Functional and signaling characterization of the neutrophil FPR2 selective agonist Act-389949

Abstract

Despite the steadily increased numbers of formyl peptide receptor (FPR) ligands identified over the years, few have been characterized in studies using animal disease models and even less have entered clinical trials in human subjects. A small-molecule compound, Act-389949, was however recently tested in a phase I clinical trial and found to be safe and well tolerated in healthy human subjects. The desired anti-inflammatory property of Act-389949 was proposed to be mediated through FPR2, one of the FPRs expressed in neutrophils, but no basic characterization was included in the study. To gain more insights into FPR2 recognition of this first-in-class compound for future utility of the agonist, we have in this study determined the receptor preference and down-stream signaling characteristics induced by Act-389949 in human blood neutrophils isolated from healthy donors. Our data demonstrate that Act-389949 is an agonist for FPR2 that triggers functional/signaling repertoires comparable to what has been earlier described for other FPR2 agonists, including neutrophil chemotaxis, granule mobilization and activation of the NADPH-oxidase. In fact, Act-389949 was found to be as potent as the prototype FPR2 peptide agonist WKYMVM and had the advantage of being resistant to oxidation by the MPO-H2O2-halide derived oxidants, as compared to the sensitive WKYMVM. The down-stream signals generated by Act-389949 include an FPR2-dependent and Gαq-independent transient rise in intracellular Ca 2+ and recruitment of β-arrestin. In summary, our data show that Act-389949 serves as an excellent tool-compound for further dissection of FPR2-regulated activities in vitro and in vivo. Potent and stable FPR ligands such as Act-389949 may therefore be used to develop the next generation of FPR signaling regulating anti-inflammatory therapeutics.

Functional selective FPR1 signaling in favor of an activation of the neutrophil superoxide generating NOX2 complex

“…3D). Taken together, these data show that RE-04-001 is a very potent stimulus that activates the neutrophil NADPHoxidase and this activation is achieved through signals specifically generated by FPR1.It is well known that the NADPH-oxidase activity triggered by FPR-specific agonists is substantially increased in TNF primed neutrophils 34,44. Accordingly, the amount of O 2 − produced by TNF primed cells was substantially increased with RE-04-001 as the activating FPR agonist(Fig.…”

“…7C), the peptides earlier shown to be ROS sensitive. 34,57 No inhibition was seen when Cmp43 and Act-389949…”

“…7) results also in agreement with earlier findings. 34,58 Compared to the FPR1 selective peptide agonist fMLF, RE-04-001 was fairly resistant to the MPO-H 2 O 2 -halide radical system (Fig. 7C).…”

“…15,37,56 In line with this, we have earlier shown that the MPO-H 2 O 2 -halide system inactivates peptide agonists such as the FPR1 selective fMLF and the FPR2 selective WKYMVM. 34,57 This inactivation is evident from the inability of the oxidized peptides to activate and trigger ROS release from neutrophils. In contrast to the two peptide agonists, the two small compound agonists Cmp43 (dual FPR agonist) and Act-389949 (FPR2 selective) are completely resistant to the MPO-H 2 O 2 -halide radical system.…”

“…In contrast to the two peptide agonists, the two small compound agonists Cmp43 (dual FPR agonist) and Act-389949 (FPR2 selective) are completely resistant to the MPO-H 2 O 2 -halide radical system. 34,58 To study the inhibitory effect on RE-04-001 of the MPO- (Fig. 7C), the peptides earlier shown to be ROS sensitive.…”