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

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.

Mass Spectrometry of Nucleic Acid Noncovalent Complexes

“…f NA bound can be used for ligand screening (section ). ,− When exclusively 1:1 complexes are formed, plots of f NA bound as a function of total added ligand ([L] total ) can be used to determine apparent K D values. − Note that the condition of exclusive 1:1 complexes was not met in each paper.…”

“…13,[591][592][593][594][595][596][597][598][599] When exclusively 1:1 complexes are formed, plots of 𝑓 as a function of total added ligand ([L]total) can be used to determine apparent KD values. [600][601][602][603][604][605] Note that the condition of exclusive 1:1 complexes was not met in each paper.…”

“…509,1008 The salt bridge interactions between the protonated NH2 groups of the aminoglycoside and the RNA make the complex more kinetically stable in the gas phase than the covalent phosphodiester bond cleavage, and thus native top-down approaches are appropriate. The Chow group studied peptide binding to the 16S A-site 601 and 23S helix 69 602,605 ribosomal RNA using direct detection of the complexes and determination of the equilibrium dissociation constants. For both of these targets as well as helix 31 and TAR (transactivation response element) RNA, they also studied the underpinnings of fluorescence displacement experiments by ESI-MS. 1017 The Nakatani group developed ligands binding specifically to RNA CUG expansion repeats, and the binding was evaluated by ESI-MS. 1147 A couple of methods papers on the direct detection of RNA-ligand interactions by ESI-MS are available, 604,1148 but despite its promises native mass spectrometry is still not widely adopted for the biophysical characterization of RNA-ligand complexes.…”

“…611,612 Another approach to determine the apparent dissociation constants is to plot 𝑓 as a function of the ligand concentration and using nonlinear curve fitting (Equation ( 9)). [600][601][602][603][604][605]619 This approach is valid only for 1:1 stoichiometries. One example of such a titration curve is shown in Figure 26.…”

A Bright Future for Antibiotics?

“…Examples are those used recently to target Helix 69 (111), the first 16 residues of the proline-rich antimicrobial peptide mammalian Bac7 (112), the thiazolyl peptide antibiotics (113), and the small peptides that were shown to inhibit translation in prokaryotes (75,114).…”