Ribosomes, macrolide antibiotics binding

Pharmacology Macrolide antibiotics reversibly bind to the P site of the SOS ribosomal subunit of susceptible organisms and inhibit RNA-dependent protein synthesis. They may be bacteriostatic or bactericidal, depending on such factors as drug concentration. 

Currently, one structure of a Irncosamide antibiotic bound to the ribosome is available for analysis [4]. like the macrolide antibiotics, drndamycin binds near the hydrophobic crevice at the entrance to the peptide exit turmel. As with the macrolide carbomycin A, dindamycin interacts not only with the hydrophobic crevice at the entrance to the peptide exit turmd, but also with the active site hydrophobic crevice. The nudeotides that surroimd the clindamydn binding site were previously implicated in binding of lincosamides based on nucleotide protection studies and on the analysis of mutations conferred by resistance (Fig. 4.4). 

Newer and more generally usefnl macrolide antibiotics include azithromycin (Zithromax) and clarithromycin (Biaxin). These too are wide-spectrum antibiotics and both are semisynthetic derivatives of erythromycin. Like the tetracyclines, the macrolide antibiotics act as protein synthesis inhibitors and also do so by binding specifically to the bacterial ribosome, thongh at a site distinct from that of the tetracyclines. 

The glycoside/aminoglycoside antibiotics, like the macrolides, exert a bacteriostatic effect due to selective inhibition of bacterial protein synthesis, with the exception of novobiocin (26). The compounds neomycin (27), spectinomycin (28) and streptomycin (29) bind selectively to the smaller bacterial 30S ribosomal subunit, whilst lincomycin (30) binds to the larger 50S ribosomal subunit (cf. macrolides). Apramycin (31) has ribosomal binding properties, but the exact site is uncertain (B-81MI10802). Novobiocin (26) can inhibit nucleic acid synthesis, and also complexes magnesium ion, which is essential for cell wall stability. 

Cethromycin (ABT-773) 39 (Advanced Life Sciences) had an NDA filed in October 2008 for the treatment of CAP.67 Advanced Life Sciences is also evaluating cethromycin 39 against other respiratory tract infections and in pre-clinical studies as a prophylactic treatment of anthrax post-exposure. Cethromycin 3968 70 is a semi-synthetic ketolide derivative of erythromycin 4071 originally synthesised by Abbott Laboratories,72 which like erythromycin 40, inhibits bacterial protein synthesis through binding to the peptidyl-transferase site of the bacterial 50S ribosomal subunit. Important macrolide antibiotics in clinical use today include erythromycin 40 itself, clarithromycin, azithromycin and, most recently, telithromycin (launched in 2001). 

Like the aminoglycosides, the binding site of the macrolide antibiotics with the large ribosomal subunit has also been determined to atomic resolution by X-ray crystallography (29, 38, 39). Key interactions between the antibiotic and the 23 S rRNA occur and are mediated through the essential desosamine sugar ... 

Figure 12 Macrolide modifying enzymes. Macrolide antibiotics such as erythromycin (shown) bind to the large ribosomal subunit through interactions with the 23 S rRNA (A). Chemical modification of the essential desosamine sugar blocks ribosome binding (B).

More than 500 different representatives of the macrolide antibiotics are known, most of which are biologically active against Gram-positive bacteria, displaying a relatively low toxicity. Clinically used are erythromycin, oleandomycin, carbomycin and leucomycin (O Fig. 5). They act as inhibitors of the bacterial protein biosynthesis by binding to the 50S-ribosomal subunit. The synthesis of the two clinically important 16-membered ring macrolide antibiotics leucomycin A3 and carbomycin B could be started from D-glucose, which was chosen because it contained three of the required stereocenters [40]. 

The mode of action of macrolide antibiotics involves the inhibition of protein synthesis of specific binding to the 50S ribosomal subunit but without a specific target at the 23S ribosomal subunit and various proteins [66]. Nevertheless, the exact interaction of the macrolide and the ribosome unit is still not fully understood. In principle, the macrolide antibiotic should inhibit also mammalian mitochondrial protein syuithesis but they are unable to penetrate the mitochondrial membrane. 

