Selection of Peptides That Target the Aminoacyl-tRNA Site of Bacterial 16S Ribosomal RNA

摘要

ABSTRACT: For almost five decades, antibiotics have been used successfully to control infectious diseasesncaused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistancenand are continually evolving new resistancemechanisms against almost every clinically used antibiotic.Novelnefforts are required for the development of new drugs or drug leads to combat these infectious diseases.nAnumber of antibiotics target the bacterial aminoacyl-tRNA site (Asite) of 16S rRNA(rRNA).Mutations innthe A-site region are known to cause antibiotic resistance. In this study, a bacterial (Escherichia coli) A-sitenrRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ andnLPLTPLP, were selected throughM13 phage display. Both peptides display selective binding to theA-site 16SnrRNA with on-bead fluorescence assays. Dissociation constants (Kd’s) of the amidated peptide HPVHHYQ-nNH2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospraynionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffernand solution conditions. HPVHHYQ-NH2 exhibits moderate affinity for the A-site RNA, with an averagenKd value of 16 μM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-sitenbinder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positionsU1495 andnG1494 and leads to increased exposure of residues A1492 and A1493.