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

Abstract

dProline-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 Lornithine or L-homoarginine increased the serum stabilities by more than 20-fold (half-life, ϳ4 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.

Structure-activity relationship study using peptide arrays to optimize Api137 for an increased antimicrobial activity against Pseudomonas aeruginosa

“…In contrast to apidaecin 1b it is also active against P. aeruginosa in diluted culture medium. Additionally, apidaecins have very low toxicities against mammalian cell lines and immune cells and are non-hemolytic against human erythrocytes [28,29]. Importantly, they are well tolerated in mice at four intraperitoneal injections of 80 mg/kg per day and highly efficient in a murine E. coli ATCC25922 infection model providing 100% survival at doses of 0.6 mg/kg (manuscript in preparation).…”

“…A promising lead structure is Api137 (gu-ONNRPVYIPRPRPPHPRL-OH; gu: N,N,N 0 ,N'-tetramethylguanidino, O: ornithine), a derivative of apidaecin 1b produced in honeybee (Apis mellifera) [27]. Api137 was optimized for high activities against Escherichia coli and Klebsiella pneumoniae and stabilized against degradation by serum proteases [28]. In contrast to apidaecin 1b it is also active against P. aeruginosa in diluted culture medium.…”

Insect‐Derived Proline‐Rich Antimicrobial Peptides Kill Bacteria by Inhibiting Bacterial Protein Translation at the 70 S Ribosome

“…[3] PrAMPs, which are expressed in mammals and insects, have been evaluated by several research groups, either in their native forms or as chemically optimized or designed lead compounds in different infection models. [4] Collectively, PrAMPs show low sequence homology but are structurally homologous (ca. 30 % proline content, one or several Pro-Arg-Pro motifs, disordered structure in solution) and appear to kill bacteria by similar mechanisms.…”

Cuantificación de la efectividad de la indolicidina y protegrina-4 por medio de análisis de parámetros cinéticos

“…Sin embargo, reportaron que la amidación en el grupo terminal carboxilo orienta perpendicularmente al péptido PMAP-23 hacia la membrana bacteriana, mientras que el péptido no modificado presenta una posición paralela respecto a la membrana, es decir, la orientación del péptido incrementa la velocidad de penetración a las membranas de las bacterias Gram negativas y, por lo tanto, resulta en una mejor permeabilización de la membrana celular. La amidación del grupo terminal carboxilo es un mejoramiento que se tiene en los AMPs sintéticos, debido probablemente a una mayor estabilidad de estos (Berthold et al, 2013).…”