candidates from

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

contributions to the field by Demain, we would limitourselves

to antibiotics of microbial origin and their derivativesreported

since 2005.

CURRENT ANTIBIOTIC PIPELINE

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

vancomycin-resistant Enterococcus faecium (VRE) andfluoroquinolone-

resistant Pseudomonas aeruginosa are rapidly increasing inUS

hospitals, and even more frightening is the recent occurrenceof

panantibiotic-resistant infections, involving Acinetobacterspecies,

MDR P. aeruginosa and carbapenem-resistant Klebsiellaspecies.8,9

Although antibiotic resistance continues to grow in hospitalsand in

the community, involving both Gram-positive andGram-negative

pathogens, the number of newly approved agents has beendecreasing,

with only six new antibiotics approved since 2003.

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

increase in MRSA, many companies redirected their attention totarget

Gram-positive pathogens, particularly MRSA, VRE andpenicillinresistant

Streptococcus pneumoniae, as evidenced by the commercial

and clinical success of linezolid and daptomycin, the onlyantibiotics

belonging to new classes introduced in clinical practice sincethe early

1960s. However, most antibiotics currently under developmentfor

Gram-positive infections are improved derivatives of existingdrugs

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

treatment of a wide range of infections, second-generationglycopep-

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) in2009.

Oritavancin, derived from the vancomycin-related glycopeptidechloroeremomycin,

is highly active against VRE strains and shows a long

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

commercialization. The long-acting glycopeptide dalbavancin, aderivative

of the teicoplanin-related glycopeptide A40926, was also not

approved by FDA, because of insufficient clinical evidence ofefficacy.

If approved, dalbavancin would be the first antibiotic to beadministered

once weekly.10

Resistance to methicillin in S. aureus is mediated by theproduction

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

most recent cephalosporins, ceftobiprole and ceftaroline (Table1),

have been specifically designed to enhance activity against MRSAand,

thanks to their oral availability, are particularly attractivefor the

community setting. Ceftobiprole is quickly bactericidal againsta wide

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

been approved in Canada and Switzerland.11 However, early in2010,

the FDA did not grant market authorization to ceftobiprole, andlater

the European authority issued a negative opinion on thiscompound.

Ceftaroline, which is active against most Gram-positivepathogens

with the exclusion of enterococci, has completed phase IIIstudies and

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

lose potency against MRSA compared withmethicillin-susceptible

S. aureus strains. The injectable carbapenem PZ-601 has shownpotent

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 arebeing

developed for Gram-positive infections. Radezolid14 andtorezolid15

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

phase I trial. Despite the fact that the use of fluoroquinoloneshas been

associated with increased incidence of MRSA,16 several newmembers

of this class are under development: delafloxacin,nemonoxacin,

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

The extensive use of fluoroquinolones and otherwide-spectrum

antibiotics such as cephalosporins, by affecting the normal gutflora,

has led to the rapid diffusion of Clostridiumdifficile-associated

diarrhea, particularly in elderly and immunocompromisedpatients.

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

completed, are microbial products under development forprevention

and treatment of C. difficile-associated diarrhea, actinglocally by

decolonizing the gut (Table 1).

Other compounds which have completed phase I clinical trials

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

inhibitor AFN-1252 targeting staphylococcal infections18 andthe

lipopeptide friulimicin (Table 1).19

The scenario is even more disappointing for compoundstargeting

Gram-negative pathogens, in which old drugs have been revampedfor

new uses, and none of them has reached phase III yet (Table1).

Ceftazidime is a marketed cephalosporin being developed incombination

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

inhibitors,20 which renders cephalosporin effective againstmost

b-lactamase-producing enterobacteria. If approved, thiscombination

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

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

a ceftazidime-like compound with improved stability againstthe

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

P. aeruginosa,22 unless administered in combination withtazobactam.

The new aminoglycoside ACHN-490, effective against pathogens

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

monobactam BAL-30072, stable against metalloenzymes, is readyto

start clinical development against difficult-to-treat Gramnegatives,

including Pseudomonas and Acinetobacter.24

The increasing spread of MDR Gram-negative pathogens,particularly

P. aeruginosa, Acinetobacter spp. and some Enterobacteriaceaehas

renewed the interest toward narrow-spectrum compounds, toavoid

other clinical conditions associated with the use ofbroad-spectrum

antibiotics. However, because of a long history of success inthe

empirical treatment of infections, many hospitals lack rapidand

effective tools for identifying etiological agents. Thislimitation poses

significant hurdles for the clinical development ofnarrow-spectrum

compounds.

