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Molecular Microbiology (2006) doi:10.1111/j.1365-2958.2006.05063.x

2006 The AuthorsJournal compilation 2006 Blackwell Publishing Ltd

Blackwell Publishing LtdOxford, UKMMIMolecular Microbiology0950-382X 2006 The Authors; Journal compilation 2006 Blackwell Publishing Ltd? 2006??Review ArticleAntibiotics and the ribosomeT.Tenson and A. Mankin

Accepted 5 January, 2006. For correspondence. *[email protected]; Tel. (+372) 7 374 844; Fax (+372) 7 374 900; orE-mail [email protected]; Tel. (+

1) 312 413 1406; Fax (

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1) 312 4139303

MicroReview

Antibiotics and the ribosome

Tanel Tenson

1

* and Alexander Mankin

2

1

Institute of Technology, University of Tartu, Nooruse 1,

Tartu 50411, Estonia.2

Center for Pharmaceutical Biotechnology m/c 870,

University of Illinois, 900 S. Ashland Ave., Chicago, IL

60607, USA.

Summary

The ribosome is one of the main antibiotic targets in

the cell. Recent years brought important insights into

the mode of interaction of antibiotics with the ribo-some and mechanisms of antibiotic action. Ribosome

crystallography provided a detailed view of the inter-

actions between antibiotics and rRNA. Advances in

biochemical techniques let us better understand

how the binding of small organic molecules can

interfere with functions of an enzyme four orders of

magnitude larger than the inhibitor. These and other

achievements paved the way for the development of

new ribosome-targeting antibiotics, some of which

have already entered medical practice. The recent

progress, problems and new directions of research of

ribosome-targeting antibiotics are discussed in thisreview.

Introduction

A great variety of natural, semisynthetic or synthetic anti-

biotics inhibit the proliferation of pathogenic bacteria by

binding to their ribosomes and interfering with translation.

For decades, protein synthesis inhibitors have been

among the most successful, clinically useful antibiotics.

Recent years brought significant progress in our under-

standing of how drugs bind to the ribosome and interfere

with translation. However, many critical aspects of the

drug action, adverse effects and mechanisms of resis-

tance remain obscure, even when it comes to the oldest

and best-studied drugs. There are even bigger gaps in

understanding the modes of action of newer classes of

ribosome-targeting antibiotics.

These and related topics were the subject of discussion

at the meeting Antibiotics and Translation that took place

in Tartu (Estonia) in June 2005. However, this brief review,

which was inspired by the talks and discussions that took

place at the meeting, is not a meeting report. Rather, our

intention is to summarize the current state of the field of

ribosomal antibiotics and to illuminate its major trends,

advances, and problems using examples of several well-

studied drugs as well as new compounds.

Interaction of antibiotics with the ribosome

The ribosome exceeds the size of an average antibiotic

by four orders of magnitude and presents multiple sites

for antibiotic binding. Before the advent of ribosome crys-

tallography, the main ways of studying drugribosome

interactions were based on characterizing resistance

mutations in the genes of ribosomal components and

mapping drug binding sites by cross-linking or footprinting

(Spahn and Prescott, 1996). These techniques provided

extremely valuable, although low-resolution, information.

They showed that most of the sites of antibiotic action

coincide with the well-recognized functional centres of the

ribosome, such as the decoding centre and tRNA bindingsites in the small subunit and the peptidyl transferase

centre, nascent peptide exit tunnel and/or GTPase-acti-

vating region in the large subunit. However, the molecular

and atomic details of the interaction of drugs with their

target were beyond the resolution power of these meth-

ods. The nuclear magnetic resonance (NMR) studies of

aminoglycosides complexed to synthetic RNA (Fourmy

et al

., 1996) provided the first detailed view of the interac-

tion of protein synthesis inhibitors with the RNA target.

More recent crystallographic studies of ribosomal sub-

units complexed with a variety of antibiotics presented an

extended panel of fascinating, detailed views of the ribo-

somedrug interactions at atomic resolution (Brodersen

et al

., 2000; Carter et al

., 2000; Ogle et al

., 2001; Schlun-

zen et al

., 2001; Hansen et al

., 2002; 2003; Tu et al

.,

2005; Yonath, 2005). Compatible with the view of the

ribosome as an RNA machine and in excellent agreement

with genetic and biochemical data, NMR and crystallog-

raphy showed that interactions of drugs with rRNA

account for most of the contacts that antibiotics establish

with their ribosomal target.


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2

T. Tenson and A. Mankin

2006 The AuthorsJournal compilation 2006 Blackwell Publishing Ltd, Molecular Microbiology

In the small ribosomal subunit, the best-studied site of

drug action is the decoding centre, which is targeted by

aminoglycosides (Fig. 1). The site comprises a segment

of an extended rRNA helix 44 that resides on the interface

surface of the small subunit (Ogle et al

., 2001). The gen-

eral organization of this rRNA element is not influenced

significantly by contacts with other ribosome elements.

Therefore, its structure is preserved even when isolated

from the ribosome. This made it possible to use study

fairly small artificial molecular complexes to understand

how aminoglycosides interact with their rRNA target

(Lynch et al

., 2003). Common aminoglycoside antibiotics

share a universal two-ring structure of neamine that

includes the 2

-deoxystreptamine moiety (ring II) (Fig. 1A

and B). Depending on the type of ring II substitution (4,5

or 4,6), the drugs fall into neomycin/paromomycin or kan-

amycin/gentamicin families respectively. The NMR and

crystallographic studies showed that the universal neam-

ine two-ring structure forms a solid lock-and-key type of

interaction with the target site (Ogle et al

., 2001; Vicens

and Westhof, 2001; 2003; Lynch et al

., 2003; Francois

et al

., 2005). The ring I is inser ted into helix 44 by stacking

against G1491 (here and throughout the article, the num-

bering of Escherichia coli

rRNA is used) and by forming

a pseudo-base pair by establishing two hydrogen bonds

with the universally conserved A1408. This interaction

helps to maintain adenines A1492 and A1493 in the

bulged-out conformation that is similar to the conformation

in the ribosome where codonanticodon interaction

occurs in the A site (Fig. 1E and F). The conserved ring

II establishes hydrogen bonds with G1494 and U1495 of

16S rRNA. The universal interactions of rings I and II of

the neamine moiety of aminoglycosides with the decodingcentre are supplemented by opportunistic (fuzzy) inter-

actions that drug-specific rings III and IV of aminoglyco-

sides establish with helix 44 of 16S rRNA. These fuzzy

contacts fine-tune placement of the drug within its RNA

target and define the drug specificity of aminoglycoside

resistance mutations.

