Design, synthesis and characterization of fMLF-mimicking AApeptides

Abstract

The tripeptide N-formyl-Met-Leu-Phe (fMLF) is a potent neutrophil chemoattractant and the reference agonist for the G protein-coupled N-formylpeptide receptor (FPR). As it plays a very important role in host defense and inflammation, there has been considerable interest in the development of fMLF analogues in the hope of identifying potential therapeutic agents. Herein we report the design, synthesis, and evaluation of AApeptides that mimic the structure and function of fMLF. The lead AApeptides induced calcium mobilization and mitogen-activated protein kinase (MAPK) signal transduction pathways in FPR-transfected rat basophilic leukemic (RBL) cells. More intriguingly, at high concentrations, certain AApeptides were more effective than fMLF in the induction of calcium mobilization. Their agonistic activity is further supported by their ability to stimulate chemotaxis and the production of superoxide in HL-60 cells. Similarly to fMLF, these AApeptides are much more selective towards FPR1 than FPR2. These results suggest that the fMLF-mimicking AApeptides might emerge as a new class of therapeutic agents that target FPRs.

Keywords: AApeptides; calcium; chemotaxis; fMLF; peptidomimetics.

Figure 1

The structure of α-peptides, α-AApeptides and γ-AApeptides. In both α-AApeptides and γ-AApeptides, half of side chains are introduced through acylation, while the other half of side chains are chiral and derived from amino acids.

Figure 2

The structures of fMLF and AApeptides.

Figure 3

AApeptide-induced activation of calcium mobilization in human FPR1 transfected RBL cells.

Figure 4

γ-AApeptide-induced activation of calcium mobilization in human FPR2 transfected RBL cells. Data shown are representative of three independent experiments.

Figure 5

Activation of ERKs by fMLF and γ-AApeptides 3, 4 and 7 in human FPR1-transfected RBL cells. These cells were stimulated with fMLF peptide (1 µM, upper-left), γ-AApeptides 3 (10 µM, upper-right), 4 (10 µM, lower-left) and 7 (10 µM, lower-right), respectively. After various time intervals the cells were harvested and the phosphorylated ERKs were determined by Western blotting with anti-phospho-ERK antibodies. Data shown are representative of three independent experiments.

Figure 6

a, effects of the lead γ-AApeptides on ROS production in differentiated HL-60 cells. b, effects of the lead γ-AApeptides on chemotaxis of differentiated HL-60 cells. Data shown are representative of three independent experiments.

Scheme 1

a, synthesis of α-AApeptides 1 and 2; b, synthesis of γ-AApeptides 3–7.