Background. Rubiscolin-6 (amino acid sequence: YPLDLF) is an opioid peptide derived from the plant enzyme Rubisco and Soymorphin-6 (peptide sequence: YPFVVA) is an opioid peptide derived from the enzymatic digestion of soy proteins. In this work we investigated the pharmacological activities of Rubiscolin-6, Soymorphin-6 and their new C-terminal amides in vitro using cells expressing the human recombinant delta and mu opioid receptors and in vivo using the mouse tail flick and formalin tests. Methods. In in vitro experiments, we evaluated the capability of Rubiscolin-6, Soymorphin-6 and their new C-terminal amides to activate the mu- and delta-human recombinant receptors permanently transfected in CHO cells. In such cells, the expression of a chimeric G protein that forces the opioid receptors to couple with the calcium pathway allows measuring receptor activation with an automated calcium mobilization assay. In in vivo experiments, the tail flick test was used to determinate antinociceptive response induced by a thermal stimulus and compounds were injected at 10 µg/10µL for intracerebroventricular (i.c.v.) administrations. In the formalin test, which measured the response to inflammatory pain, compounds were administered subcutaneously in the dorsal surface of the right hind paw of the mouse at the dose of 100 µg/20 µL. Results. In the calcium mobilization assay, Rubiscolin-6 C-amide, Soyamorphin-6 and Soyamorphin-6 C-amide stimulated calcium mobilization in mu expressing cells only at micromolar concentrations while Rubiscolin-6 was completely inactive. In cells expressing the delta receptor, all compounds showed an incomplete concentration response curve, being active only at micromolar concentrations. The pharmacological parameters obtained with these ligands in CHO cells are summarized in Table 1. In the tail flick test, Rubiscolin-6 induced a significant but transient antinociceptive effect whereas Rubiscolin-6 C-amide induced a statistically significant, prolonged antinociceptive effect (Figure 1, left panel). Soymorphin-6 and Soymorphin-6 C-amide exerted significant antinociceptive until 30 min after the administration (Figure 1, right panel). In the formalin test, Rubiscolin-6 was not able to change the nociceptive effect of formalin whereas Rubiscolin-6 C-amide reduced formalin-induced nociception in both test phases (Figure 2, left panel). Soymorphin-6 and Soymorphin-6 C-amide did not change the nociceptive effects of formalin (Figure 2, right panel). Conclusions. In conclusion, we found Rubiscolin-6 derivative C-terminal amide able to exert strong antinociceptive effect after central and subcutaneous administration despite its low agonist potency at opioid receptors. These results push us to further investigate Rubiscolin-6 amide mechanism of action in other experimental paradigms and design other derivatives more active in animal models of pain and inflammation.

Pharmacological activities of plant-derived opioid peptides rubiscolin-6, soymorphin-6 and their c-terminal amide derivatives / Minosi, P; Dimmito, Mp; Stefanucci, A; Sturaro, C; Calò, G; Pieretti, S; Mollica, A. - (2021). (Intervento presentato al convegno 40° Congresso Nazionale SIF. SIF - SOCIETÀ ITALIANA DI FARMACOLOGIA tenutosi a Modalità telematica) [10.36118/pharmadvances.03.2021.01].

Pharmacological activities of plant-derived opioid peptides rubiscolin-6, soymorphin-6 and their c-terminal amide derivatives.

Minosi P;Pieretti S;Mollica A
2021

Abstract

Background. Rubiscolin-6 (amino acid sequence: YPLDLF) is an opioid peptide derived from the plant enzyme Rubisco and Soymorphin-6 (peptide sequence: YPFVVA) is an opioid peptide derived from the enzymatic digestion of soy proteins. In this work we investigated the pharmacological activities of Rubiscolin-6, Soymorphin-6 and their new C-terminal amides in vitro using cells expressing the human recombinant delta and mu opioid receptors and in vivo using the mouse tail flick and formalin tests. Methods. In in vitro experiments, we evaluated the capability of Rubiscolin-6, Soymorphin-6 and their new C-terminal amides to activate the mu- and delta-human recombinant receptors permanently transfected in CHO cells. In such cells, the expression of a chimeric G protein that forces the opioid receptors to couple with the calcium pathway allows measuring receptor activation with an automated calcium mobilization assay. In in vivo experiments, the tail flick test was used to determinate antinociceptive response induced by a thermal stimulus and compounds were injected at 10 µg/10µL for intracerebroventricular (i.c.v.) administrations. In the formalin test, which measured the response to inflammatory pain, compounds were administered subcutaneously in the dorsal surface of the right hind paw of the mouse at the dose of 100 µg/20 µL. Results. In the calcium mobilization assay, Rubiscolin-6 C-amide, Soyamorphin-6 and Soyamorphin-6 C-amide stimulated calcium mobilization in mu expressing cells only at micromolar concentrations while Rubiscolin-6 was completely inactive. In cells expressing the delta receptor, all compounds showed an incomplete concentration response curve, being active only at micromolar concentrations. The pharmacological parameters obtained with these ligands in CHO cells are summarized in Table 1. In the tail flick test, Rubiscolin-6 induced a significant but transient antinociceptive effect whereas Rubiscolin-6 C-amide induced a statistically significant, prolonged antinociceptive effect (Figure 1, left panel). Soymorphin-6 and Soymorphin-6 C-amide exerted significant antinociceptive until 30 min after the administration (Figure 1, right panel). In the formalin test, Rubiscolin-6 was not able to change the nociceptive effect of formalin whereas Rubiscolin-6 C-amide reduced formalin-induced nociception in both test phases (Figure 2, left panel). Soymorphin-6 and Soymorphin-6 C-amide did not change the nociceptive effects of formalin (Figure 2, right panel). Conclusions. In conclusion, we found Rubiscolin-6 derivative C-terminal amide able to exert strong antinociceptive effect after central and subcutaneous administration despite its low agonist potency at opioid receptors. These results push us to further investigate Rubiscolin-6 amide mechanism of action in other experimental paradigms and design other derivatives more active in animal models of pain and inflammation.
2021
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1546610
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