A similar trend the universal interactions of the con-

served pharmacophore with the core binding site, which

is fine-tuned by interactions of drug-specific chemical

groups with the ancillary nucleotides is also seen in the

binding mode of another ancillary important class of

drugs, macrolides (Schlunzen et al


.,

2002; Berisio et al

., 2003a,b; Tu et al

., 2005; Wilson et al

.,

2005). Macrolides consist of a 14- to 16-atom lactone ring

decorated with one or several sugars and side-chains

(Fig. 2). These drugs bind at the upper segment of the

nascent peptide exit tunnel in the large ribosomal subunit

near the peptidyl transferase centre. Crystallographic

studies of macrolides complexed to the large ribosomal

subunit of archaeon Haloarcula marismortui

and of bac-

terium Deinococcus radiodurans

showed a conserved

interaction of the macrolide core with the ribosome. The

lactone ring lays flat against the RNA-based exit tunnel

wall, and the universally present monosaccharide or dis-

accharide extensions at position 5 of the lactone protrude

towards the peptidyl transferase centre (Fig. 2) and inter-

act with nucleotides A2058 and A2059. Methylation of

A2058 (and sometimes A2059) by Erm-type methyltrans-

ferases confers resistance to all classes of macrolides

(Madsen et al

., 2005). These universal contacts are

supplemented by additional idiosyncratic interactions that

side-chains of individual macrolides establish with rRNA.

These latter drug-specific contacts may contribute dra-

matically to the drug affinity for its ribosomal binding site.

For example, binding of tylosin is enhanced owing to the

interaction of the mycinose sugar extending from the C14

position of the drug with the loop of helix 35 in domain II

of 23S rRNA (Fig. 2G) (Hansen et al

., 2002). Not surpris-

ingly, methylation of nucleotides in the loop of helix 35 in

domain II of 23S rRNA can confer resistance to tylosin

(Liu and Douthwaite, 2002), and the tylosin producer,

Streptomyces fradiae

, carries a dedicated methylase thathelps it to avoid suicide by methylating G748 in the loop

of helix 35 (Douthwaite et al

., 2004).

Increased affinity owing to the interaction of a side-

chain with the loop of helix 35 is apparently also exploited

by ketolides. These drugs represent the newest genera-

tion of macrolides, which lack 3-cladinose but carry a

functionally important alkyl-aryl or quinollylallyl side-chain

that can be attached at different positions of the core

macrolide structure (Zhong and Shortridge, 2001)

(Fig. 3). Biochemical (RNA footprinting) and genetic

(resistance mutations) data obtained with E. coli

ribo-

somes agree with interaction of the ketolide side-chain with the loop of helix 35 (Hansen et al

., 1999; Xiong

et al

., 1999). Surprisingly, however, a direct interaction

between the drugs and the loop of helix 35 was not

observed in either of the explored crystalline complexes

of ketolides with archaeal (

H. marismortui

) or bacterial

(

D. radiodurans

) ribosomes (Berisio et al

., 2003a; Schlun-

zen et al

., 2003; Tu et al

., 2005). Furthermore, in the

D. radiodurans

and H. marismortui

structures, the position

of the ketolide side-chain is drastically different (Fig. 2H

and I) (Tu et al

., 2005; Wilson et al

., 2005). This discrep-

ancy raises important questions: Which of the conflicting

data should be used for rational design of newer mac-

rolides? Do any of these techniques show how drugs

interact with the ribosome in live pathogenic bacteria?

Some insights into these problems are provided by

comparative footprinting of ketolides on ribosomes of

different organisms (L. Xiong and A. Mankin, unpubl.

results). The RNA probing did not show an interaction of

telithromycin, a clinically important ketolide, with the loop

of helix 35 in ribosomes of D. radiodurans

or Halobacteria

in perfect agreement with the available crystallographic


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3


Fig. 1.

Interaction of aminoglycosideantibiotics with 16S rRNA in the smallribosomal subunit.AC. Chemical structures of (A) neomycin (B)

kanamycin and (C) streptomycin.DF. Orientation of 16S rRNA residues criticalfor monitoring codonanticodon interaction: (D)in the vacant 30S subunit; (E) during recogni-tion of accurate codonanticodon pairing; (F) in

the complexes of vacant 30S subunit with

paromomycin. Note change in orientation ofA1492 and A1493 in (E) and (F) compared with(D). According to Ogle et al

. (2001).


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4



structures. In contrast, the telithromycin side-chain effi-

ciently protected position 752 in the loop of helix 35 from

chemical modification in ribosomes of E. coli

and Staphy-

lococcus aureus

. Thus, footprinting data confirmed the

conclusion from crystallographic studies that the precise

placement of the same drug molecule can vary in ribo-

somes of different organisms. This inference is in line with

the notion that is starting to emerge from analysing the

spectra of resistance mutations in different organisms:

interactions of a drug with ribosomes of different species

can be rather specific, and they can be influenced not only

by the often conserved nucleotides of a functional sitetargeted by an antibiotic but also by less-conserved

peripheral rRNA residues. This new paradigm is appar-

ently going to replace the overly convenient generalization

that knowledge about the interaction of a drug with the

ribosomes of one model organism can be safely extrapo-

lated to understand how the drug binds to the ribosomes

of any pathogen.

