Structure–activity relationships of ketolides vs. macrolides

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Structure±activity relationships of ketolides vs. macrolides S. Douthwaite Department of Biochemistry and Molecular Biology, Odense University, Odense, Denmark ABSTRACT Since their discovery, the macrolide antimicrobials have proved clinically valuable for the treatment of respiratory tract infections, offering coverage against a broad spectrum of pathogens and excellent tolerability. However, the global increase in macrolide resistance among respiratory pathogens, particularly Streptococcus pneumoniae, threatens their future usefulness. The ketolides, of which telithromycin is the first to reach clinical development, represent a new generation of antimicrobials that have been developed with a view to overcoming the problem of macrolide resistance. Telithromycin is structurally derived from macrolides, and possesses several distinguishing features that are important for its improved microbiological profile. The L-cladinose at position C3 of the macrolactone ring has been replaced with a keto function. This modification enables telithromycin to bind to its target without tripping the inducible resistance to macrolide±lincosamide± streptograminB (MLSB) drugs that many groups of pathogens exhibit. The C6 position has been modified by the addition of a methoxy group. This helps prevent hemiketalization of the C6 position with the 3- and 9-keto groups, thereby conferring excellent acid stability, particularly at gastric pH values. Telithromycin is differentiated from other ketolide compounds by the addition of a large aromatic N-substituted carbamate extension from positions C11/C12. This carbamate extension improves binding of the drug to its target, the 50S ribosomal subunit, as demonstrated in in vitro experiments. Telithromycin binds to wild-type ribosomes with 10-fold greater affinity than erythromycin A and 6-fold greater affinity than clarithromycin; its affinity for MLSB-resistant ribosomes is 4 20 times that of both macrolides. The increased ribosomal affinity of telithromycin correlates with its superior potency against Gram-positive cocci both in vitro and in vivo, and is one of the factors determining the drug's activity against MLSB-resistant respiratory pathogens. Clin Microbiol Infect 2001: 7 (Supplement 3): 11±17

INTRODUCTION The macrolides are a major family of oral antimicrobials with established efficacy and a good tolerability profile, and are thus frequently used for the treatment of community-acquired respiratory tract infections (RTIs). The prototype macrolide, erythromycin, was discovered in the early 1950s. This drug soon proved to be a valuable new addition to the field of antiinfectives as it offered activity against emerging penicillinresistant pathogens and could also be used in those patients who showed b-lactam intolerance. However, resistance to erythromycin appeared quite rapidly in staphylococci, streptococci, pneumococci and enterococci [1]. Corresponding author and reprint requests: S. Douthwaite, Department of Biochemistry and Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark Tel.: +45 65 50 23 95 Fax: +45 65 93 27 81 E-mail: [email protected]

Ahed Bhed Ched Dhed Ref marker Fig marker Table marker Ref end Ref start

A number of semisynthetic derivatives of erythromycin, such as clarithromycin and azithromycin, have since emerged. These `second-generation' macrolides have a more favorable pharmacokinetic profile than erythromycin, induce fewer gastrointestinal side-effects and offer improved activity against both Haemophilus influenzae and atypical pathogens [2±4]. However, increasing macrolide resistance among respiratory tract pathogens, particularly Streptococcus pneumoniae, has led to a search for new agents that are more effective against macrolide±lincosamide±streptogramin group B (MLSB)-resistant strains, and have low potential to select for or induce resistance and cross-resistance. The ketolides, of which telithromycin (HMR 3647) is the first to undergo clinical development, represent a new family of antimicrobials that are derived chemically from the macrolides and have been developed for use against respiratory pathogens. This article describes how the novel alterations in the structure of this ketolide are related to its improved antimicrobial properties.

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Paper 49 Disc 12 Clinical Microbiology and Infection, Volume 7, Supplement 3, 2001

KETOLIDE AND MACROLIDE STRUCTURE The structures of all macrolides, and their ketolide-derivatives, are based on a macrolactone ring, with the therapeutically most relevant macrolides comprising a 14-, 15- or 16membered ring (Figure 1). Erythromycin A, a natural antibiotic isolated from Saccharopolyspora erythrea (formerly Streptomyces erythreus), consists of a 14-membered lactone ring with two attached sugar groups: L-cladinose at C3 and desosamine at C5. Erythromycin has been derivatized in numerous ways to improve its antimicrobial properties [5]. Some of the more successful derivatives include those synthesized via an oxime intermediate at the C9 position, in some cases retaining the 14-membered ring structure and producing, for example, roxithromycin. In another derivatization pathway, the ring structure has been altered via a Beckmann rearrangement of a C9 oxime intermediate, giving rise to the 15-membered azalide, azithromycin. Another of the semisynthetic macrolides, clarithomycin, is produced by addition of a methoxy

Macrolides 12-membered ring (methymycin)

16-membered ring (spiramycin)

14-membered ring

15-membered ring (azithromycin)

Semi-synthetic derivatives: clarithromycin dirithromycin flurithromycin roxithromycin

Erythromycin (A, B, C, etc.)

