Bifunctional antibiotics that target both bacterial RNA and resistance-causing
enzymes are disclosed. The A-site of bacterial 16S rRNA serves as the target site
for most aminoglycoside antibiotics. Resistance to this class of antibiotics is
frequently developed by microbial enzymatic acetylation, phosphorylation or ribosylation
of aminoglycosides, modifications that weaken their interactions with the target
RNA. Using surface plasmon resonance (SPR), the binding affinity and stoichiometry
of various amino-glycosides have been investigated and it was found that neamine,
the key pharmacophore of the deoxystreptamine class of amino-glycosides, binds
to the A-site in a two to one stoichiometry with a Kd of 10 M
for each binding site. A library of neamine dimers was prepared and their affinities
to 16S rRNA A-site were determined by SPR, with Kd=40 nM for the best
dimer (an 103-fold increase in affinity). Antibiotic activities
of the dimers were determined for several bacterial strains by the Kirby-Bauer
method. The most active dimer, based on antibiotic activity, also showed the highest
inhibition of in vitro translation (IC50=0.055 M). The latter
assay was developed in order to correlate the relationship between SPR-based affinity
and translation inhibition. By these combined methods, transport limitations for
the semisynthetic aminoglycosides as well as non-ribosomally based antibiotic activity
could be determined. Further analysis of these dimers as substrates for aminoglycoside
modifying-enzymes identified a neamine dimer that was a potent inhibitor (Kis=0.1
M) of the APH(2";) activity of the bifunctional enzyme AAC(6";)-APH(2";),
the primary enzyme responsible for high level gentamicin C resistance in several
bacterial strains.
