Therapeutic peptides containing fluorinated amino acids.

摘要

The development of peptide-based drugs has recently intensified, although peptide/protein drugs generally require injection and suffer from low metabolic stability. While the introduction of fluorine atoms or trifluoromethyl groups into small molecule drug candidates often improves their druggability, this approach has been underutilized in peptide-based drug candidates.;We describe here the effects of fluorination on alpha-helical peptides that have the potential as antimicrobial or anti-diabetic agents. We incorporated optically pure fluorinated amino acids prepared through multi-step synthesis into two cationic antibacterial peptides, the membrane-disruptive magainin and cell-penetrating buforin. Four fluorinated analogues in the buforin and two in the magainin series were prepared and analyzed for (1) their antimicrobial activity against both Gram-positive and Gram-negative bacterial strains, (2) their hemolytic activity, and (3) their ability to resist hydrolytic cleavage by trypsin. All but one fluorinated peptide (M2F5) showed retention, or significant enhancement, of antimicrobial activity. The peptides also showed modest increases in protease resistance, relative to the parent peptides. Only one of the six fluorinated peptides (BII1F2) was degraded by trypsin at a slightly faster rate than the parent peptide. Hemolytic activity of peptides in the buforin series was essentially null, while fluorinated magainin analogues displayed an increase in hemolysis compared to the parent peptide. The fluorinated peptides exhibited an increased propensity for adopting alpha-helical conformations in both the buforin and magainin series peptides. In the case of the buforin peptides, higher alpha-helical content is coupled closely with enhanced antimicrobial potency. In contrast, higher amounts of secondary structure correlates with increased hemolysis and formation of aggregates in the magainin series.;We then turned our attention to the fluorination of the gut hormone glucagon-like peptide-1 (GLP-1) that has immense potential as an anti-diabetic, but is limited by short lifetime in plasma due to the action of the protease dipeptidyl peptidase IV (DPP IV). Seven fluorinated analogues were prepared and evaluated for their binding affinity to the receptor, signal transduction ability, and enzymatic stability. Introduction of hexafluoroleucine into GLP-1 at strategic sites conferred resistance against its regulatory protease, DPP IV. Although the in vitro binding affinity and signal transduction activity decreased slightly, the all-important efficacy was retained in 6 out of 7 fluorinated analogues. We also found that (1) the P2' site had a significant impact on protease stability, (2) double substitutions at 8 and 9 positions turned GLP-1 into a partial agonist with retention of maximal binding ability, (3) F9 was similar to natural GLP-1 in every aspect, suggesting that a large hydrophobic side chain is well tolerated at position 9 or that the CF 3 group is able to make multipolar contacts in the binding interface. Oral glucose tolerance experiments indicated that the enhanced in vitro stability of fluorinated GLP-1s could translate into metabolic stability in vivo.;These results demonstrate that fluorinated amino acids could be potentially useful for engineering peptide drug candidates. Considering the successful introduction of fluorine into small molecule drugs, incorporation of fluorinated amino acids into naturally bioactive peptides demands further investigation.