PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
ACS Chem Biol. Author manuscript; available in PMC Jan 20, 2013.
Published in final edited form as:
PMCID: PMC3499096
NIHMSID: NIHMS347269
The Chemistry of Peptidyltransferase Center-Targeted Antibiotics: Enzymatic Resistance and Approaches to Countering Resistance
Kevin P. McCusker and Danica Galonić Fujimori*
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 600 16th St, MC2280, San Francisco, CA 94158
Department of Pharmaceutical Chemistry, University of California, San Francisco, 600 16th St, MC2280, San Francisco, CA 94158
*Corresponding Author, Fujimori/at/cmp.ucsf.edu
The continued ability to treat bacterial infections requires effective antibiotics. The development of new therapeutics is guided by knowledge of the mechanisms of action of and resistance to these antibiotics. Continued efforts to understand and counteract antibiotic resistance mechanisms at a molecular level have the potential to direct development of new therapeutic strategies in addition to providing insight into the underlying biochemical functions impacted by antibiotics. The interaction of antibiotics with the peptidyltransferase center and adjacent exit tunnel within the bacterial ribosome is the predominant mechanism by which antibiotics impede translation, thus stalling growth and proliferation. Resistance enzymes catalyze the chemical modification of the RNA that composes these functional regions, leading to diminished binding of these antibiotics. This review discusses recent advances in the elucidation of chemical mechanisms underlying resistance and driving the development of new antibiotics.
The advent of antibiotics in the early twentieth century, followed by further development of these compounds, played a critical role in the treatment of infectious diseases and the corresponding decrease in mortality and morbidity from those causes (13). Among these early antibiotics was the macrolide erythromycin, a member of a large and chemically diverse group of antibiotic compounds that exert their action by interacting with the ribosomal RNA (rRNA) component of critical functional sites within the ribosome. Perhaps the most commonly targeted of these sites are the peptidyltransferase center (PTC) and the adjacent exit tunnel. Antibiotic binding to either of these sites interferes with the process of translation, which is the basis of the antibiotic activities of these compounds (4, 5). The acquisition of resistance by pathogenic microbes jeopardizes the continued clinical utility of antibiotic compounds (1, 6). Of the common resistance mechanisms (79), target mutations and modifications that impact the binding of PTC-targeted antibiotics have a dominant role. Relatively subtle changes to the rRNA architecture—i.e. methylation of a single nucleotide—yield significant resistance phenotypes (7, 10). When combined, these target modifications and mutations can lead to unpredicted and more severe resistance phenotypes; however, even a single modification can be sufficient to elicit a multi-drug resistance (MDR) phenotype (10) owing to the overlapping binding sites of antibiotics within the PTC and its immediate vicinity (7, 1113). The enzymatic mechanisms of these resistance modifications, their attendant structural consequences within the PTC and chemical approaches to countering this resistance are the focus of this review.
The antibiotic families that interact with the PTC include macrolides, (their derivatives ketolides), lincosamides, streptogramins, oxazolidinones, phenicols and pleuromutilins (Figure 1) (4, 7, 14). The overlapping nature of the binding sites of these compounds is evidenced by multiple, distinct multi-drug resistance phenotypes involving combinations of the aforementioned drugs, each arising from a single modification (7). Compounding this is the rapid pace of genetic changes and facility of horizontal gene transfer in prokaryotic pathogens. Shortly after the introduction of the natural product erythromycin into clinical use in 1953, resistance arose in Staphylococci, presumably due to the acquisition of the indigenous resistance mechanism of the producing strain (15, 16). As the indigenous resistance mechanism to the natural product penicillins—β-lactamase activity—was known prior to the introduction of penicillin to the clinic, this indigenous resistance to erythromycin should not have come as a great surprise (1, 17). However, when microbes exhibited resistance to the entirely synthetic oxazolidinone linezolid fifty-some years later (18), acquisition of an indigenous resistance mechanism seemed an unlikely explanation. At that juncture, it was evident that microbes could become resistant to virtually every PTC-targeted antibiotic—natural product, semi-synthetic or fully synthetic—that was currently available (19), and that a more complete understanding of the interrelated mechanisms of resistance was needed.
Figure 1
Figure 1
PTC-targeting antibiotics.
The indigenous resistance mechanism to erythromycin and related macrolides mentioned above involves the post-transcriptional modification of the 23S ribosomal RNA (rRNA) by a specific N-methyltransferase enzyme encoded by an erm (erythromycin ribosome methylation) gene (8) (Figure 2a). This modification still plays a significant role in resistant bacteria (7). There are currently 34 annotated erm genes and corresponding enzymes (7), which lead to the mono- or di- methylation of N6 of A2058 and occasionally A2509 (E. coli numbering will be employed throughout) (4, 20). By itself, this modification usually results in resistance to macrolides, lincosamides and streptogramins B (7). However, an erm gene has recently been found in an operon with cfr (chloramphenicol florfenicol resistance) leading to resistance against all of the previously noted classes of PTC-targeted antibiotics (21). The cfr gene was initially identified on plasmids in methicillin-resistant Staphylococci isolated from animals exhibiting antibiotic-resistant infections (22), but it has since been found in the chromosome of clinical isolates of MRSA (methicillin-resistant Staphylococcus aureus) (23). The enzyme encoded by cfr is responsible for the installation of a methyl group at the C8 position of A2503 within 23S rRNA (24). This single modification confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramins A (18). Despite the superficial similarity of rRNA methylation, the products of the erm and cfr genes catalyze distinct chemical transformations, differing in their sites of action (heteroatom vs. carbon) and mechanisms (Figure 2).
Figure 2
Figure 2
RNA modifications and polar methylation mechanisms. a) Methylated bases, labeled with the modification and representative enzymes responsible for the transformation. Superscripts denote the position of modification on an RNA base; subscripts indicate (more ...)