APPROACHES LEADING TO NEW ANTIBIOTIC CLASSES

It is generally agreed that the best way to overcome thedecreasing

efficacy of existing antibacterial agents is to introduce intopractice

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

sources can provide a rich reservoir of such compounds, andthe

different approaches used usually aim at discovering either anovel

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 ofan

antibacterial agent belonging to a new chemical class or animproved

variant of an existing class is a rare event, and theapproaches

described below reflect strategies designed and implementedto

capture this rare event. Appropriate strategies includeretrieving

microbial strains from underexplored environments, screeningnew

microbial taxa, mining microbial genomes and usinginnovative

assays. These strategies have led to some novel chemicalclasses, as

illustrated in Figure 1.

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

sediment samples led to the discovery of abyssomicins (Figure1),

which are polycyclic antibiotics from the new marineactinomycete

taxon Verrucosispora.25 The compounds were discovered using asimple

agar diffusion assay, which involved pursuing antibiotics theaction of

which could be reverted upon addition of p-aminobenzoicacid.

Abyssomicins represent a new chemical class, and preliminarystudies

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

only abyssomicin C and its atrop stereoisomer show antibioticactivity

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 themyxobacteria

Sorangium cellulosum and Chondromyces crocatus.27 Thesecompounds

show activity against Mycobacterium tuberculosis and theirtarget

appears to be the electron transport chain.

Another successful approach has been exploring microbialgenomes

for the presence of secondary metabolite pathways. As thecorresponding

genes are organized in clusters and bioinformatic toolsallow

a reasonable prediction of the pathway product, thisbioactivityindependent

approach can directly target structural novelty. On a

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

Thallion Pharmaceuticals, Montreal, QC, Canada) identifiedECO-

02301, a linear polyene from Streptomyces aizunensis withantifungal

activity28 and ECO-0501, a glycosidic polyketide fromAmycolatopsis

orientalis with activity against Gram-positive pathogens,including

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

lipopeptide, designated orfamide (Figure 1), was identified fromthe

Pseudomonas fluorescens genome.30 In this case, thebioinformatic

prediction that the peptide contained four leucine residuessuggested

feeding with 15N-Leu, which facilitated compound purificationand

characterization. Orfamide shows a moderate antifungalactivity

against amphotericin-resistant strains of Candida albicans andmay

prove beneficial in agriculture and crop protection.

Another important strategy for discovering new classes ofantibiotics

has been the implementation of increased-sensitivity assaysin

screening programs. One such approach relied on theantisense

technology. When the level of a desired bacterial target isdepleted

by overexpression of the cognate antisense mRNA, the strainbecomes

hypersensitive to compounds acting on that target. By using atarget

against which few compounds are known to act, the increased

sensitivity of the assay should allow the identification ofcompounds

routinely missed with growth inhibition assays on thewild-type

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

fatty acid biosynthesis in bacteria. Antimicrobial activitieswere

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

was inoculated with S. aureus carrying the antisense constructand the

other plate with an S. aureus control. Different inhibitionhalos in the

two plates indicated an increased sensitivity of the ‘antisensestrain.’

After screening 4250 000 microbial product extracts, the assayled to

the identification of a new chemical class that includesplatensimycin

(Figure 1), produced by Streptomyces platensis, and relatedcompounds.

Platensimycin shows antibacterial activity againstGrampositive

pathogens, including MDR strains, and was also effective in

an experimental model of infection.32

In another increased-sensitivity assay, a high-throughputscreening

program was implemented to identify inhibitors of a cell-freetranslational

system affecting steps other than elongation. The assay made

use of a model ‘universal’ mRNA that could be translated withsimilar

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

The rationale behind the approach was to use a sensitive assayand to

discard frequently encountered compounds using a polyU-basedassay.

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

tetrapeptide produced by a Streptomyces sp., which targetsspecifically

the 30S ribosomal subunit by interfering with fMet-tRNA bindingto

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

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 leadto

novel antibiotic classes. Usually, when unexploited microbialdiversity

is accessed, there is no need for specific, high-sensitivityassays.

Whichever the approach chosen, there is no guarantee ofsuccess.

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

increase the probabilities of success.35

IMPROVED VARIANTS FROM MICROBIAL SOURCES

New variants of known classes can be found by screeningmicrobial

strains, by varying cultivation procedures or by manipulatingthe

biosynthetic pathway. There is an increasing amount ofliterature

related to pathway manipulation and this trend is likely tocontinue as

methodological advancements result in increased success rates.In

some cases, the desired variant might not be a more activecompound,

but a molecule carrying functional groups suitable for furtherchemical

modifications. As the antibiotics in clinical use belong to afew

classes, which have been extensively explored by screeningand

chemical modification, there is probably little space forfinding

improved variants within those classes. We provide selectedexamples

of microbial strains producing improved variants of chemicalclasses

not yet in clinical use.

