Tetracycline Analogs Whose Primary Target Is Not
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Apr. 1994, p. 637-640 Vol. 38, No. 4 0066-4804/94/$04.00+0 Copyright C 1994, American Society for Microbiology Tetracycline Analogs Whose Primary Target Is Not the Bacterial Ribosome IAN CHOPRAt Infectious Disease Research Section, Lederle Laboratories, Pearl River, New York 10965 INTRODUCTION TET DETERMINANTS PROVIDE POOR PROTECTION AGAINST CHELOCARDIN AND 6-THIATETRACYCLINE The tetracyclines are a group of broad-spectrum antibiotics which were discovered in 1948 following the isolation of Various early studies reported that chelocardin and 6-thia- chlortetracycline from Streptomyces aureofaciens (4). Later, tetracycline are active against tetracycline-resistant organisms other tetracyclines (Table 1) were identified; they were either (2, 3, 6, 13, 15). This has been confirmed in a comprehensive naturally occurring molecules (tetracycline, demethylchlortet- study by Oliva and Chopra (8), in which the activities of these racycline) or products of semisynthetic approaches (doxycy- tetracycline analogs were examined against Escherichia coli cline, minocycline) (4). and Staphylococcus aureus strains containing determinants for The tetracyclines listed above prevent bacterial growth by effiux [Tet(B) or Tet(K)] or ribosomal protection [Tet(M)] inhibiting protein synthesis (4, 11). They bind to a single (Table 2). E. coli strains bearing Tet(B) or Tet(M) determi- high-affinity site on the bacterial 30S ribosomal subunit and nants display at least a 256-fold increase in resistance to prevent attachment of aminoacyl-tRNA to the ribosomal ac- tetracycline. In contrast, susceptibility ratios obtained with ceptor site (4). Nevertheless, the antibiotic interaction is chelocardin and 6-thiatetracycline are always <16, and in many reversible since these agents are bacteriostatic (4, 9). cases they are 1 to 2 (Table 2). Thus, Tet(B) and Tet(M) The broad spectrum of activity and relative safety of the determinants in E. coli offer little or no protection from the tetracyclines have led to their extensive use in the therapy of analogs chelocardin and 6-thiatetracycline. In S. aureus, nei- bacterial infections in humans and animals (4). However, one ther Tet(K) nor Tet(M) confers substantial levels of resistance of the most important limitations to the continued widespread to the analogs, since susceptibility ratios of 8 or less are use of tetracyclines has been the emergence of bacterial observed, whereas susceptibility ratios of 128 or greater are resistance to these antibiotics (4). Two principal mechanisms observed for tetracycline (Table 2). The data presented in of resistance occur (4): (i) energy-dependent removal of Table 2 are based on results obtained with Tet determinants antibiotic from the cell mediated by membrane-located efflux expressed constitutively or induced by the addition of a proteins, and (ii) ribosomal protection whereby tetracyclines subinhibitory concentration of tetracycline to the growth me- are unable to prevent attachment of aminoacyl-tRNA to the dium (8). ribosomal acceptor site. The determinants encoding these resistance mechanisms EARLY HINTS THAT CHELOCARDIN AND have been grouped into classes defined by the lack of cross- 6-THIATETRACYCLINE MAY NOT ACT AS hybridization under stringent conditions (4, 6). The resistance CLASSICAL TETRACYCLINES ARE CONFIRMED BY determinants that mediate active efflux comprise Tet(A) CELL-FREE TRANSLATION AND RIBOSOMAL through Tet(F), Tet(K), and Tet(L) (4), and those that confer FOOTPRINTING STUDIES resistance by ribosomal protection are designated Tet(M) and Tet(O) (4). Many of these determinants are located on plas- It is well established that many tetracyclines (e.g., tetracy- mids or within transposable elements and, consequently, have cline, chlortetracycline, minocycline, and doxycycline) are bac- become widely distributed in pathogenic bacteria (4). teriostatic agents and that this bacteriostatic activity is associ- The identification of novel tetracycline analogs able to ated with reversible inhibition of protein synthesis (4, 11). In circumvent the efflux or ribosomal resistance mechanisms contrast, it has been known for some time that the tetracycline could considerably extend the therapeutic utility of this anti- analogs 6-thiatetracycline and chelocardin are bactericidal biotic class. Chelocardin and 6-thiatetracycline are analogs rather than bacteriostatic agents (1, 10, 12). These observa- discovered in the 1970s; they exhibit the desired profile since tions imply that chelocardin and 6-thiatetracycline might pos- they are active against strains resistant to other tetracyclines sess a mode of ribosomal binding that differs from those of (Table 2). During the course of studies