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. 2016 Sep 15;24(18):4486-4491.
doi: 10.1016/j.bmc.2016.07.050. Epub 2016 Jul 25.

The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes

Danielle N Dremann  1 , Christine S Chow  2
Affiliations

The development of peptide ligands that target helix 69 rRNA of bacterial ribosomes

Danielle N Dremann et al. Bioorg Med Chem. .

Abstract

Antibiotic resistance prevents successful treatment of common bacterial infections, making it clear that new target locations and drugs are required to resolve this ongoing challenge. The bacterial ribosome is a common target for antibacterials due to its essential contribution to cell viability. The focus of this work is a region of the ribosome called helix 69 (H69), which was recently identified as a secondary target site for aminoglycoside antibiotics. H69 has key roles in essential ribosomal processes such as subunit association, ribosome recycling, and tRNA selection. Conserved across phylogeny, bacterial H69 also contains two pseudouridines and one 3-methylpseudouridine. Phage display revealed a heptameric peptide sequence that targeted H69. Using solid-phase synthesis, peptide variants with higher affinity and improved selectivity to modified H69 were generated. Electrospray ionization mass spectrometry was used to determine relative apparent dissociation constants of the RNA-peptide complexes.

Keywords: Helix 69; Ligand binding; Peptide; Pseudouridine; Ribosomal RNA.

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Figures

Figure 1
Figure 1
A. The 70S E. coli bacterial ribosome (2AVY, 2AW4) is shown with H69 highlighted in cyan, aminoacyl-tRNA site (A site) in red, peptidyl transferase center (PTC) in purple, 30S subunit in light grey, and 50S subunit in yellow. B. Secondary structures of H69 are given (from left to right): human H69, wild-type H69 with E. coli numbering, modified H69 (ΨΨΨ), and unmodified H69 (UUU).
Figure 2
Figure 2
The base structures of uridine (left), pseudouridine (middle), and 3-methylpseudouridine (right) are shown.
Figure 3
Figure 3
The ESI mass spectrum shows RQVANHQ-NH2 (35 μM) bound to modified H69 (ΨΨΨ, 2.7 μM) RNA at a single titration point.
Figure 4
Figure 4
The titration curve for RQVANHQ-NH2 bound to ΨΨΨ is shown. The apparent dissociation constant (Kd) value is given.
Figure 5
Figure 5
The relative binding affinities and selectivities of the peptide variants for ΨΨΨ (upper) or UUU (lower) are illustrated. Each position contains either A, R, or the parent amino acid along with the remaining parent amino acid sequence, which is indicated in blue. The size of the letter indicates the relative affinity or selectivity of that amino acid variant compared to the other amino acids at that position, with larger letters indicating tighter binding or more selective species.
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