Proline-rich
antimicrobial peptides (PrAMPs) from insects and mammals have recently been evaluated for their
pharmaceutical potential in treating systemic
bacterial infections. Besides the native
peptides, several shortened, modified, or even artificial sequences were highly effective in different murine
infection models. Most recently, we showed that the 18-residue-long
peptide Api88, an optimized version of
apidaecin 1b, was efficient in two different animal
infection models using the pathogenic Escherichia coli strains ATCC 25922 and Neumann, with a promising safety margin. Here, we show that Api88 is degraded relatively fast upon incubation with mouse serum, by cleavage of the C-terminal
leucine residue. To improve its in vitro characteristics, we aimed to improve its serum stability. Replacing the C-terminal
amide by the free
acid or substituting Arg-17 with l-
ornithine or l-
homoarginine increased the serum stabilities by more than 20-fold (half-life, ∼4 to 6 h). These analogs were nontoxic to human embryonic kidney (HEK 293), human
hepatoma (HepG2), SH-SY5Y, and HeLa cells and nonhemolytic to human erythrocytes. The binding constants of all three analogs with the chaperone DnaK, which is proposed as the bacterial target of PrAMPs, were very similar to that of Api88. Of all the analogs tested, Api137 (Gu-ONNRPVYIPRPRPPHPRL; Gu is N,N,N',N'-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional
alanine and d-
amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against an E. coli strain, whereas substitutions of residues 14 to 18 decreased the activity dramatically. Based on the significantly improved resistance to proteolysis, Api137 appears to be a very promising lead compound that should be even more efficient in vivo than Api88.