Ketolides

Fig 1 Ketolide and macrolide relationship.

N

group at position C6 of erythromycin A. All of these ring modifications increase the drug stability in gastric acid, thus improving absorption by the oral route [3], and prolonging serum half-lives relative to erythromycin A. Erythromycin A is rapidly inactivated by gastric acid and is inconsistently absorbed after oral administration [6], as well as possessing a short serum half-life (*1.4 h), which necessitates the inconvenience of frequent dose administration (up to six times daily) and results in poor compliance [7]. Telithromycin is differentiated structurally from the macrolides in three ways (Figure 2), each of which is associated with specific improvement in antimicrobial properties: 1 The feature that distinguishes the ketolides from the macrolides is the presence of a 3-keto function in place of the L-cladinose moiety. This sugar residue had previously been thought to be essential for the antimicrobial activity of erythromycin. While this is in principle true, the loss of the cladinose sugar can be more than adequately compensated for by derivatizing other positions of the lactone ring (in particular at C11/C12, see below). In addition, replacement of the cladinose with a keto group improves activity against certain erythromycin-resistant strains due to the ketolides' ability to avoid induction of MLSB resistance [8]. 2 The C6-hydroxyl of erythromycin A has been blocked with a methoxy group, as in clarithromycin. In conjunction with the 3-keto group, this prevents internal hemiketalization and gives the ketolide molecule excellent acid stability (and hence gastrointestinal stability) compared with the macrolides. Both clarithromycin and azithromycin are completely inactivated after 1 h at pH 1 at 37 8C in vitro and erythromycin A is inactivated even faster under these conditions. However, telithromycin maintains full activity even after 4 h incubation under the same conditions [9].

N

N O

3-keto function Avoids MLSB resistance induction

• O

12

OCH3

11 N

Methoxy group at C6

• Improves acid stability

6

O O O

3 O

O

C11,12 carbamate side chain Increases affinity for the ribosomes

•

O

N

OH

Fig 2 Structure of the ketolide telithromycin.

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Paper 49 Disc Douthwaite Ketolides, macrolides: structure±activity relationships

3 The addition of a large aromatic N-substituted carbamate extension at C11/C12 is responsible for the improved ribosome-binding affinity of telithromycin [10], and contributes to telithromycin's improved interaction with MLSB-resistant ribosomes, compared with erythromycin A and newer macrolides [11].

(a)

l

A AA

A G T

752 A l

l

l

l

l

G mG

l

2059 A G l A 2058

C C C A C

l

G GA D A

A C A G GCΨ

A

A G C A G ACG G l

l

l

l

l

l

l

l

l

CGG A Cm C U C G mA

l l

C G U C U CC AU C U G G C Ψ l AG Ul l G A A C UU A l l A U GG C G l G A G l U Ψ G C C U G G

l

There are several known mechanisms of macrolide resistance [17±19], which can be broadly classified under the headings of efflux mechanisms, enzymatic inactivation of the drug, and target site modifications. Most relevant to this review are the target site modifications, and these generally involve changes in the structure of nucleotide A2058, thereby conferring

l

l

Resistance to macrolides and other MLSB drugs

l

l

Bacterial ribosomes consist of two subunits, 30S and 50S, each of which is composed of ribosomal RNA (rRNA) and proteins. The 50S subunit contains the peptidyl transferase center, which catalyzes the formation of peptide bonds, linking amino acids to the growing polypeptide chain during the synthesis of new proteins. Macrolides, ketolides and all other members of the MLSB class of antibiotics (which includes the lincosamides and Group B streptogramins) bind to the 50S subunit at or close to the peptidyl transferase center, and exert their antimicrobial effects by blocking protein synthesis. The structure of the 50S subunit has recently been solved at 2.4 AÊ by X-ray crystallography, enabling the molecular details to be viewed at atomic resolution [12]. The peptidyl transferase center appears to be constructed entirely from elements of 23S rRNA. This indicates that peptide bond formation is catalyzed by rRNA and not by the ribosomal proteins and shows that the catalytic center is formed mainly from structures in domain V of the 23S rRNA [13]. The complex folding of 23S rRNA brings elements of domains II and V into close proximity at the neck of the peptide exit channel, immediately adjacent to the peptidyl transferase center [12,13]. It is here that macrolides, ketolides and the structurally unrelated lincosamides and Group B streptogramins interact [14]. Some MLSB drugs directly block peptidyl transferase, whereas others, such as erythromycin and its derivatives, block the route of the newly formed peptide chain down the exit channel [15]. Erythromycin and its ketolide derivatives bind here by interacting with several positions within the 23S rRNA: the strongest contacts are at nucleotides A2058 and A2059 within domain V, and there are other less pronounced interactions including A752 within domain II [10,14,16] (Figure 3).

