Novel Apidaecin 1b Analogs with Superior Serum Stabilities for Treatment of Infections by Gram-Negative Pathogens

Abstract

ABSTRACTProline-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 pathogenicEscherichia colistrains 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 itsin vitrocharacteristics, we aimed to improve its serum stability. Replacing the C-terminal amide by the free acid or substituting Arg-17 withl-ornithine orl-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 isN,N,N′,N′-tetramethylguanidino) appeared most promising due to its high antibacterial activity, which was very similar to Api88. Positional alanine andd-amino acid scans of Api137 indicated that substitutions of residues 1 to 13 had only minor effects on the activity against anE. colistrain, 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 efficientin vivothan Api88.