Crystallography and conventional biochemical tech-

niques use fairly artificial ribosomedrug complexes that

certainly do not reproduce the complexity and sophistica-

tion of the real cell environment. Further limitations are

imposed by the fact that most of the in vitro

techniques

usually deal with the static, non-translating ribosome

whereas the ribosome undergoes cycles of conforma-

tional changes during protein synthesis in vivo

. Therefore,

monitoring interactions of antibiotics with the translating

ribosome in the living cell may provide unexpected

insights into the mode of drug binding and mechanisms

of drug action. One such example is presented by recent

studies of the action of oxazolidinones on ribosomes of

S. aureus

(Colca et al

., 2003). Oxazolidinones are a new

Fig. 2.

Interaction of macrolides with the large ribosomal subunit.AC. Chemical structures of a 14-atom lactone ring erythromycin (A),and a 16-atom lactone ring josamycin (B) and tylosin (C).D. Beginning of the nascent peptide exit tunnel (Schmeing et al

.,2002). The peptidyl transferase active site is marked by nucleotides

A2451 and C2452 (space-fill representation). Position of a Tyr-Phe-tRNA analogue (sticks representation) illustrates orientation of thenascent peptide; 3

-terminal A76 of tyrosyl-tRNA is shown in cyan,Tyr and Phe are yellow. Nucleotides A2058 and A2059 located in the

upper part of the tunnel and essential for binding of macrolides are

shown in green. Amino acid residues of proteins L4 and L22 areshown in blue and yellow respectively.E. Binding of carbomycin (a drug similar to josamycin) (Hansen et al

.,2002). Lactone ring is red and the mycaminose, mycarose and isobu-

tyrate residues extending to the peptidyl transferase centre are black.F. Binding or erythromycin to the ribosome (Schlunzen et al

., 2001).The lactone ring is red, desosamine sugar is black and cladinose isin magenta.

G. Binding of tylosin, a 16-member ring macrolide (Hansen et al

.,2002). Lactone is red, the mycaminosemycarose chain is black andthe mycinose sugar is magenta. Nucleotides belonging to the loop ofhelix 35 are shown in gold.H and I. difference in orientation of the alkyl-aryl side-chain (green)

of a ketolide telithromycin in the structure of the drug bound to thelarge ribosomal subunit of D. radiodurans

(H) (Berisio et al

., 2003a) orH. marismortui

(I) (Tu et al

., 2005).


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5


class of protein synthesis inhibitors that interfere with

translation by binding to the large ribosomal subunit

(Lin et al

., 1997; Kloss et al

., 1999; Xiong et al

., 2000).

Although mutational studies clearly pointed to peptidyl

transferase as the primary drug target, in vitro

biochemi-

cal data provided confusing results. In part, this was

owing to the fact that oxazolidinones are notoriously

sticky drugs that are prone to non-specific binding(Lin et al

., 1997). Therefore, attempts to cross-link oxazo-

lidinones to the ribosome in vitro

often resulted in the

labelling of a number of ribosomal proteins as well as

multiple sites in rRNA (Matassova et al

., 1999; A. Mankin,

unpublished). In dramatic contrast to the in vitro

experi-

ments, cross-linking of photo-active oxazolidinones in the

living S. aureus

cells resulted in very specific labelling of

only one ribosomal protein (L27) and only one nucleotide

(A2602) in rRNA of the entire ribosome (Colca et al

.,

2003). Thus, either unique intracellular ionic conditions or

one of the functionally significant conformational states of

the ribosome creates a highly specific site for oxazolidi-

none binding. This site is obviously the main site of the

drug action, as mutations of the surrounding positions

render cells resistant to oxazolidinones (Prystowsky

et al

., 2001; Tsiodras et al

., 2001; Meka et al

., 2004).

Interestingly, cross-linking of the drug to the protein L27

indicates that the N-terminus of the protein reaches deep

into the peptidyl transferase centre (Maguire et al

., 2005)

a concept that is not immediately obvious from the

available crystallographic structures of the archaeal large

ribosomal subunit (Harms et al

., 2001; Moore and Steitz,

2002). Intriguingly, a short RNA molecule of the size of

tRNA was seen cross-linked by oxazolidinone in vivo

(Colca et al

., 2003). The nature of this RNA has not been

identified, but it may provide important clues about the

still-obscure mechanism of drug action. Therefore, it

appears that in vivo

studies of the drug binding and action

may reveal very important aspects of the mechanisms ofinhibition of protein synthesis that are beyond the reach

of most in vitro

techniques.

Mechanisms of action

Even knowing the exact location of the drug binding site

in the ribosome and understanding the mode of antibiotic

binding is often not enough to understand how antibiotics

inhibit translation. The early functional studies on the

mechanisms of antibiotic action carried out in the 1960s

to 1970s elucidated the most general mechanisms of

action of the major antibiotic families. However, the lack

of appropriate experimental techniques left many ques-

tions unanswered. What makes some classes of riboso-

mal antibiotics bactericidal while most protein synthesis

inhibitors are bacteriostatic? Do drugs that bind to the

same ribosomal site have the same mode of action? What

is the relationship between the drug structure, its affinity

for the ribosome and the mechanism of protein synthesis

inhibition? Some of these questions can now be

addressed owing to advances in ribosome crystallogra-

Fig. 3.

Chemical structures of new ribosome-targeting antibiotics: ketolides (telithromycin and cethromycin), linezolid and tygacil.


6/14

6



phy, fast kinetic assays, the availability of efficient cell-free

protein synthesis systems and other new techniques.

Ribosome crystallography provided a clear view of the

mechanism of action of aminoglycoside drugs that act at

the decoding centre (Ogle et al

., 2001). The neamine

core-based aminoglycoside drugs affect the ribosome

accuracy at the initial step of aminoacyl-tRNA selection.