The canonical reaction, catalyzed by methyltransferases, in which the electrophilic S-adenosyl-L-methionine (SAM) donates a methyl group to a suitable nucleophilic site on RNA via a polar mechanism, has long been known. There are traditionally two types of sites at which this nucleophilic methylation occurs: 1) inherently nucleophilic heteroatoms (25) and 2) carbon centers rendered nucleophilic by a conjugate-addition mechanism (26) (see Figure 2 for mechanisms and modified bases). Heteroatom methylation by this mechanism accounts for a significant proportion of the modified bases in E. coli rRNA (2729), and the enzymes responsible for carrying out these reactions have largely been identified (27). The functional impacts of these methylations and other rRNA modifications have been reviewed recently (29). The polar reactions occurring at carbon centers require a more complicated mechanism than direct reaction with nucleophilic heteroatoms. These reactions modify the C5 position of the pyrimidine bases cytosine and uridine, but this position is only sufficiently nucleophilic to participate in the polar reaction subsequent to the addition of an enzyme-derived cysteine to C6 (Figure 2c) (26). While resulting in the addition of a methyl group to a carbon atom, these methyltransferase reactions are distinct from the installation of a methyl group on an electrophilic position such as the C8 of adenosine, as catalyzed by Cfr employing a significantly different mechanism.
The chemical demands of installing a methyl group onto the electrophilic C8 or C2 of adenosine are unique among RNA modification reactions. Cfr activity results in the methylation of the C8 of A2503 (24), and the related enzyme, RlmN, catalyzes the analogous installation of a methyl group at C2 of the same adenosine (30) (Figure 2). The recent identification of the enzymes responsible for these modifications in 23S rRNA has drawn attention to both the role that C8 methylation plays in a multi-drug resistance phenotype (18) and the novel catalytic mechanism employed by these enzymes (3137). The electrophilic, rather than nucleophilic, character of the C2 and C8 amidine positions of adenosine, coupled with the observation of the cysteine-rich motif (CX3CX2C), characteristic of the radical SAM superfamily, in Cfr and RlmN, implicate a radical mechanism (24).
To assess this unique mechanism, enzymes, purified anaerobically due to the presence of oxygen-sensitive iron-sulfur clusters, were assayed with intact ribosomes and individual ribosomal components to determine likely substrates. Both Cfr and RlmN were shown to act only on A2503 within naked 23S rRNA, using either full-length rRNA or truncated substrates (32). These in vitro experiments confirmed the prior in vivo observations of enzymatic activities (24, 30). Interestingly, in the in vitro experiments, Cfr was shown to modify both the C8 and C2 of A2503—i.e. 2,8-dimethyladenosine was seen as a product when rRNA with no prior modification at A2503 was used as a substrate for Cfr (32). This implies a significant degree of flexibility in the base orientation about the glycosidic linkage in A2503. In fact, both syn and anti conformations of A2503 in intact ribosomes have been observed crystallographically (3840); however, the relevance to the naked RNA substrate bound to Cfr remains unclear. It seems probable that the activity of the housekeeping RlmN was expanded in Cfr to include the second amidine carbon. The transition between the ancestral rlmN, through duplications and horizontal gene transfers, to the current cfr is incompletely defined, and it is unclear when cfr’s current role as an antibiotic resistance determinant arose (41). There is no evidence that cfr is an indigenous resistance mechanism in an organism that produces an antibiotic to which cfr confers resistance.
Further experiments were carried out to assess the role(s) of SAM in these reactions, which apparently require SAM as both a radical initiator and as a source of newly introduced carbon. Consistent with radical SAM enzymology (42, 43), 5′–deoxyadenosine (5′–dA) and methionine were produced from the reductive cleavage of SAM (32). Methylated bases and S-adenosyl homocysteine (SAH) were also observed (32), as expected from the use of SAM as a source of the newly introduced carbon (44). The canonical radical SAM mechanism predicts that the 5′–deoxyadenosyl radical (5′–dA) generated by reductive cleavage of SAM will subsequently be used to abstract a hydrogen atom from the prime substrate (45)—in this case, from one of the amidine positions on the adenosine base—in order to initiate the subsequent radical transformation. Although exceptions to this direct substrate activation mechanism have been noted (4648). However, the energetics of the abstraction of an amidine hydrogen atom (BDE ≥105 kcal mol−1) (49, 50) would appear to exceed the capacity of even as potent an oxidant as 5′–dA.
Subsequent deuterium labeling studies revealed additional unique aspects of the reaction catalyzed by these enzymes. When truncated RNA substrates bearing 2-2H adenosine (2-D A) at all positions normally occupied by adenosine were employed, the resulting methyladenosine products bore –CH2D groups, indicating the amidine hydrogen was retained in the product. Furthermore, the 5′–dA product from these reactions bore no deuterium, demonstrating that the 5′–dA was not being employed to abstract a hydrogen atom from the RNA substrate. Reciprocal studies using unlabeled RNA and [methyl-2H3]-SAM (CD3-SAM) yielded CD2H methyl groups in the methyladenosine products and monodeutero 5′–dA (31). This outcome indicated that 5′–dA activates a methyl group derived from SAM for addition into the RNA substrate, rather than activating the RNA substrate directly. Together, these observations led to the notion that these enzymes do not act as methyltransferases, but rather as methyl synthases, which assemble a methyl group from a methylene (ultimately derived from SAM) and the hydrogen atom from the substrate amidine carbon (31) (Figure 3a).
Figure 3
Figure 3
Deuterium labeling patterns observed in RlmN and the proposed RlmN mechanism. a) The observed incorporation and retention of deuterium from various labeling experiments carried out with RlmN. b) The mechanism of catalysis by RlmN proposed by Grove et (more ...)
Labeling studies carried out by a second group yielded additional evidence supporting the methyl synthase activity of Cfr and RlmN (34). When reactions were carried out using a significantly truncated RNA substrate under single turnover conditions, it was noted that the methyl group installed did not directly reflect the isotopic composition of the SAM added to the reaction (i.e. the methylated base contained a –CH3 whether CD3-SAM or unlabeled SAM was used). However, when the enzymes were produced in a methionine auxotroph supplemented with [methyl-2H3]-methionine (resulting in CD3 labeling of all methionine residues and positions methylated by SAM-dependent reactions in vivo), the RlmN methylated products bore CD2H groups (34).
