H

a-D-mannoside h a-D-mannose-

1,6-a-D-Mannose h h h Ashirnose a-D-mannose-1,6-cx-D-Mannose

= 5'-hycli'oxystreptomycin lacking ring III

Figure 6.1. The structures of streptomycin and several natural analogs of streptomycin.

which have been synthesized and tested. The aldehyde oxygen forms a hydrogen bond with a phosphate oxygen of G527.

Conversion of the aldehyde to an acid or reduction to a methyl abolishes all activity (45, 46). Numerous groups have investigated conversion of the aldehyde to its amino derivative and a variety of alkylamine derivatives, with surprising results (47-50). The amino deriva tive and short-chain alkylamine derivatives remained active up to the hexylamine analog. However, activity diminished with increasing chain length; the hexylamine derivative is only about Vioo as active as dihydrostrepto-mycin. The heptaamine derivative was not investigated. Longer alkylamines (octyl and above) were nearly as active as dihydrostrep-tomycin. This sharp inflection prompted one

Figure 6.2. Molecular interactions between streptomycin and 30S (E. caLi numbering, top) with various modifications tested for activity (bottom). Dashed lines indicate possible hydrogen bonds (some of which are salt bridges when suitably reinforced with favorable electrostatic potentials). Arrows point to permissible modifications; arrows with an X point to non-permissible modifications. (a)Ringl. (b) Ring II. Dashed arrow points to modifications that results in compounds that are active by an unknown mechanism, (c) Ring III.

All modifications

Figure 6.2. Molecular interactions between streptomycin and 30S (E. caLi numbering, top) with various modifications tested for activity (bottom). Dashed lines indicate possible hydrogen bonds (some of which are salt bridges when suitably reinforced with favorable electrostatic potentials). Arrows point to permissible modifications; arrows with an X point to non-permissible modifications. (a)Ringl. (b) Ring II. Dashed arrow points to modifications that results in compounds that are active by an unknown mechanism, (c) Ring III.

Therapeutic Agents Acting on RNA Targets

Ch2oh Structure

ch2oh ch2—nh2

ch2—nhch3

ch2—nhch2ch3 ch2 — nh(ch2)2h3 ch2—nh(ch2)5ch3 ch2—nhch2ch2ch2oh o

hoch2 Figure 6.2. (Continued.)

Therapeutic Agents Acting on RNA Targets

Figure 6.2. (Continued.)

2",-carboxy-xylo-furanose (ashimose) Removal a-D-mannose (=DM) a-DM-1,6-ce-DM

2",-carboxy-xylo-furanose (ashimose) Removal

Figure 6.2. (Continued.)

group to consider the mechanism of action of the alkylamine derivatives. The activity data suggested that the short-chain alkylamine analogs have a mechanism of action identical to that of streptomycin, whereas the long-chain alkylamine analogs operate by an unknown but ribosomally unrelated mechanism; the long-chain alkylamine analogs no longer bind to 30S.

Another alkylamino derivative tested was a conjugate of streptomycin and isoniazid, another prominent anti-tuberculosis drug. This compound termed streptohydrazid, was synthesized and found to be at least as active as combined therapy using both streptomycin and isoniazid (51).Streptohydrazid was tested long before the mechanism of action of streptomycin was known (the mechanism of isoni-

azid is not fully understood); it was reasoned that a conjugate of the two might act synergis-tically. The mechanism by which streptohy-drazid works is not known, but presumably it has streptomycin-like function, isoniazid-like function, or some combination of the two.

Correlation of the streptomycin/30S crystal structure with the various streptomycin analogs that involve aldehyde modification suggests that only a hydrogen bond between the aldehyde oxygen and a protonated phosphate oxygen (or a salt bridge for the amino derivatives) must be maintained. Reduction in binding only occurs when the modification becomes too large to be accommodated within the binding pocket. The only exception to this is when streptomycin is oxidized to streptomy-cinic acid. Although the possibility of forming the required hydrogen bond exists, the analog is inactive, presumably because of the electrostatically unfavorable close approach of the carboxylic acid to a phosphate that would occur on binding.

Several active natural streptomycin analogs, such as 5'-hydroxystreptomycin and AC4437, are hydroxylated at C5' (34, 52). Semisynthetic derivatives of this position are absent. A cursory inspection of the streptomycin/SOS structure suggests that the methyl group at C4' contributes little to ribosome binding, suggesting that modifications at this position might be tolerated.

Ring III (glucosamine) makes two direct contacts with 30S, neither of which are essential to activity (37, 52). Indeed, all of ring III can be dispensed with and cause only modest reductions in activity. However, deleterious modifications to this ring are possible. Some streptomycin-resistant bacteria harbor genes that encode proteins that either phosphory-late or adenylate the C 3 hydroxyl group of streptomycin (41). Two semisynthetic analogs, 3-epidihydrostreptomycin and 3"-deoxy-dihydrostreptomycin, were synthesized to circumvent common streptomycin resistance (53, 54). The logic is sound: both analogs should retain streptomycin-like binding affinity, yet not be substrates for inactivating enzymes. As expected, these analogs worked well against common bacterial strains and better than streptomycin against many streptomycin resistant strains. Yet, for reasons that are not clear, they never reached clinical status.

Some glycosylations have been observed at C 4 in natural streptomycin analogs, yielding somewhat less active antibiotics, yet remain active against bacteria that express enzymes that phosphylate and adenylate the C3"- OH. A limited number of natural modifications occur at C2", none of which abolish activity.

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