Binding of the universal aminoglycoside rings I and II to

the decoding centre changes the orientation of two uni-

versally conserved adenine residues at positions 1492

and 1493 of 16S rRNA (Fig. 1F). A similar conformational

switch occurs during mRNA decoding when these two

adenines monitor the accuracy of codonanticodon base

pairing at the first and second anticodon positions

(Fig. 1E). Flipping out of the adenines 1492/1493 during

decoding signals the establishment of an accurate codon

anticodon pairing. Reorientation of A1492/A1493 induced

by aminoglycosides explains the miscoding effect of these

drugs: in the presence of a bound aminoglycoside, the

decoding centre issues an approval signal, even on the

binding of a non-cognate tRNA, thereby increasing thefrequency of amino acid misincorporation (Ogle et al

.,

2001; Ogle and Ramakrishnan, 2005).

A rather special aminoglycoside is streptomycin. It is

chemically related to aminoglycosides of the neamine

type (Fig. 1C), and it produces a similar effect, miscoding.

However, in contrast to deoxystreptamine-based ami-

noglycosides, the structure of streptomycin is built around

the streptamine core, and it binds at a different site in the

small ribosomal subunit, where it bridges helices 18 and

27 (Carter et al

., 2000). Aminoglycosides of both types

the neamine type and streptomycin- induce misincorpora-

tion of amino acids into a growing polypeptide chain byscrambling communication between the decoding and

GTPase-associated centres of the ribosome. However,

the molecular mechanisms of their action are different

(Pape et al

., 2000; Ogle et al

., 2001; Gromadski and Rod-

nina, 2004). An aminoacyl-tRNA enters the ribosome in

the complex with EF-Tu and GTP. GTP hydrolysis is

required for EF-Tu dissociation and aminoacyl-tRNA

accommodation in the A site. In case of the cognate

codonanticodon interaction the induction of GTP hydrol-

ysis is faster than in case of non-cognate or near-cognate

codons (Ogle and Ramakrishnan, 2005). This difference

in the rate of activation of GTP hydrolysis contributes

significantly to the accuracy of translation. Paramomycin,

an aminoglycoside of the neamine type, increases the rate

of GTP hydrolysis in case of the near-cognate tRNA to be

closer to the cognate value (Papeet al

., 2000). In contrast,

streptomycin, which reduces the conformational flexibility

of the small ribosomal subunit, lowers the rate of GTPase

activation for cognate tRNA but increases it for near-

cognate tRNA, bringing them to a more similar value

(Gromadski and Rodnina, 2004). Thus, paramomycin and

streptomycin use different strategies to eliminate the dif-

ference in the rate of GTPase activation for cognate and

near-cognate tRNAs, resulting in reduced accuracy of

translation.

Significant progress has also been achieved in under-

standing the mode of action of macrolides. The binding of

macrolides in the exit tunnel close to the peptidyl trans-

ferase centre inhibits translation by blocking progression

of the nascent peptide through the exit tunnel (Schlunzen

et al


., 2002). Different macrolides

leave different amounts of space available for the newly

synthesized peptide. In cell-free translation systems, bulk-

ier macrolides that leave less free space between the

peptidyl transferase active site and the site of drug binding

begin to inhibit translation when the nascent peptide is still

very short, while drugs that leave more free space inhibit

translation only when the nascent peptide is already fairly

long (Tenson et al

., 2003). For example, josamycin, which

reaches very closely to the peptidyl transferase centre,

effectively inhibits formation of dipeptides and tripeptides,

whereas the smaller erythromycin molecule, which leavesmore space for the growing peptide chain, starts inhibiting

translation only when the nascent peptide is at least five

amino acids long (Tenson et al

., 2003; Lovmar et al

.,

2004). Interestingly, however, the crystal structures of the

ribosomeerythromycin complexes indicate that nascent

peptides that are only three or four amino acids long

should already reach the erythromycin molecule bound at

its ribosomal site (Schlunzen et al

., 2001; Hansen et al.,

2002). Therefore, the longer nascent peptides that appar-

ently can coexist with a bound macrolide molecule in the

ribosomal tunnel have to recoil tightly, fold back or slip by

the drug. This notion of a nascent peptide bypassing thebound macrolide molecule was emphasized by the recent

crystallographic structures of ribosomeerythromycin

complexes, which showed that the drug may not

sufficiently obstruct the tunnel opening to completely

block progression of the newly synthesized polypeptide

(Tu et al., 2005).

Hindering growth of the nascent peptide by the mac-

rolide eventually results in dissociation of peptidyl-tRNA

from the ribosome (Menninger and Otto, 1982). Accumu-

lation of peptidyl-tRNA in cell cytoplasm and its slow recy-

cling can be an important factor of the inhibitory action of

this class of antibiotics (Lovmar et al., 2004). The differ-

ence in the dissociation rate of a macrolide antibiotic from

the ribosome as compared with the rate of peptidyl-tRNA

drop-off might determine the mode of drug action (Lovmar

et al., 2004). This conclusion is illustrated by the compar-

ison of two macrolide antibiotics josamycin and erythro-

mycin. Both of these drugs have comparable affinity for

the ribosome (Kd of 5.5 nM and 10.8 nM for josamycin and

erythromycin respectively). However, their dissociation

rates differ by approximately two orders of magnitude.