Parallel experiments in Cfr revealed CH3, CDH2 and CD2H groups in the methylated product, implying significant proton exchange of an intermediate, and requiring further mechanistic evaluation in the case of Cfr (34). These results are consistent with the incorporation of a methylene fragment, rather than an intact methyl group, but they also implied that the methylene fragment was protein-derived. This was further evaluated by mass spectrometric analysis of RlmN peptides, revealing an S-methylated cysteine residue at position 355 (34), which was observed subsequently by crystallography (33).
Recently, it has been demonstrated that enzymes purified without intact iron sulfur clusters are devoid of the S-methyl group. Upon reconstitution of the clusters, the S-methyl group is formed in a SAM-dependent reaction concomitantly with SAH production, implying SAM binding at the cluster is required for the typical polar reaction of SAM with the enzyme-derived cysteine (35). This combined evidence for the methylated cysteine has led to the proposed mechanism (Figure 3b) in which Cys355 is pre-methylated by SAM. The cluster-generated 5′–dA then reacts with this S-methyl to produce the methylene fragment, which is then added to the substrate (34). This mechanism is likely to be more energetically favorable than abstracting a hydrogen atom directly from the methyl group of SAM, due to the stabilizing effects of the sulfur lone pair, which would be diminished were the adjacent sulfur positively charged, as in SAM (51). While a subsequent general base abstraction of the amidine hydrogen is proposed, the observed complete retention of this hydrogen requires that this general base be fully protected from solvent. The proposed mechanism also includes roles for two cysteine residues, unassociated with the iron sulfur cluster, that were previously noted to be required to confer antibiotic resistance in vivo (41). Cumulatively, these labeling data indicate that nature evolved a new chemical strategy to incorporate a methyl group at an electrophilic center, one where the methyl group assembly is initiated via addition of a thiomethylene into the substrate.
The rapid expansion of our mechanistic understanding of these enzymes has generated ample questions for immediate study (3137). Multiple experiments have indicated that the amidine hydrogen is retained in these reactions (31), yet the flexibility implied by Cfr’s dual specificity (32) would seem contrary to the controlled active site environment or total solvent exclusion required to achieve this retention. The reactions catalyzed by Cfr and RlmN require the input of 2 electrons; however, the timing of electron injection, whether critical microscopic steps are oxidative or reductive and the identity of the physiological reductant all remain unresolved. Further characterization of proposed intermediate species, particularly adducts or radicals, would seem the most informative in terms of understanding the critical microscopic steps involving electron transfer.
While enzymologists are well situated to continue elucidating the mechanistic aspects of this novel radical SAM methyl synthase activity, the physiological and functional roles underlying the initial evolution of C2 and C8 methylation remain elusive. The impact of C2 methylation on overall genetic fitness is minimal and any antibiotic resistance is modest (52). However, this modification may play a role in ribosome stalling during the translation of regulatory genes and subsequent activation of inducible resistance genes (38). The C8 modification is more complicated, as the duplication and mutation leading to C8 reactivity may have occurred in plants, where its activity and biological role are entirely undefined (41). This gives few clues as to why bacteria obtaining the gene by horizontal gene transfer would have maintained it outside of an antibiotic-selecting environment, despite its low fitness cost (53). Perhaps the most overarching question is whether there are additional sites modified by this mechanism, as it seems unlikely that this novel mechanism would have evolved exclusively to modify a single position.
The emergence of pathogens with multiple resistance phenotypes such as those carrying the mlr (modification of large ribosomal subunit) operon (containing both cfr and erm), which confers resistance to 7 classes of PTC targeting antibiotics (21), is certainly a cause for alarm from a public health perspective. Further, the relatively rapid emergence of resistance phenotypes in clinical strains (15, 16) highlights the need for drug development strategies that can counter these resistance modifications (1, 19). Recent work with ancient bacteria has indicated that the selective pressure exerted by clinical overuse of antibiotics may not be directly responsible for the evolution of resistance mechanisms; however, misuse of antibiotics may still hasten the spread of these resistance determinants and diminish the utility of the corresponding antibiotics (54). High-resolution structures of the bacterial ribosome with antibiotic compounds bound to the PTC has provided both a molecular-level understanding of the interactions between antibiotics and PTC residues as well as a basis for modifications to PTC-targeted drugs that may help evade resistance modifications (1113, 55).
Two varieties of structural changes (and their combination) can be envisioned to counteract perturbations introduced by target modification: elaboration of the compound to gain additional favorable interactions and removal of moieties predicted to clash with target modifications. Both of these design strategies have yielded positive results (56, 57), due, in part, to the availability of high-quality structural models (11, 13, 55). Despite this, species-dependent idiosyncratic interactions of antibiotics with target ribosomes (11) and incomplete understanding of PTC-targeted antibiotic mechanisms of action (4, 58, 59) still present challenges to drug development efforts. The most recent structural information regarding PTC-targeting antibiotics and the bacterial ribosome has served to clarify the interactions of four classes of antibiotics with the bacterial PTC, including those of a human pathogen (E. coli) (11). While largely affirming the conservation of the binding sites and interactions of these drugs with bacterial PTCs in general (11, 13), these structures also illustrated that species-dependent interactions can contribute significantly to the overall affinity and specificity of these drugs (11).
The critical interaction of macrolides with A2058, as indicated by Erm-dependent resistance mechanisms and the frequency of mutation of A2058 in resistant strains (58, 6063), has led to many attempts to counteract this resistance by introducing changes to the macrolide scaffold (64). The success of ketolides at inhibiting the growth of strains rendered resistant to erythromycin by the action of Erm was attributed to additional favorable interactions afforded by the alkyl-aryl substituent of these drugs. The combined evidence of telithromycin resistance mutations, chemical footprinting experiments, and structural data now reveal the nature of the interaction between the E. coli ribosome and the alkyl-aryl arm of telithromycin (11, 6569). A pi-stacking interaction of the aryl group with the A752:U2609 base pair, which forms an interdomain bridge not present in ribosome structures from other organisms (Figure 4), confirmed that a species-dependent interaction can indeed be responsible for increasing the affinity of these compounds several hundred-fold (13, 56). However, an alkyl-aryl arm cannot be relied upon to function identically to that of telithromycin in all cases. When a similar compound was made with the pendant group at C6 (Cethromycin, Figure 1), it exhibited encouraging preliminary results but failed during phase III trials due to lack of efficacy (64, 7072).
