title: Novel apidaecin 1b analogs with superior serum stabilities for treatment of infections by gram-negative pathogens.
abstract: 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.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany.
authors.forename: Nicole
authors.initials: N
authors.lastname: Berthold
authors.forename: Patricia
authors.initials: P
authors.lastname: Czihal
authors.forename: Stefanie
authors.initials: S
authors.lastname: Fritsche
authors.forename: Ute
authors.initials: U
authors.lastname: Sauer
authors.forename: Guido
authors.initials: G
authors.lastname: Schiffer
authors.forename: Daniel
authors.initials: D
authors.lastname: Knappe
authors.forename: Gottfried
authors.initials: G
authors.lastname: Alber
authors.forename: Ralf
authors.initials: R
authors.lastname: Hoffmann
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D000900:Anti-Bacterial Agents / Q000097:blood / Q000737:chemistry / Q000494:pharmacology; D023181:Antimicrobial Cationic Peptides / Q000097:blood / Q000737:chemistry* / Q000494:pharmacology; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003713:Dendritic Cells / Q000187:drug effects; D004912:Erythrocytes / Q000187:drug effects; D004926:Escherichia coli / Q000187:drug effects* / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006207:Half-Life; D006709:Homoarginine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009952:Ornithine / Q000737:chemistry / Q000378:metabolism; D055550:Protein Stability; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D023181:Antimicrobial Cationic Peptides; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; C061361:apidaecin; D006709:Homoarginine; D001120:Arginine; D009952:Ornithine; C063842:dnaK protein, E coli