7/14

Antibiotics and the ribosome 7


Because of this difference, josamycin dissociates from the

ribosome more than 100-fold more slowly than peptidyl-

tRNA, whereas the dissociation rate of erythromycin is

roughly the same as that of peptidyl-tRNA. Essentially

none of the ribosomes that are blocked by josamycin will

be able to synthesize a polypeptide, while half of the

ribosomes will lose erythromycin before the dissociation

of peptidyl-tRNA, and polypeptide elongation can then

resume. Once the nascent polypeptide becomes long

enough to prevent rebinding of erythromycin (more than

eight amino acids long; Tenson et al., 2003), the synthesis

of the polypeptide will be completed, despite the presence

of the drug in cell cytoplasm. Therefore, while all of the

ribosomes that carry josamycin will be inactive in protein

synthesis, only a fraction of erythromycin-bound ribo-

somes will be unable to complete synthesis of the initiated

polypeptide. This prediction was experimentally con-

firmed: in a cell-free system, josamycin could completely

inhibit synthesis of a dodecapeptide, whereas up to 40%

of its control amount could be synthesized, even at satu-

rating concentrations of erythromycin (Lovmar et al.,2004). This example shows how high-resolution kinetic

data can illuminate important aspects of drug action. It is

likely that extending detailed kinetic measurements to

studies of other antibiotics will provide important clues

about the mechanisms of action of various protein synthe-

sis inhibitors.

Cell response to protein synthesis inhibitors

Binding a drug to the active centres of the ribosome blocks

translation. This eventually leads to inhibition of cell

growth and, sometimes, to cell death. What happensbetween these two events? How does the cell respond to

the drug while it still has the capacity to do so?

All antibiotics induce cell stress response. Heat- or cold-

shock responses are usually activated when bacterial

cells are treated with inhibitors of protein synthesis (Van-

Bogelen and Neidhardt, 1990). In addition, several other

stress response pathways might be induced by antibiotics

(Shapiro and Baneyx, 2002; Fischer et al., 2004). It is

difficult, however, to correlate the type of stress response

with the mode of drug action. It is also unclear whether

stress response increases cell survival upon antibiotic

treatment. Investigation of drug effects on strains lacking

individual stress response pathways and studying the

general response of a bacterial cell to treatment with

protein synthesis inhibitors may therefore open new direc-

tions for developing better drugs (Luidalepp et al., 2005).

The DNA microarray and new proteomic technologies

make it possible to explore cellular response to antibiotics

at the level of transcription, mRNA accumulation or protein

production (Evers et al., 2001; Goh et al., 2002; Sabina

et al., 2003; Hutter et al., 2004; Tsui et al., 2004). Antibi-

otic treatment changes the expression level of many

genes. One of the expected cell responses to inhibition of

translation is an attempt to restore protein synthetic

capacity by increasing ribosome production and activating

transcription of rRNA and ribosomal protein genes. How-

ever, while rRNA can be rapidly produced by transcription,

corresponding accumulation of ribosomal proteins is

diminished as translation is inhibited. Such an imbalance

in the manufacturing of ribosomal components should

inevitably lead to defects in ribosome assembly in

response to antibiotic treatment. This effect has long been

known for chloramphenicol (Dodd et al., 1991), and more

recently, it has been described for several other inhibitors

of protein synthesis: aminoglycosides, macrolides and

others (Champney and Miller, 2002a,b; Mehta and

Champney, 2003). In the case of macrolides, the drug

binding to assembly precursors has been suggested as a

cause of assembly inhibition, and it was proposed that

interference with the ribosome biogenesis accounts for

half of the macrolide inhibitory action (Champney and

Burdine, 1996). It has to be seen, however, whetherantibiotic-induced assembly defects are caused directly

by the drug binding to the assembly intermediates or are

a mere consequence of a distorted ratio in the production

of ribosomal components.

Interestingly, even low concentrations of antibiotics pro-

voke notable cell responses at the level of gene expres-

sion (Goh et al., 2002). Transcription of 510% of cellular

genes appears to be up- or downregulated in the pres-

ence of subinhibitory concentrations of the ribosome-

targeting drugs. This response is highly drug-specific and

may be directly or indirectly linked to the mechanism of

the drug action (Tsui et al., 2004). Therefore, it can beused as a hallmark of the mechanism of action of new

antibiotics. Surprisingly, even drugs that are not consid-

ered to be inhibitors of bacterial protein synthesis (aniso-

mycin, cycloheximide) appear to elicit significant changes

in the transcription of a number of genes, suggesting that

either these drugs do, in fact, interact with the bacterial

ribosome or that their interactions with other cellular tar-

gets provoke alterations in gene expression.

Separation of the fine effects of the drug on protein

production from their gross effect on cell growth opens

interesting venues for investigating detailed mechanisms

of the drug action. Most of the current models of the

mechanisms of antibiotic action operate with the effects

of the drugs on bulk protein synthesis. However, a number

of indications suggest that the extent of inhibition of trans-

lation of different genes may vary and depend on the

nature of mRNA, tRNA or the polypeptide (Tenson and

Mankin, 1995; Lovett and Rogers, 1996; Tenson and

Ehrenberg, 2002). Thus, josamycin inhibits incorporation

of phenylalanine into dipeptide much more strongly than

incorporation of valine (Lovmar et al., 2004), whereas


8/14

8 T. Tenson and A. Mankin


erythromycin and chemically unrelated pactamycin

severely inhibit poly(A)-dependent poly(Lys) synthesis but

have little effect on poly(U)-dependent synthesis of

poly(Phe) (Picking et al., 1991; Dinos et al., 2004). The

proteome- and transcriptome-profiling approaches and

recently developed defined efficient cell-free translation

systems may be instrumental in unravelling the specific

effects of antibiotics on the synthesis of individual proteins

(Antoun et al., 2004; Freistroffer et al., 1997; Tenson et al.,

2003).

Antibiotic resistance

Appearance and spread of antibiotic resistance closely

follow the introduction of new drugs to medical practice.