Figure 4
Figure 4
Erythromycin and telithromycin bound to E. coli ribosomes. Critical residues are indicated. The RNA backbone is shown as an orange ribbon; the protein chain is shown as a gray ribbon. Erythromycin is colored fuschia and telithromycin is colored light (more ...)
Beyond the elaboration of the alkyl-aryl substituent on ketolides, the point of attachment and, to some extent, the macrolide scaffold have been modified to recover some of the interaction lost due to resistance mechanisms (64). The so-called bicyclolides introduce a second heterocycle between the 6 and 11 or 6 and 3 positions (73, 74). The 6, 11-bicyclolide modithromycin (Figure 1) exhibits improved in vitro efficacy against erm+ strains of S. pyogenes showing idiosyncratic resistance to telithromycin (75). Another area of development unrelated to resistance is the alteration of the alkyl-arm to one containing an azetidine (azetidinyl ketolides in Figure 1). Addition of this moiety diminishes the hepatotoxicity of telithromycin (76). This improvement would allow application of the drug to a wider range of infections and a more expanded patient pool—both critical criteria for broad-spectrum agents.
An indirect approach to mitigate Erm-mediated resistance and restore clinical utility of impacted macrolides would be to inhibit the Erm enzymes. Previous inhibitors of Erm enzymes were found to bind to the conserved SAM binding site (7779). However, employing the conserved binding site of a common metabolite makes selective inhibition challenging. New approaches have been used to probe the sequence and structural elements of RNA substrates critical for methylation by Erm enzymes. This work has elucidated the minimal RNA substrate for Erm enzymes, which could guide the design of RNA analogs as Erm-specific inhibitors (80). These compounds would then be co-administered with macrolides, similar to the co-administration of β-lactams with β-lactamase inhibitors (e.g. Augmentin).
Substantial development of PTC-targeted antibiotics can also be found in pleuromutilins and oxazolidinones (64). The first pleuromutilin for human clinical use (Retapamulin) was FDA-approved in 2007, although there had been extensive (greater than 30 years) prior veterinary usage (64, 81, 82). Significant development of this underrepresented class was aimed at addressing macrolide resistance, as Erm-mediated mechanisms do not generally confer resistance to pleuromutilins. As a fully synthetic (as opposed to the predominant semi-synthetic and natural product-derived) scaffold, the oxazolidinones held significant promise as a method to evade existing resistance mechanisms while avoiding new ones as there were no antibiotic producing strains serving as pools of resistance genes (83).
The emergence of Cfr-related resistance to multiple antibiotic classes including the oxazolidinones proved that a novel, fully synthetic scaffold was not a late twentieth-century magic bullet, as the oxazolidinones bind to a site already exploited by other antibiotics (8486). However, in the case of Linezolid, both the modification of the aryl pendant group to gain further favorable binding interactions and the “de-elaboration” of the acetamide to an alcohol (administered as a prodrug phosphate) led to the development of radezolid and torezolid (phosphate), respectively (87, 88). Both of these compounds exhibit activity against linezolid resistant strains, although radezolid is not as effective against Cfr-mediated resistance as it is against mutations in the 23S rRNA or ribosomal proteins L3 or L4 (57). Torezolid is active against cfr+ strains at least in part due to the removal of the bulk of the acetamide that clashes with the 8-methyl group on A2503, the site of cfr-mediated methylation (Figure 5).
Figure 5
Figure 5
Linezolid and torezolid in Deinococcus radiodurans ribosomes. The 8-methyl group on A2503 was modeled using Pymol. a) Linezolid is shown in fuschia (from PDB file 3DLL). b) Torezolid is shown in light blue; the acetamidomethyl pendant group of linezolid (more ...)
Currently, among the PTC-targeted antibiotics, the clinical candidate pool is skewed toward oxazolidinones and pleuromutilins due both to the novelty of the scaffolds to human clinical use and the ability of both families to evade (at least partially) widespread Erm-based resistance. Clearly the spread of cfr+ strains poses a challenge for the continued clinical utility of oxazolidinones and pleuromutilins. It is probable that new resistance mechanisms will continue to surface, requiring the elaboration of existing scaffolds, the development of novel scaffolds as well as efforts to rescue some drugs from obsolescence by inhibition of resistance-causing enzymes.
The critical function of the PTC in translation has led to the large number of fine-tuning modifications of the proximal rRNA; it has also made it a common target for natural product antibiotics and subsequent resistance modifications. The continuing structural work on the ribosome provides an ever more refined picture of both the critical sites within the ribosome as well as the interactions between the ribosome and its small molecule binding partners, particularly PTC-targeting antibiotics (11, 13, 55). This progress is crucial, as even small refinements can yield significant insights into biological function and drug design when interrogating a system as finely tuned and critically important as the ribosome (12). Our growing appreciation of the mechanisms (including the newly discovered methyl synthase activity of Cfr and RlmN (3135)), the timing of action, and the specificity of rRNA modifying enzymes that lead to antibiotic resistance may allow inhibition of these enzymes and the rescue of antibiotics rendered ineffective by rRNA modifying resistance mechanisms (8, 9, 19, 80). Multiple drug design approaches are yielding some ability to counter resistance mechanisms, but the overlapping binding sites of most PTC-targeted antibiotics and the relatively minor variations that can lead to resistance present significant challenges. Multi-drug resistant phenotypes are unlikely to disappear, thus continued work on these fronts is critical to ensure a supply of safe, broad-spectrum compounds to safeguard the gains in public health enabled by the use of antibiotics.
