Signaling Modulation via Minimal C-Terminal Modifications of Apelin-13

Apelin is an endogenous peptide that is involved in many diseases such as cardiovascular diseases, obesity, and cancer, which has made it an attractive target for drug discovery. Herein, we explore the penultimate and final sequence positions of [Pyr¹]-apelin-13 (Ape13) via C-terminal N _α-alkylated amide bonds and the introduction of positive charges, potentially targeting the allosteric sodium pocket, by assessing the binding affinity and signaling profiles at the apelin receptor (APJ). Synthetic analogues modified within this segment of Ape13 showed high affinity (K _i 0.12-0.17 nM vs Ape13 K _i 0.7 nM), potent Gα_i1 activation (EC₅₀ Gα_i1 0.4-0.9 nM vs Ape13 EC₅₀ 1.1 nM), partial agonist behavior disfavoring β-arrestin 2 recruitment for positively charged ligands (e.g., 49 (SBL-AP-058), EC₅₀ β-arr2 275 nM, E _max 54%) and high plasma stability for N-alkyl ligands (t _1/2 > 7 h vs Ape13 t _1/2 0.5 h). Combining the benefits of the N _α-alkylated amide bond with the guanidino substitution in a constrained ligand led to 63 (SBL-AP-049), which displayed increased plasma stability (t _1/2 5.3 h) and strong reduction of β-arrestin 2 signaling with partial maximal efficacy (EC₅₀ β-arr 864 nM, E _max 48%), significantly reducing the hypotensive effect in vivo.