When drugs act on a protein enzyme, target site muta-

tions are one of the most common resistance mecha-

nisms. Multiplicity of rRNA genes in most microorganisms

slows development of this type of resistance to the

ribosome-targeting protein synthesis inhibitors (Cundliffe,

1990). Nevertheless, other resistance mechanisms, oftenoriginating in drug-producing organisms, eventually ren-

der pathogens resistant to the currently used drugs. In

addition, several important pathogens (such as Mycobac-

terium tuberculosisor Helicobacter pylori) have only one

or two rRNA cistrons which facilitates development of drug

resistance due to rRNA mutations. The problem of rising

resistance is the main driving force for the pharmaceutical

industry to continue the effort to modify existing drugs and

to develop newer antibiotics, and it has been a subject of

research in many laboratories (Shah, 2005).

Substantial progress has been achieved in understand-

ing the mechanisms of macrolide resistance. One of themost prominent mechanisms is modification of rRNA in

the drug target site by Erm-type methyltransferases

whose expression can be either constitutive or inducible

by low concentrations of macrolides (Weisblum, 1995a;

Liu and Douthwaite, 2002; Madsen et al., 2005). Erm

enzyme attaches one or two methyl groups to the N6

position of adenine 2058 in 23S rRNA. Methylation of

A2058 sterically hinders the binding of macrolides to the

ribosome (Fig. 2) (Schlunzen et al., 2001; Tu et al., 2005).

The A2058 methylation also affects binding of two other

important groups of antibiotics, lincosamides and strepto-

gramins B, producing a so-called MLSB resistance (Weis-

blum, 1995a). Importantly, different types of Erm genes

produce an idiosyncratic species-specific resistance pat-

tern (Douthwaite et al., 2005).

Until recently, Erm studies were complicated by the

lack of convenient techniques for monitoring A2058 meth-

ylation. While dimethylation of A2058 can be easily

detected by primer extension, monomethylation was diffi-

cult to monitor (Hansen et al., 2001). A considerable step

forward was brought about by combining efficient RNA

fragmentation techniques with mass spectrometry. This

approach, which has been used by S. Douthwaite and

colleagues at the University of Southern Denmark,

helped to establish a correlation between the site and

mechanism of rRNA methylation and the resistance pat-

tern in several pathogens (Madsen et al., 2005). Thus,

whereas dimethylation of A2058 in 23S rRNA renders

cells resistant to macrolides and ketolides, monomethyla-

tion of A2058 in Streptococcus pneumoniae and Stre-

ptococcus pyogenesconfers resistance to erythromycin,

but cells remain sensitive to ketolides (telithromycin)

(Douthwaite et al., 2005). In M. tuberculosis, the relaxed

specificity of Erm(37) results in methylation of not only

A2058 but also neighbouring A2057 and A2059, which

accounts for an unusual resistance profile of this patho-

gen (Madsen et al., 2005).

An important but still poorly understood problem is the

mechanism of inducible Erm resistance. Early studies by

the B. Weisblum and D. Dubnau laboratories revealed the

basic translation attenuation scheme, which involves stall-

ing of the ribosome during translation of a leader cistronof a bicistronic ermC operon (Dubnau, 1984; Weisblum,

1995b). Stalling, which occurs at low concentrations of

inducing macrolides, results in rearrangement of the

mRNA secondary structure and activation of expression

of the downstream ermmethyltransferase cistron. Stalling

critically depends on the sequence of the leader peptide

and is mediated by specific interactions between the ribo-

some, the drug and the nascent peptide. However, the

details of the operation of this mechanism remain largely

unknown. As Erm can interact with 23S rRNA only before

or during ribosome assembly (Skinner et al., 1983;

Champney et al., 2003), the ensuing resistance is basedon an intricate kinetic interplay between induction of erm

translation owing to stalling of the sensitive ribosome on

the leader peptide open reading frame (ORF), production

of functional Erm by resistant or drug-free ribosomes, and

accumulation of newly assembled resistant ribosomes.

Better understanding of the rates of individual reactions

and kinetic modelling of the entire network involved in

induction may help to find ways of evading this major

resistance mechanism. Little is known about the molecu-

lar mechanisms of interactions leading to the ribosome

stalling. It is clear that the structure of the drug can

dramatically influence erm inducibility (Kamimiya and

Weisblum, 1988). One of the acclaimed advantages of

ketolides, the newest generation of macrolide antibiotics,

is that these drugs do not induce inducible erm (Bryskier,

2000). The lack of the cladinose sugar at position 3 of the

lactone ring in ketolides is a likely reason why ketolides

do not activate ermexpression. However, it remains to be

elucidated whether ketolides, in contrast to other mac-

rolides, are unable to cause the ribosome stalling on the

leader ORF. Progress in this area will critically depend on


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our understanding of the conformation of the nascent

peptide and its progression through the exit tunnel.

Induction of erm resistance is likely related to another

resistance mechanism that also involves non-covalent

interaction between the ribosome and the nascent pep-

tides. Production of specific small peptides can render

cells resistant to a variety of macrolide antibiotics (Tenson

and Mankin, 2001; Vimberg et al., 2004). The resistance

peptides affect only the ribosome on which they were

synthesized, a phenomenon common to several other

functionally important nascent peptides (Tenson and Man-

kin, 1995; Lovett and Rogers, 1996; Tenson and Ehren-

berg, 2002; Cruz-Vera et al., 2005). Resistance conferred

by short peptide expression is based on specific interac-

tions between the nascent peptide and the macrolide, as

different peptide sequences produce resistance against

structurally different macrolides (Vimberg et al., 2004).

The available experimental data seem to be compatible

with a bottle brush model according to which resistance

peptides eject the drug from the ribosome (M. Lovmar

et al., in preparation). Yet again, the nature of molecularinteractions in the ribosome exit tunnel leading to drug

expulsion is unknown.

We already mentioned an emerging new paradigm in

understanding drugribosome interactions the recogni-

tion that ribosomes of different organisms may vary

substantially in their structure. This view has a direct impli-

cation in studies of antibiotic resistance. The general loca-

tion of the site of antibiotic action is usually the same in

different bacteria and, as a result, resistance mutations in

different species cluster in the same rRNA segments

(Vester and Douthwaite, 2001). However, specific effects

of mutations are often organism-specific. The recent find-ings by the Boettger and Yonath laboratories indicated that

the fitness cost of the A2058G mutation that confers MLSBresistance depends on the nature of the neighbouring

polymorphic 20572611 base pair (Pfister et al., 2005).