Meanwhile, innovative techniques have allowed researchers to compete with microbes on their terms. Recent efforts in bacterial phenotypic profiling have uncovered novel interactions of gene and drug function, raising the possibility of drug-drug synergies of potential clinical value (89). Thorough validation of antibiotic targets in multiple genetic backgrounds in a cell-based assay has proven to correlate a drug to its target while revealing its method of entry, efflux sensitivity and resistance mechanism(s). Possessing this information from the outset of drug development—while also starting from compounds with empirical biological activity—would seem to provide a significant advantage (90). Animal (C. elegans) models have been employed to screen for compounds that would otherwise fail during in vitro screening, such as those that act as prodrugs, target virulence factors, or influence host immune response (91). Given the wide range of subtle factors employed by microbes to fine-tune ribosomal function and evade antibiotic activity, sustained, innovative and cooperative efforts must be made in research and drug development to counteract resistance and maintain the efficacy of antibiotics. We should also bear in mind that the public health utility of antibiotic compounds is enhanced by the increased comprehension of biological complexity and underlying bacterial biochemical function afforded by ongoing investigation of modes of antibiotic action and resistance mechanisms (92).
Acknowledgments
We would like to thank C. Fitzsimmons and F. Yan for critical comments on this manuscript. Our work on RNA modification and antibiotic resistance is supported by NIAID (R01A1095393–01) and NSF (Career 1056143), both granted to DGF.
Keywords
MethyltransferaseAn enzyme that catalyzes the transfer of an intact methyl group from the biological methyl donor, S-adenosyl-L-methionine, to a nucleophilic site on a target molecule via a polar mechanism
Methyl synthaseAn enzyme that catalyzes the installation of a methyl group at a specific site through the assembly of the methyl from constituent fragments, such as a methylene and a hydride
Radical SAM superfamilyA family of enzymes that catalyze diverse reactions initiated by a 5′–deoxyadenosyl radical formed by the reductive cleavage of S-adenosyl-L-methionine at a [4Fe-4S] cluster ligated by a conserved CX3CX2C motif
Peptidyltransferase centerThe active site of the ribosome, where peptide linkages are formed and from which the peptide is released. This is also a common site of interaction for antibiotic compounds
RibosomeThe protein biosynthetic machinery of the cell, composed of ribosomal RNA (rRNA) and protein components, commonly subdivided into the large and small subunits
AntibioticA chemotherapeutic agent employed to inhibit the growth of microbes. This term is often used interchangeably with antibacterial
Antibiotic resistanceA means by which bacteria evade the activity of an antibiotic compound. Commonly employed resistance mechanisms include active drug efflux, drug modification or inactivation, and modification of the target of the antibiotic compound

1. Wright GD. Molecular mechanisms of antibiotic resistance. Chem Commun. 2011;47:4055–4061. [PubMed]
2. Shlaes DM. Antibiotics: The perfect storm. Springer; New York: 2010. pp. 1–110.
3. Walsh C. Antibiotics: Actions, origins, resistance. ASM Press; Washington, D.C.: 2003. pp. 1–340.
4. Tenson T, Mankin A. Antibiotics and the ribosome. Mol Microbiol. 2006;59:1664–1677. [PubMed]
5. Wilson DN. The A-Z of bacterial translation inhibitors. Crit Rev Biochem Mol Biol. 2009;44:393–433. [PubMed]
6. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Jr, Gilbert D, Rice LB, Scheld M, Spellberg B, Bartlett J. Bad bugs, no drugs: No ESKAPE! An update from the infectious diseases society of america. Clin Infect Dis. 2009;48:1–12. [PubMed]
7. Roberts MC. Update on macrolide-lincosamide-streptogramin, ketolide, and oxazolidinone resistance genes. FEMS Microbiol Lett. 2008;282:147–159. [PubMed]
8. Douthwaite S, Fourmy D, Yoshizawa S. Nucleotide methylations in rRNA that confer resistance to ribosome-targeting antibiotics. In: Grosjean H, editor. Topics in Current Genetics. 2005. pp. 285–307.
9. Vester B, Long KS. Antibiotic resistance in bacteria caused by modified nucleosides in 23S ribosomal RNA. In: Grosjean H, editor. DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution. Landes Bioscience; Austin, TX: 2009. pp. 537–549.
10. Poehlsgaard J, Douthwaite S. The bacterial ribosome as a target for antibiotics. Nat Rev Micro. 2005;3:870–881. [PubMed]
11. Dunkle JA, Xiong L, Mankin AS, Cate JHD. Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc Natl Acad Sci USA. 2010;107:17152–17157. [PubMed]
12. Douthwaite S. Designer drugs for discerning bugs. Proc Natl Acad Sci USA. 2010;107:17065–17066. [PubMed]
13. Bulkley D, Innis CA, Blaha G, Steitz TA. Revisiting the structures of several antibiotics bound to the bacterial ribosome. Proc Natl Acad Sci USA. 2010;107:17158–17163. [PubMed]
14. McCoy LS, Xie Y, Tor Y. Antibiotics that target protein synthesis. Wiley Interdiscip Rev RNA. 2010;2:209–232. [PubMed]
15. Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide, and streptogramin antibiotics by target modification. Antimicrob Agents Chemother. 1991;35:1267–1272. [PMC free article] [PubMed]
16. Chabbert YA. Antagonisme in vitro entre l’erythromycine et la spiramycine. Ann Inst Pasteur (Paris) 1956;90:787–790. [PubMed]
17. Abraham EP, Chain E. An enzyme from bacteria able to destroy penicillin. Nature. 1940;146:837. [PubMed]
18. Long KS, Poehlsgaard J, Kehrenberg C, Schwarz S, Vester B. The Cfr rRNA methyltransferase confers resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, and streptogramin A antibiotics. Antimicrob Agents Chemother. 2006;50:2500–2505. [PMC free article] [PubMed]
19. Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol R. 2010;74:417–433. [PMC free article] [PubMed]
20. Morić I, Savić M, Ilić-Tomić T, Vojnović S, Bajkić S, Vasiljević B. rRNA Methyltransferases and their role in resistance to antibiotics. J Med Biochem. 2010;29:165–174.
