Design and Synthesis of New Aminoglycoside Antibiotics Containing Neamine as an Optimal Core Structure:  Correlation of Antibiotic Activity with in Vitro Inhibition of Translation

Abstract

The structure and activity of the pseudodisaccharide core found in aminoglycoside antibiotics was probed with a series of synthetic analogues in which the position of amino groups was varied around the glucopyranose ring. The naturally occurring structure neamine was the best in the series according to assays for in vitro RNA binding and antibiotic activity. With this result in hand, neamine was used as a common core structure for the synthesis of new antibiotics, which were evaluated for binding to models of the Escherichia coli 16S A-site ribosomal RNA, in vitro protein synthesis inhibition, and antibiotic activity. Analysis of RNA binding revealed some correlation between the relative affinity and specificity of RNA binding and antibacterial efficacy. However, the correlation was not linear. This result led us to develop the in vitro translation assay in an effort to better understand aminoglycoside−RNA interactions. A linear correlation between in vitro translation inhibition and antibiotic activity was observed. In addition, IC50s in the protein synthesis assay were typically lower than the K ds obtained for RNA binding, suggesting that binding of these compounds to intact ribosomes is tighter in these cases than binding to the model RNA oligonucleotides. This reflects possible differences in RNA conformation between intact ribosomes and the free RNA of the model system, or possible high-affinity ribosomal binding sites in addition to the A-site RNA.

Rational Drug Design and High-Throughput Techniques for RNA Targets

“…Similar findings were reported from screening of designed neomycin B analogs targeting the A site of eubacterial 16S rRNA [93]. The A site [94] and HIV-1 RRE [101] were used for binding studies of aminoglycoside mimetics obtained by conventional [94] and combinatorial [101] synthetic derivatization of the neamine core with amino acids [101] and various amines [94]. In an attempt to explore the RNA binding capacity of amino sugar compounds by simplifying the structure of the aminoglycosides, a chemical library was synthesized by adding amino acid and amine side chains to a 2-aminoglucopyranoside synthon [91].…”

“…Among these methods, surface plasmon resonance (SPR) has extensively been used to study the binding of aminoglycoside derivatives to HIV-1 RRE [90] and oligonucleotides representing the bacterial 16S rRNA A site [91][92][93][94]. In order to perform SPR experiments, the RNA was biotinylated and immobilized on streptavidin-coated chips [90].…”

Vacuuming the Periplasm

“…Clearly it would be very useful for the cell if the transporter were able to catch even those molecules that had escaped periplasmic vacuuming. However, aminoglycosides appear to stimulate proton transport at concentrations (30 to 70 M) that are several orders of magnitude higher than those (20 to 30 nM) needed to inhibit protein synthesis (14).…”

Defining the Molecular Forces That Determine the Impact of Neomycin on Bacterial Protein Synthesis: Importance of the 2′-Amino Functionality

“…CR-Neo exhibits reduced bactericidal potency and affinity for the 16S rRNA A site relative to Neo (3,8,9,48). However, differential affinity for the A site is not the likely basis for the differential antibacterial activities of the two drugs, since these two parameters are poorly correlated (1,21,25,44). Our results indicate that the enhanced bactericidal potency of Neo relative to that of CRNeo reflects a correspondingly enhanced antitranslational activity, which, in turn, correlates with an enhanced drug-induced restriction of 1492 and 1493 base mobilities.…”