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.
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]GTPyS receptor binding assays. Antinociceptive properties of compounds were measured by in vivo assays in C57Bl6 mice after intravenous or intrathecal applications.
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 radio-probes [3H]DAMGO and [3H]DIDI (pKi of 9.52 and 7.86, respectively) and potently stimulated [35S]GTPyS binding, proving full agonism at both receptor types. In the [35S]GTPyS 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 μ and δ receptor subtypes revealed μ-receptor selectivity.
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.
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Bojnik E, Magyar A, Tóth G, Bajusz S, Borsodi A, Benyhe S: Binding studies of novel non-mammalian en-kephalins, structures predicted from frog and lungfish brain cDNA sequences. Neuroscience, 2009, 158, 867–874.
Cegielska-Perun K, Bujalska-Zadro¿ny M, Makulska-Nowak HE, Modification of morphine analgesia by venlafaxine in diabetic neuropathic pain model. Pharmacol Rep, 2012, 64, 1267–1275.
Chang KJ, Blanchard SG, Cuatrecasas P: Unmasking of magnesium-dependent high-affinity binding sites for [DAla2, DLeu5]enkephalin after pretreatment of brain membranes with guanine nucleotides. Proc Natl Acad Sci USA, 1983, 80, 940–944.
Checler F, Vincent JP, Kitabgi P: Degradation of neuro-tensin by rat brain synaptic membranes: involvement of metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases. J Neurochem, 1983, 41, 375–384.
Dhawan BN, Cesselin F, Raghubir R, Reisine T, Bradley PB, Porthogese PS, Hamon M: International Union of Pharmacology. XII. Classification of opioid receptors. Pharmacol Rev, 1996, 48, 567–592.
Dickenson AH: Peptides. In: Neurotransmitters, Drugs and Brain function. Ed. Webster RA. John Wiley & Sons Ltd., New York, 2001, 251–264.
Keil B: Specificity of proteolysis. Springer-Verlag, Berlin, 1992.
Kitabgi P, De Nadai F, Rovère C, Bidard JN: Biosynthesis, maturation, release and degradation of neurotensin and neuromedin N. Ann NY Acad Sci, 1992, 668, 30–42.
Kleczkowska P, Kosson P, Ballet S, Van den Eynde I, Tsuda Y, Tourwé D, Lipkowski AW: PK20, a new opioid-neurotensin hybrid peptide that exhibits central and peripheral antinociceptive effects. Mol Pain, 2010, 6, 86.
Lipkowski AW, Cooperative reinforcement of opioid pharmacophores. Pol J Pharmacol Pharm, 1987, 39, 153–164.
Lipkowski AW, Carr DB: Rethinking opioid equivalence. Pain-Clinical Updates, 2002, 10, 1–7.
McDonald J: Opioid receptors. Contin Educ Anaesth Crit Care Pain, 2005, 5, 22–25.
McQuay H: Opioids in pain management. Lancet 1999, 353, 2229–2232.
Misterek K, Maszczynska I, Dorociak A, Gumulka SW, Carr DB, Szyfelbein SK, Lipkowski AW: Spinal co-administration of peptide substance P antagonist potentiates antinociceptive effect of opioid peptide. Life Sci, 1994, 54, 939–944.
Mizoguchi H, Bagetta G, Sakurada T, Sakurada S: Dermorphin tetrapeptide analogs as potent and long-lasting analgesics with pharmacological profiles distinct from morphine. Peptides, 2011, 32, 421–427.
Nevin ST, Kabasakal L, Ötvös F, Tóth G, Borsodi A: Binding characteristics of the novel highly selective delta agonist, [3H]Ile5,6-deltorphin II. Neuropeptides, 1994, 26, 261–265.
Nonaka T, Dohmae N, Hashimoto Y, Takio K: Amino acid sequences of metalloendopeptidases specific for acyl-lysine bonds from Grifola frondosa and Pleurotus ostreatus fruiting bodies. J Biol Chem, 1997, 272, 30032–30039.
Rosenbaum JS, Sadee W: Opiate receptor binding affected by guanine nucleotide. J Biol Chem, 1983, 258, 1419–1422.
Sato T, Sakurada S, Sakurada T, Furuta S, Chaki K, Kisara K, Sasaki Y, Suzuki K: Opioid activities of D-Arg2-substituted tetrapeptides. J Pharmacol Exp Ther, 1987, 242, 654–659.
Tóth G, Kramer M, Sirokman F, Borsodi A, Ronai A: Preparation of [7,8,19,20-3H]naloxone of high specific activity. J Labelled Comp Rad, 1982, 19, 1021–1030.
Vadivelu N, Whitney ChJ, Sinatra RS: Pain pathways and acute pain processing. In: Acute pain management. Eds. Sinatra RS, de Leon-Casasola OA, Viscusi ER, Ginsberg B, Cambridge University Press, New York, 2009, 3–20.
Yaksh TL, Rudy TA: Chronic catheterization of the spinal subarachnoid space. Physiol Behav, 1976, 17, 1031–1036.
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Kleczkowska, P., Bojnik, E., Leśniak, A. et al. Identification of Dmt-D-Lys-Phe-Phe-OH as a highly antinociceptive tetrapeptide metabolite of the opioid-neurotensin hybrid peptide PK20. Pharmacol. Rep 65, 836–846 (2013). https://doi.org/10.1016/S1734-1140(13)71064-8
- in vivo analgesia
- PK20 metabolite
- opioid receptor binding