designed to probe the tetracyclines with bacteriostatic properties or that the analogs molecular basis of action of these analogs (8, 9, 11), new have a separate, distinct mode of action that is unrelated to insights into the mode of action of these tetracyclines have direct inhibition of protein synthesis at the ribosome. been obtained. This article reviews these findings and con- Recent data generated by using cell-free translation systems cludes that the tetracycline group contains some analogs that from E. coli and Bacillus subtilis indicate that both analogs are do not directly inhibit protein synthesis at the level of the very poor inhibitors of protein synthesis (11). Chemical prob- bacterial ribosome. ing methods recently introduced by Stern and coworkers (14), (This minireview is based on a lecture presented at the 32nd which permit the ligand interactions involving individual bases Interscience Conference on Antimicrobial Agents and Chemo- in rRNA to be determined, are also broadly consistent with the therapy, Anaheim, Calif., 11 to 14 October 1992.) conclusion that the ribosome is not the primary target for chelocardin or 6-thiatetracycline (4, 11). By using these tech- niques, which can be applied if an antibiotic binds to rRNA by t Present address: SmithKline Beecham Pharmaceuticals, Brock- stable interaction with specific bases, the sites of drug interac- ham Park, Betchworth, Surrey RH3 7AJ, United Kingdom. tion can be identified by their diminished or enhanced reactiv- 637 638 MINIREVIEWS ANTIMIcROB. AGENTS CHEMOTHER. TABLE 1. Principal tetracyclines used for the therapy of infectious diseases Chemical name Yr Generic name Trade name discoveryof 7-Chlortetracycline Chlortetracycline Aureomycin 1948 5-Hydroxytetracycline Oxytetracycline Terramycin 1948 Tetracycline Tetracycline Achromycin 1953 6-Demethyl-7-chlortetracycline Demethylchlortetracycline Declomycin 1957 6-Methylene-5-hydroxytetracycline Methacycline Rondomycin 1965 6-Deoxy-5-hydroxytetracycline Doxycycline Vibramycin 1967 7-Dimethylamino-6-demethyl-6-deoxytetracycline Minocycline Minocin 1972 ities toward chemical probes such as dimethyl sulfate (DMS) cline, but not chelocardin, stimulated the reactivity of U-1052/ or kethoxal. C-1054 (Table 3). In a typical experiment, 70S ribosomes, either alone or complexed with antibiotics, are incubated with a chemical THE PRIMARY TARGET FOR CHELOCARDIN AND probe under conditions which give rise to only a few stops 6-THIATETRACYCLINE IS THE BACTERIAL within 300-nucleotide stretches of a given rRNA molecule. CYTOPLASMIC MEMBRANE-A MOLECULAR Control and antibiotic-complexed RNAs are extracted and EXPLANATION FOR THE ACTIVITIES OF then hybridized to a synthetic DNA oligomer complementary THESE COMPOUNDS AGAINST TETRACYCLINE to a site downstream of the target sequence. The primer is RESISTANT STRAINS extended with reverse transcriptase, and the cDNA band patterns produced by primer extension of probed control Following the ribosomal interaction studies described by RNAs and antibiotic-complexed RNAs are compared. Nucle- Rasmussen et al. (11), a series of additional experiments was otides exhibiting reduced or enhanced reactivities in the drug- conducted to explore the molecular basis by which chelocardin treated complex are identified. and 6-thiatetracycline inhibit bacterial growth and prevent Utilization of the footprinting technique has established that incorporation of precursors into macromolecules. Direct evi- tetracycline protects base A-892 from reactivity toward DMS dence that the cyloplasmic membrane is the primary target for and stimulates U-1052/C-1054. In contrast, neither chelocardin chelocardin and 6-thiatetracycline was obtained from enzyme nor 6-thiatetracycline protected A-892, although 6-thiatetracy- leakage studies and examination of inhibited cells by electron microscopy (9). Interaction of these analogs with the mem- brane is lethal, resulting in cellular lysis, possibly by stimulation TABLE 2. Levels of resistance to tetracyclines conferred by Tet(B), of murein (peptidoglycan) hydrolases (9). Tet(K), and Tet(M) determinantsa Elucidation of the modes of action of chelocardin and 6-thiatetracycline provides a rational molecular explanation for the activities of those compounds against strains expressing Relative level of resistance Tet(B), Tet(K), or Tet(M) determinants. Thus, tetracycline conferred by resistance mechanisms that protect ribosomes [Tet(M)] or that remove antibiotic from the cytoplasm [Tet(B), Tet(K)] are not expected to confer resistance to agents that attack the cyto- Compound Tet B TetM Tet K TetM plasmic membrane. (E.l boli) (Saureus) IDENTIFICATION OF ADDITIONAL ATYPICAL CH3 OH N(CH3)2 TETRACYCLINES AND DEFINITION OF STRUCTURE- ACTIVITY RELATIONSHIPS WITHIN THE CONH2 256 512 512 128 TETRACYCLINE