Structure–Activity Relationship Study of Opiorphin, a Human Dual Ectopeptidase Inhibitor with Antinociceptive Properties

Toward developing new potential analgesics, this first structure–activity relationship study of opiorphin (H-Gln-Arg-Phe-Ser-Arg-OH), a human peptide inhibiting enkephalin degradation, was performed. A systematic Ala scanning proved that Phe3 is a key residue for neprilysin and aminopeptidase N (AP-N) ectoenkephalinase inhibition. A series of Phe3-halogenated analogues revealed that halogen bonding based optimization strategies are not applicable to this residue. Additional substituted Phe3 derivatives showed that replacing l-Phe3 for d-Phe3 increased the AP-N inhibition potency by 1 order of magnitude. NMR studies and molecular mechanics calculations indicated that the improved potency may be due to CH−π stacking interactions between the aromatic ring of d-Phe3 and the Hγ protons of Arg2. This structural motif is not possible for the native opiorphin and may be useful for the design of further potent and metabolically stable analogues.