Microbial resistance to currently available antibiotics has become an increasing threat to public health. The cyclic dodecapetide tyrocidine A produced by Bacillus brevis is an attractive template for drug development to counter the resistance because it disrupts cell membranes in its bactericidal function and, as a result, unlikely to provoke new resistance. However, this natural product kills both bacteria and mammalian cells indiscriminately and is limited from direct application in treatment of resistant microbes. A novel method for parallel synthesis of cyclic peptides was previously developed to increase the cell-differentiating ability of the template tyrocidine A. In this thesis, this synthetic method is studied with circular dichroism (CD) spectroscopy and applied to optimize t...[ Read more ]

Microbial resistance to currently available antibiotics has become an increasing threat to public health. The cyclic dodecapetide tyrocidine A produced by Bacillus brevis is an attractive template for drug development to counter the resistance because it disrupts cell membranes in its bactericidal function and, as a result, unlikely to provoke new resistance. However, this natural product kills both bacteria and mammalian cells indiscriminately and is limited from direct application in treatment of resistant microbes. A novel method for parallel synthesis of cyclic peptides was previously developed to increase the cell-differentiating ability of the template tyrocidine A. In this thesis, this synthetic method is studied with circular dichroism (CD) spectroscopy and applied to optimize the biological functions of tyrocidine A.

Comparison of the CD spectra of the linear and cyclic tyrocidine A found that the linear precursor adopts a β-pleated sheet structure closely resembling the cyclic product which is thermally stable in various media. This provides strong evidence that the linear precursor is predisposed to a favorable conformation that facilitates the head-to-tail ring closure observed in the synthetic method. Subsequently, two approaches were undertaken to optimize the natural antibiotic. First, using the conformation-dependent cyclization method, constituent amino acids of tyrocidine A were individually substituted with alanine and screening of the resulting analogues revealed that both antibacterial and hemolytic activities of the tyrocidine A are most susceptible to substitution at glutamine-5. Display of a variety of side chains at this position resulted in successful dissociation of the two intimately associated properties and identification of analogues with a therapeutic index up to 140. In a second approach, a 192-membered library was designed on the basis of the structural differences between the natural cyclic antibiotics tyrocidines, streptocidines, and loloatins, and successfully constructed with the developed method. Screening of the library identified three analogues with 10 to 90-fold increase in therapeutic index and concurrent increase in antibacterial activity. These newly discovered analogues are promising leads for further drug development to combat the microbial resistance.