l

U A A UC GCC A A A

mG

MECHANISMS OF MACROLIDE RESISTANCE Target for MLSB drugs on the ribosome

U

AUU A G CGG l

Ψ

13

Ψ CU G l G 2505 G C

(b)

Fig 3 (a) The drug binding site depicted on a portion of the 23S rRNA secondary structure model (Escherichia coli sequence). Nucleotides with which macrolide and ketolide drugs interact in domain II (upper) and domain V (lower) of the rRNA are circled. (b) The threedimensional configuration of the drug binding site projected from the crystallographic coordinates of the 50S subunit (12). The encircled nucleotides in (a) are high-lighted in the portions of domain II (upper) and domain V (lower) shown here. The relative sizes of erythromycin (ery) and telithromycin (tel) illustrate how telithromycin can simultaneously interact with A752 in domain II and A2058 in domain V.

resistance to all MLSB antimicrobials. The MLSB phenotype is one of the most prevalent forms of resistance observed in S. pneumoniae, and is mediated by an erm gene, which encodes a methyltransferase that methylates the N6 position of A2058

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Paper 49 Disc 14 Clinical Microbiology and Infection, Volume 7, Supplement 3, 2001

[20]. Numerous erm determinants of the MLSB resistance phenotype have been classified [20,21]. These are generally carried on plasmids or transposons, and are exhibited in a wide range of bacteria including streptococci (including S. pneumoniae), Staphylococcus aureus, Enterococcus spp., Mycoplasma pneumoniae and Legionella spp. Expression of the erm gene can be constitutive or inducible by subinhibitory macrolide concentrations, the latter being the primary mechanism that occurs in Gram-positive cocci [20]. In other organisms, mutation of A2058 or adjacent nucleotides also alters the structure of the drug target, with A2058 substitutions conferring MLSB resistance [22]. RIBOSOMAL INTERACTION OF TELITHROMYCIN Macrolides and ketolides bind at the same target site close to the peptidyl transferase center of the 50S subunit, and presumably interrupt protein synthesis in the same manner. Despite the many similarities, there are subtle differences in their mode of interaction with the 23S rRNA, and these differences are reflected by how avidly the drugs interact with their target on the ribosome.

carbamate extension at C11/C12 in common with the ketolides telithromycin and HMR 3004 (Table 1). After incubation with saturating concentrations of ketolide and macrolide antibiotics, ribosomes were probed with the chemical reagents dimethyl sulfate and kethoxal. Chemical footprinting patterns within the 23S rRNA were analyzed by reverse transcriptase primer extension to reveal bases protected by the various antibiotics within domains II and V of wildtype 23S rRNA. The results are summarized in Table 2. All the ketolides and macrolides, irrespective of whether they had a C11/C12-carbamate side chain, interacted similarly at the same positions in domain V (A2058, A2059, A2062 and G2505). However, only those compounds with the C11/C12-carbamate extension protected the A752 base in domain II against modification: this included the two ketolides (telithromycin and HMR 3004) and the macrolide RU 66252. The ketolide lacking the C11/C12-carbamate extension (RU 56006) did not interact with A752, indicating that the C11/C12-carbamate side chain mediates binding to domain II of 23S rRNA. In footprinting experiments with erythromyTable 1 Modifications in the lactone ring structure of ketolides and macrolides

Interaction of telithromycin with wild-type ribosomes Chemical footprinting experiments were conducted in vitro on Escherichia coli ribosomes with a range of ketolide and macrolide antibiotics to determine the sites and strength of drug±ribosome interactions [10]. The antibiotics studied included erythromycin A as the reference macrolide, several ketolides (telithromycin, RU 56006 and HMR 3004) and RU 66252. The latter is classified as a macrolide as it retains the L-cladinose group at the C3-position, but has an alkyl±aryl

Antibiotic

Class

Lactone ring position ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ C3 C6 C11,12

