3-(1H-Indol-3-yl)-2-[3-(4-nitrophenyl)ureido]propanamide Derivatives are Agonists of Human Formyl Peptide Receptor 2

N-formyl peptide receptor (FPR1) and FPR2 are G protein-coupledreceptors (GPCR) involved in host defense and sensing cellular dysfunction. Previously we found that antagonists of gastrin-releasing peptide/neuromedin B receptors (BB1/BB2), PD168368 and PD176252, were potent mixed FPR1/FPR2 agonists [Mol. Pharm. (2011) 79: 77-90]. In the present studies, we screened 13 structural derivatives of PD176252 for their ability to activate human neutrophils and HL-60 cells transfected with human FPR1 or FPR2. While none of the compounds had BB2 antagonist activity, five of the compounds stimulated Ca2+ flux in HL-60 cells expressing FPR2, but not in HL-60 cells expressing FPR1, suggesting they were selective for FPR2. The most potent compounds EMY-96 [(R)3-(1H-indol-3-yl)-2-(3-(4-nitrophenyl)ureido)-N-((1-(pyridin-2-yl)cyclohexyl) methyl)propanamide] and its S-isomer ML-16 induced Ca2+ flux with EC50 values in the low micromolar range. Furthermore, pretreatment of FPR2/HL-60 cells with specific the FPR2 antagonist WRW4 prevented Ca2+ flux activated by EMY-96 and ML-16. Interestingly, neither EMY-96 nor ML-16 was able to induce Ca2+ flux in human neutrophils; however, EMY-96 desensitized human neutrophils and FPR2/HL-60 cells to subsequent activation by the hexapeptide WKYMVM. In addition, all five active compounds dose-dependently stimulated human neutrophil chemotaxis. Lastly, these compounds induced β-arrestin binding to FPR2. Thus, these 3-(1H-Indol-3-yl)-2-[3-(4-nitrophenyl)ureido] propanamide derivatives represent unique FPR2 agonists and exhibit novel properties in human neutrophils and FPR-transfected HL-60 cells. This work was supported in part by National Institutes of Health grant P20 RR-020185, National Institutes of Health contract HHSN266200400009C, an equipment grant from the M.J. Murdock Charitable Trust, and the Montana State University Agricultural Experimental Station