The aminoglycosides are bacteriocidal. Other antibiotics whose mechanism of action involves inhibition of protein synthesis (tetracycline, the macrolides, lincomycin, etc.) are invariably bacteriostatic. The reason for this difference is not known. In fact, the reason that protein inhibition by aminoglycosides should be a cell-killing process has not been satisfactorily addressed. The accumulation of nonsense proteins due to misreading of mRNA has been shown not to be the reason. If ribosomal binding were an irreversible process, lethality might be comprehensible SM does not bind irreversibly. 

It is widely accepted that MLS antibiotics inhibit protein synthesis by binding to closely related sites on the 508 subunit of the 70S ribosome of bacteria [4], despite being structurally different from each other (see Figs. 1 and 2 in a later section). That is the reason why, when inducible resistant Staphylococcus aureus cells are exposed to a low concentration of the drug (0.05 tg erythromycin/ml - 6.8 x 10 M), they show resistance against not only erythromycin but also other macrolide antibiotics as well as lincosamide and type B streptogramin antibiotics. Erythromycin has been widely used and has been the object of extensive molecular and biological studies. 

In this chapter, the molecular-biological mode of action of macrolide antibiotics and the biochemical and genetic mechanisms of resistance to MLS antibiotics are reviewed. Based on a recent X-ray crystallographic study on a 50S ribosomal subunit from Haloarcula marismortui and the finding of intracellular macrolide accumulation, the mode of action from the viewpoint of a new hypothetical concept, deposition binding, and mechanisms of drug resistance in clinically isolated bacteria are discussed. In addition, recent major developments in macrolide antibiotics are briefly described. 

Despite having functional differences, all macrolide antibiotics appear to compete mutually for binding to ribosome, suggesting the presence of almost the... 

It is well known that nonprotonated molecules of a macrolide antibiotic are the active species of an inhibitor of protein synthesis [39], In our experimental condition (pH 7.6), nonprotonated molecules of rokitamycin can be calculated to be about 10 times as numerous as those of erythromycin from values of 7.6 and 8.7, respectively (Table I) [34]. Despite having the advantage in the number of nonprotonated molecules, the ratio of rokitamycin bound per ribosome (41%) is only about half as much as that of bound erythromycin (76%), when the molecules of the antibiotics each are incubated with the same amount of ribosome molecules. This suggests that rokitamycin s mode of binding to ribosomes may be noticeably different from that of erythromycin. 

This suggests that one macrolide molecule is able to arrive at a binding site of ribosome and that, after the drug is bound to the site of the ribosome, we can determine, in terms of the water solubility of macrolide antibiotics, whether reversible or cohesive (probably irreversible) binding occurs. In other words, the solubility of an antibiotic depends primarily on the difference between intracellular pH and values and could determine the extent of the antibiotic s ability to deposit at a 508 ribosomal binding site. 

In contrast, in S. aureus carrying the erm gene responsible for ML8 resistance—the so-called A ,/V -dimethylation of a specific adenine residue in 23rRNA, drag accumulation is easily decreased to about one-tenth of that in susceptible cells when they are washed five times with chilled salt solution. That is why a macrolide antibiotic present in their cells can hardly bind to their 508 ribosomes even at the highest permissible concentration of the drag [6, 54]. 

Macrolide antibiotics neither restrain the binding of aminoacyl-tRNA to ribosomes, nor do they inhibit ribosome-dependent hydrolysis of GTP in the presence... 

Menninger and Otto [101] proposed a major inhibitory mechanism common to probably all macrolide antibiotics. In E. coli mutants with temperature-sensitive peptidyl-tRNA hydrolase (aminoacyl-tRNA hydrolase EC 3.1.1.29), they observed that peptidyl-tRNA accumulates at a nonpermissive temperature (40°C) and that the cells die. The accumulation at a high temperature was enhanced when the cells were pretreated with erythromycin, carbomycin, or spiramycin at doses sufficient to inhibit protein synthesis in wild-type cells but not sufficient to kill either mutant or wild-type cells at the permissive temperature (30°C). Based on their observations, they suggested that stimulated dissociation of peptidyl-tRNA from ribosomes is the major mechanism of action of macrolide antibiotics. Their observations agree with recent results showing that a macrolide antibiotic binds to peptidyltransferase in ribosome. 

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