Tetracycline Analogs Whose Primary Target Is Not

Home , Chlortetracycline

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 membrane 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 SERIES OH 0OH 0 In addition to chelocardin and 6-thiatetracycline, several Tetracycline other tetracycline analogs that appear to be atypical molecules, i.e., membrane active, bactericidal agents, have been discov- CH, NH2 ered (8, 9, 11) (Table 3). This information, together with the results from 16S rRNA footprinting studies, permits conclu- CH,C 1111 II ~~~8-168-16 1 1 2 sions on structure-activity relationships for the tetracycline OH OH 0O 0 series as a whole to be made (Table 3). Exposure of ribosomes to tetracyclines that inhibit protein Chelocardin synthesis produced the following two 16S rRNA footprinting patterns: (i) protection of A-892 and stimulation of U-1052/C- N(CH,)2 1054, and (ii) failure to protect A-892, but stimulation of U-1052/C-1054. Although and (NiIH1lIIi4II2i~ONH 4 2-16 8 8 chlortetracycline tetracycline protect A-892, the ribosomal interaction leading to this re- OH 0tOHOHO sponse is not directly correlated to inhibition of protein synthesis, since minocycline and doxycycline, which are effec- 6-Thiatetracycline tive ribosomal inhibitors, do not protect A-892 (Table 3). Examination of molecular structures (Table 3) indicates that a Levels of resistance are based on the data of Oliva and Chopra (8). the ability to protect A-892 may relate more to possession of a VOL. 38, 1994 MINIREVIEWS 639

TABLE 3. Structure-activity relationships within the tetracycline series Effect on reactivity of bases in Type of inhibition 16S rRNA to DMS MIC(ilg/ml) Ribosome as Protection of Stimulation of Analog Structure for E.colia Bacteristatic Bactericidal primary target A892 U1052C1054 Tetracycline CH2 OH 0 (CH rrrmr ~~~~~~~0.5+ + + +

OHN0OH 0

Chlortetracycline Ci OH3 OH N(CH3)2 OH 0.25 + + + +

Minocycline N(CH3)2 NH3)2 I|hIXhII3II1IhIXIGONH . 0.25 + | + +

OH0 OH0

Doxycycline CH3 OH N(025H O~H Anhydrote[racyclinehIi..,JCONH2H | 0.25 + + | + OH0 OH0

Anhydrochbor- Cl CH3 N(CH312 + + tetracycline O 4 0

OH OH 0 0

J C3; g-~~~~CONH2 CT OH 0O 0 CH3-)2T I + I : T 1 6-Thiatetracycline 0.0 s~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~OH3CH32+

Chelocardin CO H 0.5

OH OH °O O

Cl CH3 N(CHS)2 4-Epi-anhydrochlortetracycline 80

CX OQ NX HO H 2

OH OH ° O I a E. coli K-12 MC 4100 grown in M63 minimal medium.

pseudoaxial hydroxy group at carbon 6 rather than to direct synthesis at the ribosome. However, it is obvious that several activity as an inhibitor of protein synthesis. Therefore, the atypical tetracyclines that do not cause primary inhibition of enhanced reactivity of U-1052/C-1054 may be more closely protein synthesis nevertheless enhance the reactivity of correlated with the ability of tetracyclines to inhibit protein U-1052/C-1054 to DMS (Table 3). A complete explanation of 640 MINIREVIEWS ANTIMICROB. AGENTS CHEMOTHER. this phenomenon is lacking, but the affinities of these tetracy- resistant to typical tetracyclines. However, the atypical tetra- clines for the ribosome may be adequate to alter the chemical cyclines cannot be considered therapeutic candidates because reactivities of bases in 16S rRNA to DMS, yet may be of their potential for causing adverse side effects. It remains to insufficient to prevent protein synthesis. Moreover, in terms of be seen whether tetracycline analogs which retain the ability to interaction with the whole cell, it is likely that bacterial death inhibit bacterial protein synthesis at the ribosome, while truly occurs by membrane disruption before any potential binding of circumventing effilux-based or ribosomal resistance mecha- these analogs to ribosomes within the cell can take place. nisms, can be generated. Finally, there is a set of tetracyclines (chelocardin and 4-epi- anhydrochlortetracycline) that are not ribosomal inhibitors ADDENDUM IN PROOF and that do not alter the reactivity of A-892, U-1052, or C-1054 to DMS (Table 3). These features are likely related to substi- Novel tetracycline analogs that inhibit bacterial protein tution at C-4 with moieties in the epi (i) configuration (Table synthesis and circumvent effilux-based and ribosomal resistance 3). mechanisms have recently been described (R. T. Testa, P. J. Which structural features of tetracycline analogs render Petersen, N. V. Jacobus, P.-E. Sum, Ving J. Lee, and F. P. them effective membrane perturbants? At physiological pH, Tally, Antimicrob. Agents Chemother. 37:2270-2277, 1993). many tetracyclines exist as an equilibrium mixture of two free base forms: a low-energy, lipophilic nonionized species and a REFERENCES high-energy, hydrophilic zwitterionic structure (4, 5, 11, 12). 1. Bakhtiar, M., and S. Selwyn. 1983. Antibacterial activity of a new The equilibrium between these two forms is influenced by the thiatetracycline. J. Antimicrob. Chemother. 11:291. 2. Burdett, V., J. Inamine, and S. Rajagopalon. 1983. Heterogeneity polarity of the environment, the lipophilic form allowing of tetracycline resistance determinants in Streptococcus. J. Bacte- passage through the cytoplasmic membrane, whereas adoption riol. 149:995-1004. of the hydrophilic, zwitterionic structure is probably important 3. Chabbert, Y. A., and M. R. Scavizzi. 1976. 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