Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20

Patrycja Kleczkowska, Engin Bojnik, Anna LeAniak, Piotr Kosson, Isabelle Van Den Eynde, Steven Ballet, Sandor Benyhe, Dirk Tourwe, Andrzej W. Lipkowski

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Background: Recently, we presented a novel compound (PK20, Dmt-D-Lys-Phe-Phe-Lys-Lys-Pro-Phe-Tle-Leu-OH) that targets single entity opioid and neurotensin pharmacophores. This endomorphin-2-like opioid peptide was introduced as a highly active analgesic because it elicited a strong dose- and time-dependent antinociceptive response when administered centrally and peripherally. Its pain-relieving activity was observed as rapidly as 5 min after drug injection. Such promising results led us to perform further studies, such as determining the resistance to enzymatic degradation, which resulted in obtaining a very stable opioid pharmacore PK20 metabolite. Methods: The synthesis of PK20 and its N-terminal tetrapeptide fragment has been accomplished using solid phase peptide chemistry. The biological stability of peptides has been measured in human serum and analyzed by HPLC/MS. Peptides were pharmacologically characterized in in vitro MOP and DOP receptor binding as well as [35S]GTP?S receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications. Results: Dmt-D-Lys-Phe-Phe-OH (PK20M), an N-terminal tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20, is characterized by a long duration of action, as demonstrated by a preserved, long-lasting analgesic effect even 2 h post-injection (average % MPE = 69.33). In rat brain membranes, PK20M efficiently displaced both the MOP and DOP receptor selective radioprobes [3H]DAMGO and [3H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [35S]GTP?S binding, proving full agonism at both receptor types. In the [35S]GTP?S assay, which measured the agonist-mediated G protein activation, PK20M together with PK20 and Met-enkephalin were potent stimulators of the regulatory G proteins. The relative affinities of PK20M for the ? and receptor subtypes revealed ?-receptor selectivity. Conclusion: The novel MOP receptor selective metabolite has been shown to possess opioid subtype receptor selectivity, high potency, and effective analgesic activities as measured in various bioassays.

Original languageEnglish
Pages (from-to)836-846
Number of pages11
JournalPharmacological Reports
Issue number4
Publication statusPublished - 2013



  • Antinociception
  • In vivo analgesia
  • Opioid receptor binding
  • PK20 metabolite
  • [35S]GTP?S

ASJC Scopus subject areas

  • Pharmacology

Cite this

Kleczkowska, P., Bojnik, E., LeAniak, A., Kosson, P., Van Den Eynde, I., Ballet, S., Benyhe, S., Tourwe, D., & Lipkowski, A. W. (2013). Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20. Pharmacological Reports, 65(4), 836-846.