The Effect of Pro2 Modifications on the Structural and Pharmacological Properties of Endomorphin-2

Endomorphins (EM-1 and EM-2) are selective, high affinity agonists of the mu-opioid (MOP) receptor, an important target in pain regulation. Their clinical use is impeded by their poor metabolic stability and limited entry to the central nervous system. In this study, the Pro(2) residue of EM-2 was modified systematically through substitution by hydroxyproline (Hyp), (S)-beta-homoproline beta Pro), 2-amino-cyclopentene-1-carboxylic acid (Delta Acpc), or 2-aminocyclohexene-1-carboxylic acid (Delta Achc) to obtain stable MOP active compounds. Both Hyp(2) and beta Pro(2) substitution decreased receptor affinity. Analogues incorporating alicyclic beta-amino acids exhibited diverse receptor binding properties, depending on the configuration of the substituent side-chain. (1S,2R)Delta Acpc(2)-EM-2 was shown to have MOP affinity and selectivity comparable to those of EM-2 and proved to act as agonist while being resistant to proteolysis. NMR and molecular dynamics (MD) studies revealed that bent backbone structures are predominant in the most potent analogues, while their presence is less pronounced in ligands of lower receptor affinity.