As a result, the same resistance mutation may have a

substantially different cost of fitness in different organ-

isms. An even more striking example of organism-specific

resistance mutations comes from studies of oxazolidino-

nes (reviewed in Meka and Gold, 2004). In clinical isolates

of streptococci, enterococci and staphylococci, oxazolidi-

none resistance is usually associated with a mutation at

position 2576 of 23S rRNA. In the laboratory-selected

resistant E. coli strains, resistance resulted from a

mutation at position 2032. In Mycobacterium smegmatis,

resistance was conferred by the G2447U mutation.

In the archaeon Halobacterium halobium, the resistance

resulted from several mutations in domain V of 23S rRNA,

with U2500C providing the highest resistance (Kloss

et al., 1999; Xiong et al., 2000; Prystowsky et al., 2001;

Sander et al., 2002). Although all of these mutations clus-

ter in the peptidyl transferase centre, thus revealing it as

the primary site of oxazolidinone action, their diversity

suggests either that the binding of the drug is somewhat

different in ribosomes of different organisms or that the

fitness cost of mutations varies significantly between dif-

ferent species.

The fitness cost of resistance mutations is an important

factor that affects appearance, fixation and maintenance

of the resistance. Acquisition of resistance is often asso-

ciated with a certain loss of fitness that can be manifested,

among other things, in hypersensitivity to several stress

conditions. For example, Salmonellastrains that became

resistant to fusidic acid owing to acquisition of the P413L

mutation in the translation factor EF-G are hypersensitive

to oxidative stress, UV light and several antibiotics

(Macvanin et al., 2004; Macvanin and Hughes, 2005).

A secondary mutation in the EF-G allele, T423I, partially

restores cell fitness (Nagaev et al., 2001). Compounds

targeting ribosomal sites where resistance mutations will

have high cost of fitness and could not be compensated

by the second site mutations should make good antibiot-

ics. Approaches are being developed to identify such sitesin the ribosome (Laios et al., 2004).

Adverse antibiotic effects mediated by inhibition of

mitochondrial protein synthesis

Most clinically useful inhibitors of protein synthesis act on

functionally important sites of the ribosome, such as

the decoding centre, peptidyl transferase or GTPase-

activating region (Spahn and Prescott, 1996). These sites

are highly conserved between ribosomes of different

domains of life, and only minor differences in the struc-

tures of bacterial and eukaryotic cytoplasmic ribosomesusually account for antibiotic selectivity.

An even bigger problem is avoiding inhibition of mito-

chondrial protein synthesis. Evolutionarily, bacterial

ribosomes are much closer to mitochondrial than to

cytoplasmic eukaryotic ribosomes. In spite of the much

smaller size of mitochondrial rRNA as compared with that

of bacteria, structural similarity of the functionally critical

regions of the mitochondrial and bacterial rRNA allows

some antibiotics to inhibit mitochondrial translation

(Bottger et al., 2001). One example is chloramphenicol, a

peptidyl transferase inhibitor that was widely used in the

past (Spahn and Prescott, 1996). Inhibition of mitochon-

drial protein synthesis by chloramphenicol (Denslow and

OBrien, 1978) seems to parallel the myelosuppression

induced by the drug, and it was the main reason why the

use of this antibiotic was discontinued in most developed

countries. The new class of ribosome-targeting antibiotics,

oxazolidinones, that, similarly to chloramphenicol, act on

the peptidyl transferase centre of the large ribosomal sub-

unit (Bobkova et al., 2003) can cause myelosuppression

upon prolonged administration (Kuter and Tillotson, 2001).


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Studies by Leach and co-workers at Pharmacia and

Upjohn (now Pfizer) indicated that inhibition of mitochon-

drial synthesis is a likely cause of oxazolidinone adverse

effects and is mediated by the drug binding to the large

subunit of mammalian mitochondrial ribosomes (Nagiec

et al., 2005).

Other drugs, such as aminoglycosides, that act on the

small ribosomal subunit can also interfere with mitochon-

drial protein synthesis. The unfortunate consequence

is neural damage, resulting in hearing loss (Fischel-

Ghodsian et al., 2004; Li et al., 2005). As toxic effects are

seen only in a limited number of patients, it seems that

the toxicity depends on the genetic variations between

individuals. Some mutations in mitochondrial rRNA that

appear to increase the similarity of the mitochondrial

rRNA to its eubacterial homologue have been shown to

correlate with the aminoglycoside-induced hearing loss

(Fischel-Ghodsian et al., 2004; Li et al., 2005). Other

mitochondrial mutations can potentially decrease the gen-

eral performance of mitochondrial ribosomes so that even

limited inhibition of their activity by antibiotics may bringthe level of protein synthesis to below the critical threshold

(Jacobs, 2003). It would be important to establish a

correlation between the polymorphism of human mito-

chondrial rRNA and the sensitivity of mitochondrial

protein synthesis to inhibitors of bacterial translation.

Such a pharmacogenomic approach may help to optimize

antibiotic regimens on the basis of the patients genetic

background.

