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Rat sialorphin (Gln-His-Asn-Pro-Arg) is a natural blocker of neprilysin (NEP) that belongs to the family of endogenous opioid peptide-degrading enzymes. Studies have confirmed the efficiency of sialorphin in blocking the activity of NEP, both in vitro and in vivo. It has been demonstrated that this inhibitor has a strong analgesic, anti-inflammatory, immunological and metabolic effect either directly or indirectly by affecting the level of Met/Leu-enkephalins. In this work, sialorphin and their 12 analogues were synthesised using the solid-phase method. The effect of the peptides on the degradation of Met-enkephalin by NEP and metabolic degradation in human plasma was investigated in vitro. We show that the change in the N-terminal amino acid configuration from L to D in almost all peptides, except D-Arg-His-Asn-Pro-Arg (peptide XI), led to the abolition of their inhibitory activity. With molecular modelling technique we explained the structural properties of the L and D-arginine located on the N-terminal part of the peptide. The detailed analysis of the protein binding pocket allowed us to explain why D-arginine is so unique among all D residues. Peptide XI showed the highest stability among the tested peptides in human plasma. For instance sialorphin after a 2-hour incubation in human plasma was almost completely decomposed, while the level of peptide XI dropped to 45% after 48 h under these conditions.

Enkephalins are involved in a number of physiological processes. However, these peptides are quickly degraded by peptidases, e.g. the neutral endopeptidase (NEP). Inhibition of the enzymatic degradation of enkephalins is one of the possible approaches to prolong their activity. Selective inhibitor of NEP, sialorphin, is the attractive lead compound for enkephalins degradation studies. In this work, an alanine scan of sialorphin and a series of its hybrids with opiorphin, synthesised by the solid phase method, were performed. The effect of the peptides on degradation of Met-enkephalin by NEP in vitro was investigated. Molecular modelling technique was used to identify residues responsible for protein–ligand interactions. We showed that substitution of amino acids Gln1, Pro4 and Arg5 of sialorphin for Ala significantly reduced the half-life of Met-enkephalin in the presence of NEP. [Ala3]sialorphin displayed a higher inhibitory potency against NEP than sialorphin. Substitution of His2 for Ala led to a compound which was as active as lead compound. Sialorphin has a structure which hardly tolerates substitution in its sequence at positions 1, 4 and 5. The conversion of His2 for alanine in sialorphin is tolerated very well. The higher inhibitory potency of [Ala3]sialorphin than sialorphin against NEP is caused by removal of the hydrophilic residue (Asn) and a better fit of the peptide to the enzyme-binding pocket. The role of side chains of sialorphin in degradation of enkephalin by NEP has been explored. This study also provides an important SAR information essential for further drug design.