JAC Advance Access published online on February 25, 2004
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkh125
© 2004 by The British Society for Antimicrobial Chemotherapy
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Original articles
1 Institut für
Mikrobiologie, Biochemie und Genetik, Universität Erlangen,
Erlangen, Germany;
* Corresponding author. E-mail: whillen{at}biologie.uni-erlangen.de.
Received 10 December 2003
; revised 19 December 2003
; accepted 21 December 2003
Objectives: The new antibiotic tigecycline
(9-t-butylglycylamido-minocycline; GAR-936) overcomes
most of the known tetracycline resistance mechanisms. Here we analyse
its mode of antibiotic action by probing 70S ribosomes of Escherichia
coli with dimethylsulphate (DMS) and Fe2+-mediated
cleavage to identify binding sites of tetracycline and tigecycline. Methods: Fe2+-mediated cleavage
makes use of the ability of Fe2+ to replace
the Mg2+ ion complexed with tetracyclines. After
addition of H2O2, Fe2+ generates
short-lived, highly reactive hydroxyl radicals that can cleave RNA
close to the tetracycline binding sites. Results: We identified three prominent Fe2+-mediated
cleavage sites in helices 29 and 34, and in the internal loop of
helix 31 of 16S rRNA in the presence of tetracycline or tigecycline.
Qualitatively, these sites are modified identically by both antibiotics,
but quantitative differences observed in the cleavage intensities indicate
that the drugs bind in slightly different orientations. These results
are supported by DMS modification, mutational analysis of 16S rRNA
and structural modelling of tigecycline at a tetracycline-binding
site in the 30S ribosomal subunit. Conclusions: Both derivatives bind to identical
or overlapping sites and probably share the same mode of antibiotic
action. The fact that tigecycline overcomes most of the known tetracycline
resistance mechanisms is interpreted as a result of steric hindrance
due to the large substituent at position 9.
Keywords: glycylcyclines, mode of action, tetracycline
resistance
Comparison of tetracycline and tigecycline binding
to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage
of
16S rRNA
2 Wyeth Research, Cambridge, MA,
USA
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