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

Abstract

For almost five decades, antibiotics have been used successfully to control infectious diseases caused by bacterial pathogens. More recently, however, two-thirds of bacterial pathogens exhibit resistance and are continually evolving new resistance mechanisms against almost every clinically used antibiotic. Novel efforts are required for the development of new drugs or drug leads to combat these infectious diseases. A number of antibiotics target the bacterial aminoacyl-tRNA site (A site) of 16S ribosomal RNA (rRNA). Mutations in the A-site region are known to cause antibiotic resistance. In this study, a bacterial (E. coli) A-site rRNA model was chosen as a target to screen for peptide binders. Two heptapeptides, HPVHHYQ and LPLTPLP, were selected through M13 phage display. Both peptides display selective binding to the A-site 16S rRNA with on-bead fluorescence assays. Dissociation constants (K d s) of the amidated peptide HPVHHYQ-NH 2 to various A-site RNA constructs were determined by using enzymatic footprinting, electrospray ionization mass spectrometry (ESI-MS), and isothermal titration calorimetry (ITC) under a variety of buffer and solution conditions. HPVHHYQ-NH 2 exhibits moderate affinity for the A-site RNA, with an average K d value of 16 μM. In addition, enzymatic footprinting assays and competition ESI-MS with a known A-site binder (paromomycin) revealed that peptide binding occurs near the asymmetric bulge at positions U1495 and G1494 and leads to increased exposure of residues A1492 and A1493.It has been half a century since the first antibiotic penicillin was discovered and successfully applied to the treatment of infectious diseases; however, infectious diseases still remain the third-leading cause of death in the United States and the second-leading cause of death worldwide (1). Widespread and intensive use of antibiotics in hospitals and agriculture has led to significant levels of resistance (2,3). The emergence of antibiotic resistance, especially multidrug resistance, is a growing threat to human health (4). Therefore, a significant driving force exists for the development of new antimicrobial agents, especially those to combat multidrug-resistant pathogens.The majority of antibiotics used in the clinic target protein synthesis, which occurs at the core of the ribosome (5). High-resolution X-ray crystal structures of rRNA fragments, 30S and 50S subunits, and whole ribosomes, either free or complexed with antibiotics, have provided important information that will help in the design of new anti-infective compounds (6-10). † This work was supported by NIH grant AI061192. SUPPORTING INFORMATION AVAILABLEFigures S1-S4 show MALDI-TOF characterization of HPVHHYQ-NH 2 , ESI-MS data (salt dependence), and binding curve for RNase footprinting data (A1493). This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 September 8. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH...

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