21. Smith LK, Mankin AS. Transcriptional and translational control of the mlr operon, which confers resistance to seven classes of protein synthesis inhibitors. Antimicrob Agents Chemother. 2008;52:1703–1712. [PMC free article] [PubMed]
22. Schwarz S, Werckenthin C, Kehrenberg C. Identification of a plasmid-borne chloramphenicol-florfenicol resistance gene in Staphylococcus sciuri. Antimicrob Agents Chemother. 2000;44:2530–2533. [PMC free article] [PubMed]
23. Toh S-M, Xiong L, Arias CA, Villegas MV, Lolans K, Quinn J, Mankin AS. Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid. Mol Microbiol. 2007;64:1506–1514. [PMC free article] [PubMed]
24. Kehrenberg C, Schwarz S, Jacobsen L, Hansen LH, Vester B. A new mechanism for chloramphenicol, florfenicol and clindamycin resistance: Methylation of 23S ribosomal RNA at A2503. Mol Microbiol. 2005;57:1064–1073. [PubMed]
25. Al-Arif A, Sporn MB. 2′-O-Methylation of adenosine, guanosine, uridine, and cytidine in RNA of isolated rat liver nuclei. Proc Natl Acad Sci USA. 1972;69:1716–1719. [PubMed]
26. Santi DV, Hardy LW. Catalytic mechanism and inhibition of tRNA (uracil-5-) methyltransferase: Evidence for covalent catalysis. Biochemistry. 1987;26:8599–8606. [PubMed]
27. Purta E, O’Connor M, Bujnicki JM, Douthwaite S. YgdE is the 2′-O-ribose methyltransferase RlmM specific for nucleotide C2498 in bacterial 23S rRNA. Mol Microbiol. 2009;72:1147–1158. [PubMed]
28. Andersen NM, Douthwaite S. YebU is a m5C methyltransferase specific for 16S rRNA nucleotide 1407. J Mol Biol. 2006;359:777–786. [PubMed]
29. Chow CS, Lamichhane TN, Mahto SK. Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications. ACS Chem Biol. 2007;2:610–619. [PMC free article] [PubMed]
30. Toh S-M, Xiong L, Bae T, Mankin AS. The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA. RNA. 2008;14:98–106. [PubMed]
31. Yan F, Fujimori DG. RNA methylation by radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift. Proc Natl Acad Sci USA. 2011;108:3930–3934. [PubMed]
32. Yan F, LaMarre JM, Röhrich R, Wiesner J, Jomaa H, Mankin AS, Fujimori DG. RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA. J Am Chem Soc. 2010;132:3953–3964. [PMC free article] [PubMed]
33. Boal AK, Grove TL, McLaughlin MI, Yennawar NH, Booker SJ, Rosenzweig AC. Structural basis for methyl transfer by a radical SAM enzyme. Science. 2011;332:1089–1092. [PMC free article] [PubMed]
34. Grove TL, Benner JS, Radle MI, Ahlum JH, Landgraf BJ, Krebs C, Booker SJ. A radically different mechanism for S-adenosylmethionine-dependent methyltransferases. Science. 2011;332:604–607. [PubMed]
35. Grove TL, Radle MI, Krebs C, Booker SJ. Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: Methyl transfer by SN2 displacement and radical generation. J Am Chem Soc. 2011 doi: 10.1021/ja207327v. [PubMed] [Cross Ref]
36. Fontecave M. Methylations: A radical mechanism. Chem Biol. 2011;18:559–561. [PubMed]
37. Stubbe J. The two faces of SAM. Science. 2011;332:544–545. [PubMed]
38. Vazquez-Laslop N, Ramu H, Klepacki D, Kannan K, Mankin AS. The key function of a conserved and modified rRNA residue in the ribosomal response to the nascent peptide. EMBO J. 2010;29:3108–3117. [PubMed]
39. Petry S, Brodersen DE, Murphy FV, Dunham CM, Selmer M, Tarry MJ, Kelley AC, Ramakrishnan V. Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon. Cell. 2005;123:1255–1266. [PubMed]
40. Jenner L, Rees B, Yusupov M, Yusupova G. Messenger RNA conformations in the ribosomal E site revealed by X-ray crystallography. EMBO Rep. 2007;8:846–850. [PubMed]
41. Kaminska KH, Purta E, Hansen LH, Bujnicki JM, Vester B, Long KS. Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria. Nucleic Acids Res. 2010;38:1652–1663. [PMC free article] [PubMed]
42. Fontecave M, Mulliez E, Ollagnier-de-Choudens S. Adenosylmethionine as a source of 5′-deoxyadenosyl radicals. Curr Opin Chem Biol. 2001;5:506–511. [PubMed]
43. Frey PA, Magnusson OT. S-adenosylmethionine: A wolf in sheep’s clothing, or a rich man’s adenosylcobalamin? Chem Rev. 2003;103:2129–2148. [PubMed]
44. Fontecave M, Atta M, Mulliez E. S-adenosylmethionine: nothing goes to waste. Trends Biochem Sci. 2004;29:243–249. [PubMed]
45. Frey PA, Hegeman AD, Ruzicka FJ. The radical SAM superfamily. Crit Rev Biochem Mol Biol. 2008;43:63–88. [PubMed]
46. Frey M, Rothe M, Wagner AFV, Knappe J. Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen-atom—Studies of H-2 glycine substituted enzyme and peptides homologous to the glycine-734 site. J Biol Chem. 1994;269:12432–12437. [PubMed]
47. Mulliez E, Fontecave M, Gaillard J, Reichard P. An iron-sulfur center and a free-radical in the active anaerobic ribonucleotide reductase of Escherichia coli. J Biol Chem. 1993;268:2296–2299. [PubMed]
48. Sun XY, Ollagnier S, Schmidt PP, Atta M, Mulliez E, Lepape L, Eliasson R, Gräslund A, Fontecave M, Reichard P, Sjöberg BM. The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681. J Biol Chem. 1996;271:6827–6831. [PubMed]
49. Kim S, Meehan T, Schaefer HF. Hydrogen-atom abstraction from the adenine-uracil base pair. J Phys Chem A. 2007;111:6806–6812. [PubMed]