Erythromycin A Clarithromycin RU 66252 RU 56006 Telithromycin HMR 3004

Macrolide Macrolide Macrolide Ketolide Ketolide Ketolide

Cladinose Cladinose Cladinose Keto Keto Keto

±OH ±OCH3 ±OCH3 ±OCH3 ±OCH3 ±OCH3

±OH ±OH Carbamate ±OH Carbamate Carbamate

Table 2 Accessibility of key nucleotides in 23S rRNA affected by drug bindinga

Position

Accessibility (mean + SD)b ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ No drug Erythromycin Telithromycin RU 56006 RU 66252 HMR 3004

Domain II A752

1.00

1.57 + 0.24

0.36 + 0.15

0.98 + 0.07

0.35 + 0.11

0.18 + 0.10

Domain V A2058 A2059 A2062 G2505

1.00 1.00 1.00 1.00

0.08 + 0.03 0.16 + 0.04 1.21 + 0.09 0.28 + 0.12

0.08 + 0.03 0.15 + 0.02 1.55 + 0.16 0.31 + 0.18

0.09 + 0.02 0.19 + 0.05 1.30 + 0.12 ND

0.07 + 0.02 0.18 + 0.04 1.80 + 0.25 0.21 + 0.12

0.08 + 0.02 0.18 + 0.05 1.63 + 0.18 0.34 + 0.14

a

Drugs at saturating concentrations (0.1±1 mM) were bound to wild-type E. coli ribosomes (0.1 mM), probed with dimethyl sulfate or kethoxal to modify unprotected bases, and site modification was analyzed by reverse transcriptase primer extension. Adapted from [10]. b Values 5 1.00 indicate protection from chemical modification and values 41.00 (highlighted in bold) indicate enhancement of chemical modification. The degree of protection is taken to correspond to the proportion of ribosomes binding the drug. ND, not determined.

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Paper 49 Disc Douthwaite Ketolides, macrolides: structure±activity relationships

cin, the accessibility of A752 was enhanced, suggesting that the cladinose mediates a different type of interaction to domain II of the rRNA [10,16]. Interaction of telithromycin with A2058G mutant (MLSB-resistant) ribosomes These in vitro binding studies have been extended to study both wild-type 23S rRNA and its A2058G mutant, which confers a strong, constitutive MLSB-resistant phenotype [11]. The binding curves and dissociation constants of various ketolides and macrolides over a concentration range from 1 nM to 1 mM were determined for wild-type and A2058G mutant ribosomes. Chemical footprinting patterns were determined as outlined above, and the results of four independent experiments are summarized in Figure 4 with the dissociation constants derived from these binding curves shown in Table 3. The two ketolides, telithromycin and HMR 3004, as well as the macrolide with the C11/C12-carbamate extension, showed 6-fold higher binding affinity for wild-type ribosomes than clarithromycin and approximately 10-fold higher affinity than erythromycin A. The ketolide RU 56006 (descladinosyl clarithromycin derivative), which lacks the C11/C12carbamate extension and does not interact at the A752 position in domain II, exhibited a 100-fold reduction in ribosomal binding affinity compared with its parent compound, clarithromycin. Mutation of A2058 to a G disrupts the main site of interaction for the drugs in domain V, and results in an enormous drop in the drug binding affinities to the ribosome (Table 3; Figure 4). However, the binding is reduced to different extents for the different drugs. Binding by the macrolides erythromycin A and clarithromycin is reduced by about four orders of magnitude, while binding of the ketolides and macrolides with the C11/C12-carbamate side chain is also Table 3 Dissociation constants (Kdiss) of ketolide and macrolide antibiotics for wild-type and A2058G mutant (MLSB-resistant) ribosomes

Antibiotic

Mean (+ SD) Kdiss (M)a ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ Wild-type Mutant A2058G

Erythromycin Clarithromycin HMR 3004 Telithromycin RU 66252 RU 56006

1.4 + 0.261078 9.5 + 1.461079 1.6 + 0.361079 1.3 + 0.261079 2.9 + 0.261079 9.8 + 1.161077

1.9 + 0.361074 1.7 + 0.461074 6.9 + 1.461076 7.9 + 1.961076 2.7 + 1.261076 42.561072

Kdiss ratio (mutant/ wild-type) 14 000 18 000 4300 6100 9300 425 000

a Kdiss values are derived from the drug binding curves shown in Figure 4. Lower values indicate stronger binding (higher binding affinity).