Lantibiotics, which are ribosomally synthesized peptidesthat

undergo posttranslational modifications to yield the activestructures

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

mostly from strains belonging to the Firmicutes and, to alesser

extent, to the Actinobacteria. Their antimicrobial activity islimited to

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

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

with antibacterial activity are divided into two classesaccording to

their biogenesis: lanthionine formation in class I compoundsrequires

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

enzyme carries both activities for class II lantibiotics. Untilrecently,

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

below). Although compounds from both classes exert theirantimicrobial

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 thataffected by

vancomycin and related glycopeptides, they are active againstMDR

Gram-positive pathogens and have attracted attention aspotential

drug candidates. The compound NVB302, a derivative ofdeoxyactagardine

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 veryeffective

in identifying lantibiotics from actinomycetes.38 It consistedof identifying

extracts active against S. aureus but inactive againstisogenic

L-forms, discarding extracts the activity of which was abolishedby

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

new lantibiotics identified, the most active compound wasNAI-107

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

the first example of a class I lantibiotic produced byactinomycetes. It

is currently a developmental candidate for the treatment ofnosocomial

infections by Gram-positive pathogens.40 The same screening

program led to the identification of additional class Ilantibiotics from

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

structurally related to NAI-107,41 afforded improved derivativesby

chemical modification.42 Another interesting advancement inthe

lantibiotic field has been the discovery of two-componentlantibiotics

produced by members of the class Bacilli. The bestcharacterized

compound is haloduracin43,44 (Figure 2a), whereas lichenicidinhas

been proposed from genomic studies but has not yet confirmedby

structural elucidation.45 Although their antimicrobialactivities have

not been described in detail, recent work suggests similaractivities for

haloduracin and nisin.44

Thiazolylpeptides are highly modified, ribosomallysynthesized

peptides that inhibit bacterial protein synthesis by affectingeither

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

compounds; or the loops defined by 23S rRNA and the L11protein,

exemplified by thiostrepton. Most thiazolylpeptides showpotent

activity against Gram-positive pathogens, yet their poorsolubility

has limited clinical progress, and only a derivative of GE2270has

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

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

belong to the subgroup targeting EF-Tu, with an antibacterialprofile

similar to GE2270; thiazomycin48 and philipimycin,49 whichtarget the

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

similar profile to thiostrepton.

For ribosomally synthesized peptides, such as lantibioticsand

thiopeptides, new representatives can be generated bysite-directed

mutagenesis of the corresponding structural genes. Libraries ofnew

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

actagardine50 and thiocillin,51 retained antibiotic activitiescomparable

with those of the parent molecule.

CHEMICAL DERIVATIVES

Many papers have been published in the past 5 yearsreporting

chemical programs aimed at overcoming the prevailingresistance

mechanisms and/or to improve the drug profile of knownmicrobial

products. Novel approaches included the use of new tools, suchas

click chemistry and total synthesis. For the classical approachof semisynthesis,

we will limit the examples to selected compounds not yet in

clinical use.

Click chemistry is a new synthetic approach that can acceleratedrug

discovery by using a few practical and reliable reactions. A‘click’

reaction must be of wide scope, giving consistently high yieldswith

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 approachwas

used to produce new lipophilic teicoplanin and ristocetinaglycons

with improved activity against Gram-positive bacteria,including

VRE.53 For aminoglycosides, which usually require multipleprotection–

deprotection steps to selectively manipulate the desiredamino

and hydroxyl groups, click chemistry allowed thetransformation

of neomycin B into several novel building blocks that were usedfor

the specific modification of the ring systems, thus generatingnew

neomycin analogs the biological activity of which is currentlyunder

investigation.54

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

become available and will be useful to design preliminary SARfor new

classes of antibiotics (such as platensimycin) or to accessnew

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

the novel scaffold and intriguing biological property ofplatensimycin

captured the interest of several research groups, whichreported

different elegant total syntheses.55 In addition, medicinalchemistry

studies have been conducted, and the design, synthesis andbiological

evaluation of several platensimycin analogs incorporatingvarying

degrees of molecular complexity have been reported.56–58Preliminary

data indicate that certain modifications of the intricate cageregion can

be made without detrimental effects on potency, whereas evensmall

modifications of the benzoic acid region result in a drasticloss of

activity (Figure 1). Another remarkable chemical improvement inthe

synthesis of natural product analogs was a short andenantioselective

synthetic route to a diverse range of 6-deoxytetracyclineantibiotics

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

group of structures with the D ring as a site of structuralvariability.