50. Zierhut M, Roth W, Fischer I. Dynamics of H-atom loss in adenine. Phys Chem Chem Phys. 2004;6:5178–5183.
51. Menon AS, Henry DJ, Bally T, Radom L. Effect of substituents on the stabilities of multiply-substituted carbon-centered radicals. Org Biomol Chem. 2011;9:3636–3657. [PubMed]
52. LaMarre JM, Howden BP, Mankin AS. Inactivation of the indigenous methyltransferase RlmN in Staphylococcus aureus increases linezolid resistance. Antimicrob Agents Chemother. 2011;55:2989–2991. [PMC free article] [PubMed]
53. LaMarre JM, Locke JB, Shaw KJ, Mankin AS. Low fitness cost of the multidrug resistance gene cfr. Antimicrob Agents Chemother. 2011;55:3714–3719. [PMC free article] [PubMed]
54. D’Costa VM, King CE, Kalan L, Morar M, Sung WWL, Schwarz C, Froese D, Zazula G, Calmels F, Debruyne R, Golding GB, Poinar HN, Wright GD. Antibiotic resistance is ancient. Nature. 2011;477:457–461. [PubMed]
55. Davidovich C, Bashan A, Yonath A. Structural basis for cross-resistance to ribosomal PTC antibiotics. Proc Natl Acad Sci USA. 2008;105:20665–20670. [PubMed]
56. Denis A, Agouridas C, Auger JM, Benedetti Y, Bonnefoy A, Bretin F, Chantot JF, Dussarat A, Fromentin C, D’Ambrières SG, Lachaud S, Laurin P, Le Martret O, Loyau V, Tessot N, Pejac JM, Perron S. Synthesis and antibacterial activity of HMR 3647 a new ketolide highly potent against erythromycin-resistant and susceptible pathogens. Bioorg Med Chem Lett. 1999;9:3075–3080. [PubMed]
57. Locke JB, Finn J, Hilgers M, Morales G, Rahawi S, Kedar GC, Jose Picazo J, Im W, Shaw KJ, Stein JL. Structure-activity relationships of diverse oxazolidinones for linezolid-resistant Staphylococcus aureus strains possessing the cfr methyltransferase gene or ribosomal mutations. Antimicrob Agents Chemother. 2010;54:5337–5343. [PMC free article] [PubMed]
58. Mankin AS. Macrolide myths. Curr Opin Microbiol. 2008;11:414–421. [PubMed]
59. Polacek N, Mankin AS. The ribosomal peptidyl transferase center: Structure, function, evolution, inhibition. Crit Rev Biochem Mol Biol. 2005;40:285–311. [PubMed]
60. Vester B, Douthwaite S. Macrolide resistance conferred by base substitutions in 23S rRNA. Antimicrob Agents Chemother. 2001;45:1–12. [PMC free article] [PubMed]
61. Schlünzen F, Zarivach R, Harms J, Bashan A, Tocilj A, Albrecht R, Yonath A, Franceschi F. Structural basis for the interaction of antibiotics with the peptidyl transferase centre in eubacteria. Nature. 2001;413:814–821. [PubMed]
62. Tu D, Blaha G, Moore PB, Steitz TA. Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell. 2005;121:257–270. [PubMed]
63. Weisblum B. Erythromycin resistance by ribosome modification. Antimicrob Agents Chemother. 1995;39:577–585. [PMC free article] [PubMed]
64. Butler MS, Cooper MA. Antibiotics in the clinical pipeline in 2011. J Antibiot. 2011;64:413–425. [PubMed]
65. Canu A, Malbruny B, Coquemont M, Davies TA, Appelbaum PC, Leclercq R. Diversity of ribosomal mutations conferring resistance to macrolides, clindamycin, streptogramin, and telithromycin in Streptococcus pneumoniae. Antimicrob Agents Chemother. 2002;46:125–131. [PMC free article] [PubMed]
66. Xiong LQ, Shah S, Mauvais P, Mankin AS. A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre. Mol Microbiol. 1999;31:633–639. [PubMed]
67. Garza-Ramos G, Xiong LQ, Zhong P, Mankin A. Binding site of macrolide antibiotics on the ribosome: New resistance mutation identifies a specific interaction of ketolides with rRNA. J Bacteriol. 2001;183:6898–6907. [PMC free article] [PubMed]
68. Xiong LQ, Korkhin Y, Mankin AS. Binding site of the bridged macrolides in the Escherichia coli ribosome. Antimicrob Agents Chemother. 2005;49:281–288. [PMC free article] [PubMed]
69. Novotny GW, Jakobsen L, Andersen NM, Poehlsgaard J, Douthwaite S. Ketolide antimicrobial activity persists after disruption of interactions with domain II of 23S rRNA. Antimicrob Agents Chemother. 2004;48:3677–3683. [PMC free article] [PubMed]
70. Or YS, Clark RF, Wang SY, Chu DTW, Nilius AM, Flamm RK, Mitten M, Ewing P, Alder J, Ma ZK. Design, synthesis, and antimicrobial activity of 6-O-substituted ketolides active against resistant respiratory tract pathogens. J Med Chem. 2000;43:1045–1049. [PubMed]
71. Ma ZK, Clark RF, Brazzale A, Wang SY, Rupp MJ, Li LP, Griesgraber G, Zhang SM, Yong H, Phan LT, Nemoto PA, Chu DTW, Plattner JJ, Zhang XL, Zhong P, Cao ZS, Nilius AM, Shortridge VD, Flamm R, Mitten M, Meulbroek J, Ewing P, Alder J, Or YS. Novel erythromycin derivatives with aryl groups tethered to the C-6 position are potent protein synthesis inhibitors and active against multidrug-resistant respiratory pathogens. J Med Chem. 2001;44:4137–4156. [PubMed]
72. Hammerschlag MR, Sharma R. Use of cethromycin, a new ketolide, for treatment of community-acquired respiratory infections. Expert Opin Invest Drugs. 2008;17:387–400. [PubMed]
