Studies on structural design, chemical synthesis, conformational properties and the mode of action of peptide antibiotics, magainin (Mag) are the focus of this program. A detailed study of organism viability in cultures of Streptococcus faecalis, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa when exposed to various concentrations of D-and L-Mag analogues and PGLa showed insignificant potency differences between corresponding D- and L-peptides. Unlike D-Mag F, D-PGLa was susceptible to pronase and was not capable of sterilizing a culture containing S. faecalis at any attainable concentration. HPLC examination of the culture revealed that PGLa was slowly deamidated into the carboxy desamino peptide and then to fragments. This clearly suggests the lack of importance of metabolism of these peptides in survival of the organisms, whether or not such a mechanism exists. Based on the determination of the minimum inhibitory and minimum bacteriocidal concentrations of Mag A, PGLa or their mixture against four bacteria, we demonstrated 2.5 to 6 fold synergism against E. coli, Klebsiella pneumoniae and P. aeruginosa, but only marginal synergism against S. aureus. Anti-tumor activity of potent Mag A and G was tested against growth of six small cell lung cancer cell lines. Despite their widely different sensitivities toward common chemotherapeutic agents, these cells showed little variation in sensitivity toward Mag A and G. Similarly, these analogues displayed the same level of growth inhibition against various hematopoietic tumor and solid tumor cell lines. Notably, Mag G was not cytotoxic toward peripheral blood lymphocytes. These studies indicate that Mag analogues have a practical therapeutic value in the treatment of infection as well as neoplasms in man and animals. Circular dichroic studies of Mag B revealed an ellipticity ratio at 222 nm/208 nm of >1.0, indicative of double-stranded coil structure when the peptide concentration was increased to more than 300 muM. Natural Mags, PGLa and antimicrobially inactive Mag H under the same conditions did not even form a helical structure. Dimerization of Mag B through the disulfide bridge of an extended Gly-Gly-Cys at the carboxyl terminus allowed the formation of a double-stranded coiled coils at 0.5-10 muM peptide. Both monomer and dimer showed a random structure in pure water and a single stranded helix in 40% trifluoroethanol. Such evidence suggests that hydrophobic interactions of side chains facilitate this helix coiled-coil formation. A new program for an automatic peptide synthesizer was developed and applied to the synthesis of difficult or large peptides including CRF, endothelin, the angiotensin II antagonist CGP 42112A, and a neurogranin fragment.