A late-stage, diastereoselective C-ring construction was used tocouple

structurally varied D-ring precursors with an AB precursorcontaining

much of the essential functionality for binding to thebacterial

ribosome. Results of antibacterial assays and preliminarydata

obtained from a murine septicemia model show that many ofthe

novel tetracyclines synthesized have potent antibioticactivities. This

synthetic platform gives access to a broad range oftetracyclines that

would be inaccessible by semi-synthesis and provides apowerful

engine for the discovery of new tetracyclines.59,60

Even on larger molecules, semi-synthetic and syntheticchemistry

has been successfully applied to study and optimize leadcompounds.

The lipoglycodepsipeptide ramoplanin (Figure 3b) is 2–10 timesmore

active than vancomycin against Gram-positive bacteria andmaintains

full activity against VRE and all MRSA strains. However, itssystemic

use has been prevented by its low tolerability at the injectionsite,

apparently related to the length of the fatty acid sidechain.

To overcome this problem, the fatty acid side chain wasselectively

removed and replaced with different carboxylic acids. Manyderivatives

showed an antimicrobial activity similar to that of theprecursor,

and a significantly improved local tolerability.61 Therecently

described, fully synthetic lactam analog of ramoplanin showedthe

same biological activity as the natural product. Moreover, a setof

alanine analogs, obtained by total synthesis, has providedinsights into

the importance of individual amino-acid residues onramoplanin

activity. The MICs of each alanine-containing analog parallelsits

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

tolerate alanine substitutions, MICs increased 415-fold uponalanine

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

12. The new data thus confirm the importance of theornithine

residues at positions 4 and 10, with the latter directlyinvolved in

target binding, most likely by ion pairing with the diphosphateof

Lipid II.62,63

The mannopeptimycins, which were originally isolated in thelate

1950s from Streptomyces hygroscopicus, have been recentlyrevived

because of their promising activity against clinically importantGrampositive

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

also bind to Lipid II, but in a manner different fromramoplanin,

mersacidin and vancomycin. Multiple approaches have been usedto

optimize the mannopeptimycin activity profile, includingselective

chemical derivatization, precursor-directed biosynthesis andpathway

engineering. The SAR data have shown that substitution of ahydrophobic

ester group on the N-linked mannose or serine moieties

suppressed antibacterial activity, whereas hydrophobic acylationon

either of the two O-mannoses, particularly the terminalmannose,

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

optimized lead obtained by adamantyl ketalization of acyclohexyl

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 thesynthesis of

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

than the parent antibiotic.65 The cyclic heptapeptide GE23077 isa

potent and selective inhibitor of bacterial RNA polymerase

that, probably because of its hydrophilicity, is unable tocross

bacterial membranes. New derivatives obtained by modifyingdifferent

moieties were reported. Although many of them retainedactivity

on the enzyme, none showed a significant antibacterialactivity

apart from marginal inhibition of Moraxella catarrhalisgrowth

(Figure 3b).66

FUTURE PERSPECTIVES

This brief and nonexhaustive excursus on the present andfuture

pipeline of antibacterial agents for treating human diseasesprovides

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

antibiotics under clinical development (Table 1), 67% arenatural

products themselves, or natural product-derived compounds, apercentage

perfectly in line with that found with exisiting drugs.67

The second consideration is that the major players inantibacterial

development are small companies, which are not deterred by thesmall

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

significant number of the compounds listed in Table 1 werenot

discovered by small companies, but actually represent projectsabandoned

by large pharmaceuticals companies. Thus, it remains to be

seen whether small biotechs will dedicate sufficient resourcesand be

successful in discovering and developing novel antibacterialagents.

In this relatively grim scenario, microbial products continueto

provide new chemical classes or unexpectedly active variantsof

chemical classes already known to science. New technologiescan

now provide access to unexplored microbial diversity or tohypersensitive

assays to detect bioactive compounds. Furthermore, theinformation

derived from rapidly accessing the genome of many microbial

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

making more effective traditional, bioassay-based screeningefforts.

In our opinion, no single technology will represent the magicbullet

for antibiotic discovery, but only the painstaking integrationof a

multidiscplinary team with profound knowledge ofmicrobiology,

chemistry and bioinformatics will ultimately lead to newantibacterial

agents of medical relevance and commercial success.