Abstract
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.
Highlights
Enkephalins, including Met-enkephalin (Tyr–Gly–Gly– Phe–Met) and Leu-enkephalin (Tyr–Gly–Gly–Phe–Leu) are involved in the pain modulating mechanism in the spinal cord (Hughes et al 1975; Noble et al 1992) and in the immune modulating system (Hiddinga et al 1994; Marksteiner et al 1993; Stefano et al 1998)
Sialorphin attenuated acute, semichronic as well as relapsing 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice after systemic administration, its anti-inflammatory action being associated with the mu and kappa opioid receptors (Kamysz et al 2016; Salaga et al 2017)
Systematic Ala scanning of sialorphin (Table 2) showed that substitution of G ln1, Pro4 and A rg5 by Ala led to compounds with significantly reduced half-lives (t1/2) of Met-enkephalin. [Ala2]sialorphin displayed equivalent inhibitory potency for neutral endopeptidase (NEP) as compared to that of sialorphin. [Ala3]sialorphin
Summary
Enkephalins, including Met-enkephalin (Tyr–Gly–Gly– Phe–Met) and Leu-enkephalin (Tyr–Gly–Gly–Phe–Leu) are involved in the pain modulating mechanism in the spinal cord (Hughes et al 1975; Noble et al 1992) and in the immune modulating system (Hiddinga et al 1994; Marksteiner et al 1993; Stefano et al 1998). Pentapeptide sialorphin (Gln–His–Asn–Pro–Arg), was found in submandibular glands and prostate of rats, encoded by rat Vcsa gene It is a selective inhibitor of NEP (Rougeot et al 2003; Messaoudi et al 2004; Davies et al 2007). The compound is active in vivo, showing a pain-suppressive potency similar to morphine in chemical and mechanical rat models of pain (Wisner et al 2006; Tian et al 2009; Rougeot et al 2010) These actions were not accompanied by side effects typical of opioids, such as development of tolerance or addiction (Popik et al 2010; Rougeot et al 2010). Sialorphin attenuated acute, semichronic as well as relapsing 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice after systemic administration, its anti-inflammatory action being associated with the mu and kappa opioid receptors (Kamysz et al 2016; Salaga et al 2017)
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