been observed in proximal tubular kidney cells and within sensory hair cells, with significant localization to mitochondria (79). (5) In vitro measurements demonstrate that
neomycin aminoglycosides bind poorly to the human mitochondrial decoding A-site, but bind to the RNA containing the poly- morphic sequence with an affinity similar to that of RNA with the bacterial sequence (6) Paromomycin has been observed to shut down mitochondrial activity in Leishmania
A direct model of aminoglycoside ototoxic- exists for neomycin-like and
like compounds, in which aminoglycosides bind to mitochondrial ribosomes containing the mutation more tightly than they do to the wild-type ribosomes. Ring I of both subclasses stacks atop the Watson-Crick base pair formed by and This base pair and the adjacent base pair form mis- matched pairings
.
andin human mitochondrial ribosomes, and the second pair is thought to disrupt the stabilizing stacking interaction between ring I and nucleotides 1409 and 1491. In individuals with the polymorphism (analogous to A1490 in the E. coli small subunit one
of the two mismatches is converted to a Watson-Crick base pair which may help restore the stacking interaction be- tween ring I and 1409 and 1491. In vitro bind- ing studies of common aminoglycosides and both mitochondrial RNA sequences confirm that aminoglycosides bind the poly- morphic sequence more tightly than the type (79). If the direct model is correct, then there is optimism that agents with enhanced specificity for the bacterial sequence will nec- essarily be less ototoxic.
One obvious weakness in the theory of involvement in aminoglycoside in- toxication is that streptomycin doesn't share a binding site with gentamicirdneomycin-like
However, streptomycin does interact with the backbone of (10). It is possible that the polymorphism po- tentiates streptomycin biding against
I chondrial ribosomes, either directly or
redly. A second weakness is that tissue 3 specificity for aminoglycoside toxicity is poorly
understood, but that is true for all competing theories of aminoglycoside ototoxicity.
7 TETRACYCLINE AND ANALOGS
Tetracyclines were the first broad-spectrum antibiotics and have been used successfully for decades to treat both gram-positive and gram negative bacterial infections (82).
cycline was the first tetracycline to be isolated, in 1948, from Streptomyces aureofaciens Other common tetracyclines, such as
racycline and tetracycline, were isolated from Streptomyces sources in subsequent years. The abundance of natural, active tetracy- clines, coupled with extensive synthetic alter- ations, provides a rich collection of compounds from which to build meaningful
tivity relationships. As was found for the noglycosides, previous observations of tetracy- cline structure and activity can be rationalized
from recent crystal struc-
tures (11, 12).
Although tetracycline was not the first agent of its class discovered, it nonetheless provides the basis for tetracycline nomencla- ture (Fig. 6.6). Numerous reviews exist that report on the biosynthesis and use of tetracy- clines 86).
Extensive efforts to identify tetracycline analogs from both natural and semisynthetic sources that possess greater efficacy, lower toxicity, and greater chemical stability have produced superior drugs such as
cline and doxycycline, but newer
analogs may be on the way. Members of a new tetracycline class, known as
clines, are superior to all existing clinically useful agents against several types of tetracy- cline-resistant bacteria
7.1 Mechanism of Action
Biochemical probing identified multiple tetra- cycline binding sites within one of which resides in the decoding A-site and seems to be responsible for the antibiotic effect of tetracy- cline (19). Two groups have independently
solved structures by X-ray
crystallography The Berlin group re- fined the positions of six tetracycline binding sites, ranging in occupancies from 0.41 to 1.0,
Therapeutic Agents Acting on RNA Targets R4 Tetracycline H OH H Chlortetracycline OH H Demeclocycline OH H H 6-Dernethyltetracycline (Dernecycline) H OH H H Oxytetracycline H OH OH 6-Deoxytetracycline H H H Minocycline H H Doxycycline H H OH
Figure 6.6. The structures of tetracycline and several analogs of tetracycline.
to a resolution of 4.5 while the Cam- bridge group identified two tetracycline sites, which they refined to (11). The two sites identified by the Cambridge group are a subset of the six sites reported by the Berlin group, with both groups tetracycline bound in a pocket formed by helices 31 and 34 as the dominant and most relevant site. Only the structure of the
complex will be discussed here, al-
though other tetracycline binding sites could contribute to ribosome inhibition.
It has been accepted for some time that tet- racycline interferes with proper tRNA binding to the A-site (57). A refinement of this model has the elongation factor complex docking correctly into the A-site, allowing the codon-anticodon interaction to take place, and the subsequent hydrolysis of GTP by (11). At this point a rota- tion of the A-site tRNA is blocked by tetracy- cline, leading to the ejection of tRNA from the A-site without bond formation. In this scenario, tetracycline extracts two
from the cell, inhibition of protein and the unproductive hydrolysis of GTP. 7.2 Structure-Activity Relationship
Tetracyclines contain four fused rings
one of which, D, is aromatic; rings A and B
contain sites of (Fig. 6.7). Essen- tially all alterations to the general tetracycline ring skeleton are deleterious; breaking any ring, disrupting aromaticity in ring D, or matization of ring A or C destroys all tetracy- cline activity Modifications to the func- tional groups on ring A and the bottoms of rings B, C, and D are generally not tolerated, but extensive modification is possible else- where. Maintenance of the ketones at and is essential, as are the hydroxyls at C3, C12, and All of these groups are involved in an extensive network of RNA bind- ing interactions composed of hydrogen bonds and shared coordination to Neither the nor the methyl at C6 is essential for activity; no direct ribosome interaction is ob- served for the C6 substituents. Removal of the dimethylamino group at C4 diminishes in
vitro activity and abolishes in activity
(85). Reduced binding is expected with the re- moval of this group; it interacts electrostati- cally with the phosphate of The
group at C2 is rarely substituted, but a 2-acetyl moiety has been observed (85). Either chemical entity is able to hydrogen bond to the ribose of
Tetracycline occupies a pocket formed by helices 31 and 34 of (11). As with most ribosome targeting antibiotics, the
Therapeutic Agents Acting on RNA Targets
tacts are to RNA only. The structures explain virtually all of the absolutely required tetracy- cline groups. Extensive hydrogen bonding net- works are observed between the drug and backbone phosphate and ribose oxygen atoms. Similar to streptomycin, no hydrogen bonds are formed between tetracycline and any nucleobases-sequence selective binding comes from the proper presentation of
pendent groups. Tetracycline binding has two interactions that are unusual inter- actions: binding through a shared which coordinates several from tetracycline and phosphate groups from the ribosome, and aromatic stacking between ring D and
All
attempts to modify tetracycline groups in- volved in the intricate interactions detailed above are deleterious. The remaining perimeter of tetracycline makes no close interactions with Indeed, the edge of tetracycline is directed away from any nearby which ex- plains why extensive modifications at C6, C7, and and the methoxy derivative are pos- sible.Removal of one or both of the common C6 substituents, hydroxyl and methyl, is com- mon, and virtually all substitutions at C6 are permitted, even for groups as large as zylmercaptan and glycosides (85). Both elec- tron donating and electron withdrawing groups are tolerated at C7 methoxy derivatives are occasionally observed and are active Numerous substitutions at are also permissible. Recently, amido substitu- tions yielded compounds with enhanced ribo- some binding that escape recognition by cer- tain efflux pumps These derivatives, known as glycylcylines, remain active against a wide variety of tetracycline-resistant bacte- ria. Of particular interest here is the fact that many of these derivatives have greatly en- hanced ribosome binding, indicating that the amido substitutions are making additional unique, productive interactions with
More work will be needed before the identities of these new interactions are revealed.