H H Oh

N^oX>oh

H bH 125 Trehazolin

Compounds 125 and 126

126 is another trehalase inhibitor [125], isolated from Streptomyces albus. These natural products are inhibitors of glycosidases, which play critical roles in intra- and intercellular processes including cell adhesion, membrane transportation, and signal transduction.

III. ENZYMATIC MODIFICATION OF AMINOGLYCOSIDES

Resistance to antibiotics manifests itself in different ways. The target of the antibiotic may be altered such that affinity for the drug may be reduced [126,127,128]. Or, the rate of the uptake of the drug into the bacterium may be attenuated such that the critical concentration necessary for saturating the binding site cannot be reached [129,130,131]. Alternatively, specific enzymes may evolve to alter the structure of the drug in bacterium, whereby the site for the binding shows lower affinity for the modified drug, thus manifesting resistance to the original drug. This third method for acquired resistance is the most common for aminoglycoside antibiotics [132,133,134,135].

Three classes of enzymes have evolved for aminoglycoside modification for the manifestation of the resistance phenotype. These enzymes are aminoglycoside acetyltransferases (Aac), aminoglycoside nucleotidyltransferases (Ant), and aminog-lycoside phosphotransferases (Aph). The acetyltransferases transfer the acetyl group from acetyl coenzyme A to specific amines in aminoglycosides. The sites of these modifications are 1-, 3-, 2'-, and 6'-amino groups. The other two classes of enzymes transfer the adenyl or the phosphoryl group from ATP to hydroxyl groups in ami-noglycosides. The common sites of adenyl transfer are hydroxyl groups at 2"- and 4'-hydroxyls of kanamycins and 3"- and 6-hydroxyls of streptomycin. The sites of phosphotransferase reactions are the hydroxyl groups at positions 3' and 2" of kanamycins and 3" and 6 of streptomycin. The products of these enzymatic modifications are invariably poorer antibiotics because their binding to their ribosomal target sites has been impaired. The nature of these enzymes and state of the art in their mechanistic understanding were reviewed comprehensively in 1998 [134].

IV. MECHANISM OF ACTION OF AMINOGLYCOSIDE ANTIBIOTICS

Aminoglycosides exhibit their antibiotic action primarily by entering the cell irreversibly by active transport, binding to the ribosomal RNA (rRNA), interfering with protein synthesis (translation process), and causing membrane damage resulting in leakage of the cell membrane [134,136,137]. These actions result in disruption of

the integrity of the bacterial cell, ultimately leading to cell death. There are exceptions, however, to this mode of action, and a few of the aminoglycosides such as spectinomycin and kasugamycin are bacteriostatic; that is, they stop the growth of bacteria rather than killing the cell, although they too interact with rRNA. It is now well established that aminoglycosides bind to the RNA major groove at various important sites such as the A site (ribosomal acceptor site) and hammerhead regions (see below).

Aminoglycosides enter bacteria in a biphasic and energy-dependent process. The initial phase (phase I) allows for transport of small quantities of aminoglycoside, although the amount that is required for antibacterial action is not known [134,138]. Phase I has been described as an energy-dependent active transport and entails a slow, linear uptake process [138,139,140]. Phase II of uptake is both energy and concentration dependent, and it shows a behavior similar to the diffusion of small molecules through channels. After uptake during phase I, aminoglycoside binds to the chain-elongating ribosomes and causes misreading of mRNA, resulting in malformed proteins. A few of these miscoded proteins become part of the cell surface and disrupt the integrity of the membrane. The damaged membrane subsequently allows more antibiotic molecules to enter the cell, where ultimately they reach a concentration that can inhibit the initiation of the translation process. Eventually, the combination of disrupted membrane and irreversible blocking of the protein biosynthesis kills the organism [136].

Ribosome plays an important role in selecting the correct EF-Tu-GTP-ami-noacyl-tRNA ternary complex at the A site, which supplies the next amino acid to the elongating polypeptide during translation [141]. In the A-site region of ribosomal RNA, the highly conserved A1492 and A1493 point toward the minor groove in the A site [142]. It was proposed that when a complementary tRNA binds to the codon, the 2'-OH moieties on mRNA may interact with the N1-position of the conserved adenines, and perhaps this process is a beginning step in the communication needed for the codon-anticodon complex. It was suggested that binding of aminoglycosides to the rRNA decreases the dissociation rates of aminoacyl-tRNA, and stabilizes a high-affinity conformation of tRNA-mRNA complex [142]. This in turn may result in inhibition of the translation process and/or favor miscoding. In fact, in early 1960s, ribosomes were identified as the target sites of action for aminoglycosides such as streptomycin. However, the limitations of scientific knowledge at the time prevented the establishment of the relationship between the ribosome and the bactericidal activity [136]. A few years later, it was understood that binding to the ribosome of small molecules such as aminoglycosides can influence not only the catalytic activity of the ribosome but also its specificity; that is, such binding can lead to improper information processing in translation, transcription, and replication.

The interactions of aminoglycosides with ribosome are concentration dependent [140]. At lower concentrations, streptomycin blocks the abundant polysomal ribo-somes (chain elongating), whereas at higher concentrations, these molecules can bind all ribosomes, including the ones involved in initiation [136]. It was proposed that binding of streptomycin to the ''initiating ribosome'' is irreversible. However, this may be due to the irreversible uptake of aminoglycoside into the cell as well. The bactericidal activity of aminoglycosides is now supported by the involvement of almost every step of the mechanism given above [134]. However, there are still several unanswered questions relating to, for example, the nature of the miscoded proteins that are transported to the membrane, the enzymes/channels in the active transport of aminoglycosides into the cell, the nature of miscoding, and their effect on protein folding and membrane integration.

Lately, there is increasing evidence of inhibition of self-splicing of group I introns by a number of aminoglycosides such as streptomycin, tobramycin, neomycin B, kanamycin, neamine, 5-e^i-sisomicin and gentamicin [143,144,145,146]. It was shown that at reasonably low (micromolar) concentrations, several of these aminoglycosides can inhibit noncompetitively the thymidylate synthase group I intron RNA splicing in vitro. It appears that the ability of various aminoglycosides to inhibit intron self-splicing is purely structure dependent; and depending on the concentration of the aminoglycoside, one or both steps in the catalysis of group I intron self-splicing are inhibited. It is very interesting, however, to learn that new activities are being discovered for these older antibiotics, and perhaps these findings will shed more light on the mechanism of action of aminoglycosides and their toxicity mechanisms. The similarities in the binding of aminoglycosides to ribosomal RNA and the catalytic self-splicing introns have led to the hypothesis that perhaps aminogly-cosides were modulators of RNA activity in biologically evolving systems. Furthermore, during the coevolution of RNA and aminoglycosides, modern ribosomes have formed on which aminoglycosides exhibit similar binding properties [146,147].

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