RESUMO
Temporin-1DRa (HFLGTLVNLAKKIL.NH(2)), first isolated from the skin of the California red-legged frog Rana draytonii, shows broad-spectrum antimicrobial activity but its therapeutic potential is limited by its toxicity against mammalian cells. The cytolytic properties of cationic alpha-helical peptides are determined by a complex interaction between cationicity, hydrophobicity, conformation, and amphipathicity. This study has investigated the cytolytic properties of conformationally constrained analogs of temporin-1DRa containing alpha-aminoisobutyric acid (Aib) substitutions. Cytolytic activity was determined against the bacteria Escherichia coli and Staphylococcus aureus, the opportunistic yeast pathogen, Candida albicans, human erythrocytes, HepG2 hepatoma-derived cells, and L929 fibroblasts. Aib substitutions at Gly(4), Asn(8), and Ala(10) increased both % helicity, determined in methanol solution, and hydrophobicity resulting in increases in both antimicrobial potencies and toxicities against the mammalian cells. Substitution at Leu(6) resulted in an appreciable decrease in cytolytic activity against all cells whereas the substitutions at His(1), Phe(2), Leu(3), Thr(5), and Val(7) had only minor effects on activity. Substitutions at Leu(9), Ile(13), Leu(14) produced analogs with decreased helicity and hydrophobicity that retained activity against microorganisms but showed appreciably lower cytolytic activities against mammalian cells. In particular, the fourfold increase in therapeutic index [ratio of LC(50) against erythrocytes to minimum inhibitory concentration (MIC) against microorganisms] of [Aib(13)]temporin-1DRa identifies it as a compound with potential for development as a therapeutically valuable anti-infective agent.
Assuntos
Ácidos Aminoisobutíricos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/química , Linhagem Celular Tumoral , Dicroísmo Circular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Testes de Sensibilidade Microbiana , Conformação Proteica , RanidaeRESUMO
The Cascades frog Rana cascadae belongs to the Amerana (or Rana boylii) group that includes six additional species from western North America (R. aurora, R. boylii, R. draytonii, R. luteiventris, R. muscosa, and R. pretiosa). R. cascadae is particularly susceptible to pathogenic microorganisms in the environment and populations have declined precipitously in parts of its range so that the protection afforded by dermal antimicrobial peptides may be crucial to survival of the species. Peptidomic analysis of norepinephrine-stimulated skin secretions led to the identification of six peptides with differential cytolytic activities that were present in high abundance. Structural characterization showed that they belonged to the ranatuerin-2 (one peptide), brevinin-1 (one peptide), and temporin (four peptides) families. Ranatuerin-2CSa (GILSSFKGVAKGVAKDLAGKLLETLKCKITGC) and brevinin-1CSa (FLPILAGLAAKIVPKLFCLATKKC) showed broad spectrum antibacterial activity (MIC=32microM against Escherichia coli and Staphylococcus aureus) but only brevinin-1CSa was strongly hemolytic against human erythrocytes (LC(50)=5microM). The taxonomy of ranid frogs is currently in a considerable state of flux. The ranatuerin-2 gene is expressed in all members of the Amerana group studied to-date and cladistic analysis based upon a comparison of the amino acid sequences of this peptide indicates that R. cascadae, R. muscosa and R. aurora form a clade that is distinct from one containing R. draytonii, R. boylii, and R. luteiventris. This conclusion is consistent with previous analyses based upon comparisons of the nucleotide sequences of mitochondrial genes.