Abstract
Orexigenic hormone ghrelin is secreted in stomach at fasting and targets growth hormone secretagogue receptor (GHSR) in hypothalamus and brainstem for its orexigenic effect. Recently, liver enriched antimicrobial peptide-2 (LEAP2) was identified as an endogenous high affinity GHSR antagonist. LEAP2 is a 40-peptide with two disulfide bridges in the central part and GHRS affinity in the N-terminal hydrophobic part. In this study, we tested modified LEAP2 N-terminal 14-peptide and its lipidized analogs for their affinity to and activation of GHSR and for anorexigenic effect after acute peripheral administration. The following substances were designed, synthesized and employed: ([Nle1, βAla15, K16-NH₂]LEAP2(1-14)) and its myristoylated ([Nle1, βAla15, K (εN-myr)16-NH₂]LEAP2(1-14)), palmitoylated ([Nleu1, βAla15, K (εN-palm)16-NH₂]LEAP2(1-14)) and stearoylated ([Nle1, βAla15, K (εN-stear)16-NH₂]LEAP2(1-14)) analogs. LEAP2 14-peptide and its lipidized analogs bound to GHSR with affinity similar to natural LEAP2, and lipidization enhanced affinity of LEAP2 14-peptide to GHSR significantly. Using beta-lactamase reporter gene response, GHSR activation was studied. GHSR natural agonist ghrelin activated the receptor with nanomolar EC50. LEAP2 14-peptide and its lipidized analogs behaved as inverse agonists of GHSR and suppressed internal activity of the receptor with EC50 in 10^(-8) M range. Unlike myristoylated LEAP2 14-peptide, non-lipidized, palmitoylated and stearoylated ones lowered significantly acute food intake in overnight fasted mice. In free fed mice, all LEAP2 analogs significantly decreased orexigenic effect of stable ghrelin analog [Dpr3]ghrelin. Palmitoylated LEAP2 14-peptide analog had the most pronounced anorexigenic effect, stability in rat plasma, and affinity to GHSR. Such properties make palmitoylated LEAP2 14-peptide a promising substance for anti-obesity treatment.