4'-O-substitutions determine selectivity of aminoglycoside antibiotics

Abstract

Clinical use of 2-deoxystreptamine aminoglycoside antibiotics, which target the bacterial ribosome, is compromised by adverse effects related to limited drug selectivity. Here we present a series of 4',6'-O-acetal and 4'-O-ether modifications on glucopyranosyl ring I of aminoglycosides. Chemical modifications were guided by measuring interactions between the compounds synthesized and ribosomes harbouring single point mutations in the drug-binding site, resulting in aminoglycosides that interact poorly with the drug-binding pocket of eukaryotic mitochondrial or cytosolic ribosomes. Yet, these compounds largely retain their inhibitory activity for bacterial ribosomes and show antibacterial activity. Our data indicate that 4'-O-substituted aminoglycosides possess increased selectivity towards bacterial ribosomes and little activity for any of the human drug-binding pockets.

Figure 1. Interaction of ring I with residues in the A site loop of 16S rRNA.

(a) Secondary structure of the aminoglycoside-binding pocket in helix 44 of 16S rRNA. Key polymorphic residues determining the selectivity of aminoglycosides are residues 1408 and 1491, highlighted in bold green. (b) Overview of paromomycin bound to the bacterial A site. Detailed view of the 6′OH paromomycin ring-I stacking interaction with G1491 and hydrogen bonding with A1408 and A1493 (ref. 10). Hydrogen bonds between aminoglycoside ring I and A1408 are shown as red dotted lines, as is hydrogen bonding between 4′OH and O2P of A1493. (c) Ring I interaction with A1408. Left paromomycin (Pm), right neomycin (Neo). Hydrogen bond interaction between the 6′-subsituent (6′OH, 6′NH₂) and the N1 of A1408 is indicated by a red dotted line, as is hydrogen bond interaction between O5′ and N6 of A1408. (d) Ring I interaction with G1408 (model). Left paromomycin, right neomycin. Possible hydrogen bond interactions are indicated by red dotted lines. The positive charge of neomycin’s 6′-ammonium group would create repulsion against the N1 and N2 amino groups of G1408, indicated by red arrows. (e) General chemical structure of 4′,6′-O-acetals and 4′-O-ethers in comparison to paromomycin, the 6′ and 4′ positions that were target for substitution are indicated.

Figure 2. Drug-induced inhibition of protein synthesis in ribosomes.

Inhibition of protein synthesis depicted as IC₅₀ (μM); IC₅₀ values represent the drug concentrations required to inhibit in vitro synthesis of firefly luciferase to 50%. (a) y axis: IC₅₀ mitohybrid ribosomes, x axis: IC₅₀ rabbit reticulocyte ribosomes; (b) y axis: IC₅₀ mitohybrid deafness ribosomes, x axis: IC₅₀ rabbit reticulocyte ribosomes.

Figure 3. In vivo activity of aminoglycoside compounds and comparators in a murine septicaemia model.

(a) Bacterial burden in kidney, CFU per g tissue. (b) Bacterial burden in blood, CFU ml⁻¹. The CFU values for the individual animals are indicated by dots, the mean of the group (five mice per treatment group) by the bar. The log reduction compared with the vehicle control as well as the P-values (non-parametric Kruskal–Wallis using pairwise comparisons) are indicated as numbers above each group. Colour coding—pre-treatment: light grey; vehicle control: dark grey; 30: red; 39: orange; 37: green; amikacin: light blue; paromomycin: dark blue; linezolid: violet. LOD, limit of detection.

Figure 4. Structures of antibiotics bound to the decoding center of the 30S ribosomal subunit.

(a) 3F_o—2F_c difference Fourier density around compound 1 (green) contoured at 1.0σ. (b) Superposition of antibiotics in the decoding center, colour coding as indicated by the numbering. (c) Decoding center of the 30S subunit (helix 44 of 16S RNA, orange; protein S12, light green), showing compound 1 in green. The conformation of A1492 and A1493 (grey) is shown. The benzyl ring is in proximity to the nucleobase of the flipped-out A1492. Also shown is a superposition with paromomycin (Par, salmon). (d) Superposition of helix 44 of 16S RNA for structures of 30S-compound 1 (green) and 30S-paromomycin (salmon, Protein Data Bank ID code 1FJG); the orientations of A1492 and A1493 that are flipped-out from helix 44 are tilted relative to the paromomycin structure. (e) View of compound 1 and loss of a hydrogen bond with O2P of A1493 compared with paromomycin. The benzyl ring is in proximity to the nucleobase of the flipped-out A1492.

Figure 5. Aminoglycoside-induced misreading.

Dose–response curves of aminoglycoside-induced misincorporation of amino acids using the H245R near-cognate mutant F-luc mRNA as template. Shown is luciferase activity upon translation of mutant template relative to wild-type F-luc mRNA (mean±s.d.; n=3). (a) Bacterial ribosomes; (b) rabbit reticulocyte ribosomes. 1 (filled circles), paromomycin (open circles) and apramycin (open triangles).