Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue. |
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Authors: | Melinda Ligeti Szilvia Bsze Antal Csmpai
zge GündÜz Mahmud Al‐khrasani Andrs z Rnai Hedvig Medzihradszky‐Schweiger Sndor Benyhe Anna Borsodi Ferenc Hudecz Anna Magyar |
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Institution: | Research Group of Peptide Chemistry at E?tv?s Loránd University, Hungarian Academy of Sciences, Budapest, Hungary. |
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Abstract: | Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue ...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change. |
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Keywords: | carbamic acid residue enzymatic stability aminopeptidase M rat brain membrane homogenate nociceptin NOP receptor radioligand binding GTPγS assay mouse vas deferens bioassay |
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