73. Liang J-H, Dong L-J, Wang Y-Y, Yao G-W, An M-M, Wang R. Synthesis and antibacterial activity of 2, 3-dehydro-3-O-(3-aryl-E-prop-2-enyl)-10, 11-anhydroclarithromycin derivatives. J Antibiot. 2011;64:333–337. [PubMed]
74. Tang D, Gai Y, Polemeropoulos A, Chen Z, Wang Z, Or YS. Design, synthesis, and antibacterial activities of novel 3,6-bicyclolide oximes: Length optimization and zero carbon linker oximes. Bioorg Med Chem Lett. 2008;18:5078–5082. [PubMed]
75. Sato T, Tateda K, Kimura S, Iwata M, Ishii Y, Yamaguchi K. In vitro antibacterial activity of modithromycin, a novel 6,11-bridged bicyclolide, against respiratory pathogens, including macrolide-resistant gram-positive cocci. Antimicrob Agents Chemother. 2011;55:1588–1593. [PMC free article] [PubMed]
76. Magee TV, Ripp SL, Li B, Buzon RA, Chupak L, Dougherty TJ, Finegan SM, Girard D, Hagen AE, Falcone MJ, Farley KA, Granskog K, Hardink JR, Huband MD, Kamicker BJ, Kaneko T, Knickerbocker MJ, Liras JL, Marra A, Medina I, Nguyen T-T, Noe MC, Obach RS, O’Donnell JP, Penzien JB, Reilly UD, Schafer JR, Shen Y, Stone GG, Strelevitz TJ, Sun J, Tait-Kamradt A, Vaz ADN, Whipple DA, Widlicka DW, Wishka DG, Wolkowski JP, Flanagan ME. Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections. J Med Chem. 2009;52:7446–7457. [PubMed]
77. Hajduk PJ, Dinges J, Schkeryantz JM, Janowick D, Kaminski M, Tufano M, Augeri DJ, Petros A, Nienaber V, Zhong P, Hammond R, Coen M, Beutel B, Katz L, Fesik SW. Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis. J Med Chem. 1999;42:3852–3859. [PubMed]
78. Alvesalo JKO, Siiskonen A, Vainio MJ, Tammela PSM, Vuorela PM. Similarity based virtual screening: A tool for targeted library design. J Med Chem. 2006;49:2353–2356. [PubMed]
79. Feder M, Purta E, Koscinski L, Cubrilo S, Vlahovicek GM, Bujinicki JM. Virtual screening and experimental verification to identify potential inhibitors of the ErmC methyltransferase responsible for bacterial resistance against macrolide antibiotics. ChemMedChem. 2008;3:316–322. [PubMed]
80. Hansen LH, Lobedanz S, Douthwaite S, Arar K, Wengel J, Kirpekar F, Vester B. Minimal substrate features for Erm methyltransferases defined by using a combinatorial oligonucleotide library. ChemBioChem. 2011;12:610–614. [PubMed]
81. Novak R, Shlaes DM. The pleuromutilin antibiotics: A new class for human use. Current Opinion in Investigational Drugs. 2010;11:182–191. [PubMed]
82. Hu C, Zou Y. Mutilins derivatives: From veterinary to human-used antibiotics. Mini-Rev Med Chem. 2009;9:1397–1406. [PubMed]
83. Brickner SJ, Barbachyn MR, Hutchinson DK, Manninen PR. Linezolid (ZYVOX), the first member of a completely new class of antibacterial agents for treatment of serious gram-positive infections. J Med Chem. 2008;51:1981–1990. [PubMed]
84. Wilson DN, Schluenzen F, Harms JM, Starosta AL, Connell SR, Fucini P. The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and effect tRNA positioning. Proc Natl Acad Sci USA. 2008;105:13339–13344. [PubMed]
85. Colca JR, McDonald WG, Waldon DJ, Thomasco LM, Gadwood RC, Lund ET, Cavey GS, Mathews WR, Adams LD, Cecil ET, Pearson JD, Bock JH, Mott JE, Shinabarger DL, Xiong L, Mankin AS. Cross-linking in the living cell locates the site of action of oxazolidinone antibiotics. J Biol Chem. 2003;278:21972–21979. [PubMed]
86. Leach KL, Swaney SM, Colca JR, McDonald WG, Blinn JR, Thomasco LM, Gadwood RC, Shinabarger D, Xiong L, Mankin AS. The site of action of oxazolidinone antibiotics in living bacteria and in human mitochondria. Mol Cell. 2007;26:393–402. [PubMed]
87. Im W, Choi S, Rhee J. Structure-activity relationship of substituted pyridyl phenyl oxazolidinone derivatives, including TR-700 (DA-7157) Abstr Intersci Conf Antimicrob Agents Chemother. 2007;47:249.
88. Skripkin E, McConnell TS, DeVito J, Lawrence L, Ippolito JA, Duffy EM, Sutcliffe J, Franceschi F. Rχ-01, a new family of oxazolidinones that overcome ribosome-based linezolid resistance. Antimicrob Agents Chemother. 2008;52:3550–3557. [PMC free article] [PubMed]
89. Nichols RJ, Sen S, Choo YJ, Beltrao P, Zietek M, Chaba R, Lee S, Kazmierczak KM, Lee KJ, Wong A, Shales M, Lovett S, Winkler ME, Krogan NJ, Typas A, Gross CA. Phenotypic landscape of a bacterial cell. Cell. 2011;144:143–156. [PMC free article] [PubMed]
90. Wang HC, Vaillancourt DJP, Roemer T, Meredith TC. High-frequency transposition for determining antibacterial mode of action. Nat Chem Biol. 2011;7:720–729. [PubMed]
91. Moy TI, Ball AR, Anklesaria Z, Casadei G, Lewis K, Ausubel FM. Identification of novel antimicrobials using a live-animal infection model. Proc Natl Acad Sci USA. 2006;103:10414–10419. [PubMed]
92. Falconer SB, Czarny TL, Brown ED. Antibiotics as probes of biological complexity. Nat Chem Biol. 2011;7:416–424. [PubMed]