15

reduced, but to a lesser degree (Table 3). The ketolides telithromycin and HMR 3004 bound to MLSB-resistant ribosomes with an affinity that remained at least 20 times higher than for erythromycin A or clarithromycin. Notably, the ketolide RU 56006 which lacks the C11/C12-carbamate side chain exhibited the most drastic reduction in binding to mutant ribosomes, emphasizing the importance of a domain II interaction with the rRNA. The ability of telithromycin to maintain some degree of binding to MLSB-resistant ribosomes is linked with its improved interaction with the A752 residue in domain II, which is mediated by the C11/C12-carbamate side chain (Figure 3). LACK OF MLSB RESISTANCE INDUCTION BY KETOLIDES VS. MACROLIDES In vitro studies of S. pneumoniae have shown that ketolides do not induce MLSB resistance [8]. The specific potential of telithromycin to induce MLSB resistance in Gram-positive cocci has also been studied recently in vitro [23]. Three antimicrobials were included in the latter study: erythromycin A, telithromycin and RU 69874, the 3-cladinose equivalent of telithromycin. Induction of MLSB resistance was evaluated using subinhibitory concentrations of the different antimicrobials according to the method of Hyder and Streitfeld [24]. Briefly, four bacterial strains with inducible MLSB-resistance phenotype ± S. aureus, Streptococcus pyogenes and two S. pneumoniae strains ± were grown with subinhibitory concentrations of telithromycin, RU 69874, erythromycin A (as a positive control) or no drug (negative control). Each of the cultures was then challenged with an inhibitory concentration of erythromycin A. All cultures that had been incubated with subinhibitory concentrations of the macrolides (erythromycin A or RU 69874) exhibited a significantly higher growth rate than unpretreated control following challenge with erythromycin A. Thus pretreatment of these strains with erythromycin A or RU 69874 resulted in induction of resistance, indicating that both of these macrolides are strong inducers of MLSB resistance. In contrast, S. pneumoniae cultures that had been incubated with subinhibitory concentrations of telithromycin showed similar growth to the uninduced control, indicating that the ketolide had not induced MLSB resistance. These results strongly suggest that the replacement of the L-cladinose group of the macrolides with a keto group is linked to telithromycin's ability to avoid induction of MLSB resistance. CONCLUSION Telithromycin possesses several structural features that differentiate it from erythromycin. Both drugs interact with two

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Paper 49 Disc 16 Clinical Microbiology and Infection, Volume 7, Supplement 3, 2001

Wild-type ribosomes A2058G ribosomes Erythromycin A

RU 66252 100

80

80

% base protection

% base protection

Telithromycin 100

60 40 20

60 40 20

0

0 1 nM

10 nM

100 nM

1 µM

10 µM

100 µM

100 mM

1 nM

10 nM

1 µM

10 µM

100 µM

100 mM

100 µM

100 mM

Drug concentration

Drug concentration

Clarithromycin

RU 56006

100

100

80

80

% base protection

% base protection

100 nM

60 40 20

60 40 20

0

0 1 nM

10 nM

100 nM

1 µM

10 µM

100 µM

100 mM

Drug concentration

1 nM

10 nM

100 nM

1 µM

10 µM

Drug concentration

Fig 4 Binding curves for ketolides and macrolides to wild-type and A2058G mutant (MLSB-resistant) ribosomes. Mean (+ SD)% base protection measured at A2058 and A2059 for wild-type ribosomes and at A2059 for A2058G mutant ribosomes. The degree of protection is taken to correspond to the proportion of ribosomes binding the drug, i.e. 0% = no binding; 100% = complete binding.

regions of the 23S rRNA within the 50S subunit of the bacterial ribosome. However, telithromycin shows a different and stronger mode of interaction with position A752 of domain II, which is mediated by its C11/C12-carbamate extension. Telithromycin binds 10-times more tightly to wildtype ribosomes than erythromycin A and 6-fold more tightly than clarithromycin. This may be one of the factors determining the greater potency of telithromycin against Gram-positive cocci both in vitro and in vivo [25]. The interaction of telithromycin with A752 in domain II of 23S rRNA enhances the binding of the drug to wild-type

ribosomes. In addition, telithromycin's A752 interaction partially ameliorates the reduction in binding caused by a mutation at A2058 in domain V that confers constitutive MLSB resistance. Telithromycin binds to MLSB-resistant ribosomes with more than 20 times higher affinity than either erythromycin A or clarithromycin. The keto substitution in the C3 position of the lactone ring, which distinguishes the ketolides from the macrolides that possess an L-cladinose moiety at this position, enables telithromycin to avoid induction of MLSB resistance in inducibly resistant Gram-positive cocci, such as S. pneumoniae.

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Paper 49 Disc Douthwaite Ketolides, macrolides: structure±activity relationships

With the growing spread of MLSB resistance in respiratory tract pathogens, and in particular its emergence in S. pneumoniae, ketolide antibiotics such as telithromycin are likely to offer a distinct advantage over the clinically available macrolides for empirical therapy of RTIs.

12.

13. 14.

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