Bacterial resistance to antibiotics has become a world-wide health crisis. Organisms that cause diarrhea, urinary tract infection, and sepsis are now resistant to many of the older antibiotics. The resulting health threat has prompted the search for structurally unique antibacterial agents with novel modes of action. Bicyclomycin is one such commercially available drug. We have discovered that this architecturally distinctive antibiotic has a novel activation and bonding mechanism and that the primary site for drug function in Escherichia coli is the essential cellular protein, transcription termination factor rho. In this proposal, we outline an integrated approach to further the understanding of the mechanism of bicyclomycin expression. Research goals include (1) identifying the site, region, and stoichiometry of bicyclomycin-rho bonding (2) determining structural and chemical and biophysical properties of the bycyclomycin-rho complex, (3) elucidating the effect of bicyclomycin and bicyclomycin derivatives or rho-dependent processes, (4) determining the role of key residues in rho on bicyclomycin binding and bonding transformations, and (5) designing second generation bicyclomycin analogues with improved activity, and determining the pathway of bicyclomycin function in other Gram-negative organisms. The methodologies used to meet these objectives include mass spectrometric and amino acid analyses of chemical and enzymatic digests of bycyclomycin-rho and bicyclomycin affinity ligand-rho complexes to identify the site and region of drug bonding. In addition, enzymatic assays and thermochemical measurements will be used to determine the stoichiometry of rho-drug bonding and the energetics of these transformations. X-ray crystal structure and limited tryptic digestion investigations will be conducted to elucidate the structure and conformation of the bicyclomycin-rho complex. Sensitive enzymatic assays (e.g., poly (C)-stimulated ATPase activity, RNA binding, rho-dependent processes. Site and random-directed mutagenesis experiments are designed to identify the key catalytic sites in rho necessary for bicyclomycin function. These collective experiments will provide the molecular basis for the rational design of new bicyclomycin analogues.