New drugs

In recent years, we saw several impressive breakthroughsin the development of new, clinically useful ribosomal anti-

biotics (Sutcliffe, 2005). Significant progress has been

achieved with macrolides and oxazolidinones. Develop-

ment of newer macrolides was driven by the need for

drugs that can inhibit the growth of pathogens that devel-

oped macrolide resistance. Ketolides, which represent a

prominent new class of macrolide antibiotics, can do

exactly that (Zhong and Shortridge, 2001). Different

ketolides, including telithromycin developed by Aventis

and approved by the US Food and Drug Administration

(FDA) in 2004, cethromycin from Abbott, bridged ketolides

introduced by Enanta, and other ketolides have a cladi-

nose sugar at position 3 of the lactone ring replaced by a

ketone function (Fig. 3). In addition, ketolides usually

carry a carbamate cycle at positions 11,12 of the lactone,

and, most importantly, they all have an extended aromatic

side-chain whose site of attachment differs in different

drugs (Fig. 3). As mentioned earlier, the aromatic side-

chain may establish new interactions in the ribosome,

facilitating ketolide binding (even though it remains to be

seen which contacts the aromatic side-chain establishes

with ribosomes of real pathogens in vivo). Other interac-

tions of ketolides with the ribosome may also be different

from those of older macrolides (Garza-Ramos et al.,

2001). Differences in the drugribosome contacts account

for the higher affinity of ketolides for the ribosome and

help these drugs to overcome, at least partly, the resis-

tance caused by Erm methylation of rRNA (Douthwaite

et al., 2005). In addition, the lack of 3-cladinose is thought

to prevent ketolides from activating expression of inducible

Erm genes (reviewed in Bryskier, 2000).

A new generation of tetracyclines, glycylcyclines,

entered the market in June 2005 with FDA approval of the

first drug of this class, tigecycline (Tygacyl), developed by

Weyth. Tigecycline (Fig. 3) carries a glycylamido substitu-

tion at position 9 of the tetracycline ring structure and

shows activity against many resistant strains. Most

importantly, in contrast to the tetracyclines of previous

generations, tigecycline is not affected by conventional

tetracycline resistance mechanisms mediated by efflux or

ribosomal protection proteins (Bauer et al., 2004).

After more than 25 years during which all of the newantibiotics were a mere improvement of old antibiotic plat-

forms, a principally new class of antibacterials was intro-

duced in 2001 (reviewed in Bozdogan and Appelbaum,

2004). Linezolid (Fig. 3), a protein synthesis inhibitor rep-

resenting a new oxazolidinone class of antibiotics, was

developed by Pharmacia and Upjohn. The drug shows

excellent activity against many Gram-positive pathogens

and was not affected by existing mechanisms of ribosomal

resistance. Oxazolidinones bind at the A site of the ribo-

somal peptidyl transferase centre and inhibit its activity

(although in a not fully understood fashion) (Shinabarger

et al., 1997; Kloss et al., 1999; Polacek et al., 2002; Bobk-ova et al., 2003; Colca et al., 2003; Ippolito et al., 2005).

Several lines of evidence suggest that translation initiation

may be the main target of the drug action (Swaney et al.,

1998); however, other scenarios cannot be discarded

(Thompson et al., 2002) and more work is needed to sort

out the exact mechanism of translation inhibition by

oxazolidinones. It is most regrettable therefore that most

pharmaceutical companies developing oxazolidinones (or,

for that matter, other ribosome-targeting antibiotics) do not

appear to express much interest in the mechanisms of the

drug action. The university laboratories will apparently

need to pick up the slack to unravel the mode of action of

this important class of antibacterials.

Several other ribosome-targeting antibiotics are cur-

rently at different stages of development and characteriza-

tion. These include pleuromutilins (Schlunzen et al., 2004),

TAN-1057 (Limburg et al., 2004), evernimicin (Belova

et al., 2001) and several others. An example of structure-

based drug design potential is represented by a series of

compounds being developed by Rib-X, a start-up biotech-

nology company that uses ribosome crystallography and


11/14



computational modelling as the main guides for develop-

ment of new drugs (Ippolito et al., 2005). The proximity of

the binding sites of linezolid and sparsomycin (Hansen

et al., 2003) in the large ribosomal subunit provided direc-

tion for the design of Rx-01 series of oxazolidinone com-

pounds whose affinity to the ribosome is improved due to

additional interactions in the sparsomycin site (Lawrence

and Sutcliffe, 2005). One of these compounds has entered

phase I clinical trials at the end of 2005.

Concluding remarks

In this review, we briefly touched on several aspects that,

in our opinion, represent the major trends in the develop-

ment of the field of ribosome-targeting antibiotics. Inevita-

bly, this view is skewed by our own research interests, and

we realize that we might have left out some exciting lines

of research. However, what we have tried to communicate

is the recent rapid progress in the field that was brought

about by high-resolution crystallographic structures of the

ribosome and its complexes with antibiotics and wasfuelled by important developments in other techniques

and approaches. New antibiotics, inhibitors of protein syn-

thesis that are already used in medical practice as well as

drugs in the pipelines of several pharmaceutical compa-

nies will hopefully keep the field going for the foreseeable

future. However, a general pullout of the Big Pharma from

the antibiotic field and the continuous merging of pharma-

ceutical companies into bigger conglomerates eliminated

many important lines of antibiotic research, including stud-

ies of ribosome-targeting drugs. Thus, the influence of

academic science in the near future on the general direc-

tion of ribosome antibiotic research is expected to grow.We believe that studies of the fundamental aspects of the

drug action that are currently funded primarily by non-

industrial sources will generate new important venues for

drug development that will eventually be exploited by the

pharmaceutical industry. Importantly, these studies will

also advance our understanding of the important aspects

of protein synthesis and will provide new tools for studies

of translation.

Acknowledgements

We want to thank the participants of the meeting Antibioticsand Translation for stimulating discussions that inspired us

for writing this review. Our special thanks are to Stephen

Douthwaite, Joyce Sutcliffe, Karen Leach, Peter Moore,

Martin Lovmar and Julian Davies for the comments on the

manuscripts. We thank Shannon Foley for proofreading. The

antibiotic-related work in the T. Tenson laboratory is sup-

ported by The Wellcome Trust International Senior Fellowship

(070210/Z/03/Z) and grant from Estonian Science Founda-

tion (5311) and in the A. Mankin laboratory by NIH Grant U19

AI56575.

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