Structure–Function Characteristics and Signaling Properties of Lipidated Peptidomimetic FPR2 Agonists: Peptoid Stereochemistry and Residues in the Vicinity of the Headgroup Affect Function

Abstract

Formyl peptide receptor 2 (FPR2) plays important roles in inflammation. In the present study, 20 analogues of the FPR2-selective lipidated α-peptide/β-peptoid agonist Lau-[(S)-Aoc]-[Lys-βNPhe] 6 -NH 2 were generated, which allowed two novel subclasses of more potent FPR2 agonists to be distinguished. Critical factors influencing FPR2 recognition comprise the presence of β-peptoid phenylalanine-like residues (i.e., βNPhe, βNspe, or βNrpe) in the peptidomimetic tail, configuration of the 2-aminooctanoic acid (Aoc) in the headgroup, and the length of the N-terminal fatty acid. Intriguingly, a single βNrpe residue in the vicinity of the N-terminus (i.e., Lau-[(S)-Aoc]-Lys-βNrpe-[Lys-βNPhe] 5 -NH 2 ) proved to increase the agonist potency, whereas the βNspe-containing analogue was a weak FPR2-selective antagonist. Another subclass displaying potent agonism comprised analogues possessing two α-amino acids vicinal to the headgroup. The optimized FPR2-activating lipidated peptidomimetics exhibited biased signaling: PLC-PIP 2 -Ca 2+ signaling was activated, but without recruitment of β-arrestin or induction of chemotaxis. These FPR2-interacting compounds are considered to be useful tools in future studies of receptor−ligand interactions.

The putative Gαq-inhibiting pepducin P4Pal10 distinctly modulates function of the Gαi-coupled receptors FPR2 and FFA2R in neutrophils

“…The P4Pal 10 pepducin abolished WKYMVM-induced O 2 − production, and the inhibition was P4Pal 10 concentration dependent with an IC 50 -value of ∼ 0.7 µM (Fig 3A). To further investigate whether the inhibitory effect of P4Pal 10 is restricted to the agonist WKYMVM or achieved on the level of FPR2, we used several other FPR2 selective agonists including the Staphylococcus aureus -derived PSMα2 peptide (28), the mitochondrial-derived MCT-ND4 peptide (29), the lipidated peptidomimetic agonist Compound 14 (13), and the activating FPR2 specific pepducin F2Pal 10 (12, 30). All these FPR2 agonists activate neutrophils and P4Pal 10 inhibited the response (Fig 3B).…”

“…The pepducin F2Pal 10 (12) and the palmitoylated ten amino acids long pepducin P4Pal 10 (Pal-SGRRYGHALR) were synthesized by CASLO Laboratory (Lyngby, Denmark). The FPR2 peptidomimetic agonist Compound 14 (13) and the FPR2 peptidomimetic antagonist CN6 (14) were kind gifts from Henrik Franzyk (Copenhagen, Denmark). MCT-ND4 was a kind gift from Hidehito Mukai (Nagahama Institute of Bio-Science, Japan).…”

Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis

“…To study the effect of Barbadin on agonist-induced FPR2 internalization, we used the peptide agonist WKYMVM and two lipopeptides, the peptidomimetic Cmp 14 and the pepducin F2Pal10 that are functionally biased (structures are shown in Fig 1). All three agonists (WKYMVM, Cmp 14 and F2Pal10) are potent in inducing an FPR2-mediated transient rise in [Ca 2+ ]i, ERK1/2 phosphorylation, and ROS production in human neutrophils, but Cmp 14 and F2Pal10 are biased away from -arrestin recruitment and chemotaxis [9][10][11]. By using an enhanced bystander bioluminescence resonance energy transfer (ebBRET) assay system, we here confirmed that Cmp 14 and F2Pal10 are poor inducers of arrestin recruitment (Fig 2A).…”

“…a large number of ligands belonging to different chemical classes [5][6][7]. Depending on the agonists examined, the signals generated by FPR2 mediate primarily pro-inflammatoryactivities, yet anti-inflammatory activities have also been described [8][9][10][11]. Based on the important roles of FPR2 in host defense and regulation of inflammation, this GPCR has been considered as a promising drug target for inflammatory conditions including cardiovascular diseases [12,13].…”

“…We have previously proposed that the dynamic transfer of agonist-occupied FPR2 from an active signaling state to a non-signaling state (i.e., receptor desensitization) is not directly associated with the ability of the receptoragonist complex to recruit -arrestin [9], but appears to involve receptor coupling to the actin 5 cytoskeleton [11,[15][16][17][18]. Nevertheless, the ability to recruit -arrestin is of functional importance also in neutrophils as demonstrated by the finding that FPR2 agonists that are biased away from -arrestin recruitment induce ROS production without concomitant induction of chemotactic migration [9][10][11].In the present study, we investigated the role of β-arrestin in regulating FPR2 endocytosis and the NADPH-oxidase activation in neutrophils by using Barbadin as a tool compound. Barbadin has been shown to bind the clathrin adaptor protein AP2 and by that prevent -arrestin/AP2mediated endocytosis of agonist-occupied GPCRs [19,20].…”