NOVEL BIVALENT PEPTIDES DERIVED FROM Β-ENDORPHIN ANALOGUES TARGET THE ΜU-OPIOID RECEPTOR WITH SIMILAR IN-VITRO POTENCY TO FENTANYL

Aim: Opioids are used for pain management albeit limited by tolerance and addiction potential. Endogenous opioid peptides (EOP), i.e., β-endorphin 1-31 (BE1-31) targeting μ-opioid receptors (MOP), are considered potential safer alternatives. Bivalent peptide design (connecting two ligands via a linker), complemented by D-amino acid substitution, is expected to enhance pharmacological activity and stability. β-endorphin 1-7 (BE1-7), the functional fragment of BE1-31, is selected for bivalent peptide synthesis which are evaluated their activity on MOP and stability in biological media.
Methods: Peptides were synthesized using solid-phase peptide synthesis (SPPS). The activity of peptides were evaluated by determining inhibition of cyclic adenosine monophosphate (cAMP) signaling in HEK293 cells overexpressing MOP. Stability of peptides in rat blood plasma were measured via liquid chromatography mass spectrometry.
Results: BE1-7 shows significantly lower potency compared to fentanyl (0.95±0.02μM and 9.92±1.53nM, n=4; t-test, P<0.05). Bivalent peptides BVE7-523 and BVE7-523D (11.23±1.17nM and 20.55±1.24nM, n=4; t-test, P<0.05), show no significant difference to fentanyl. In stability assays, BVE7-523D demonstrate significant improvement compared to BVE7-523 (t₁/₂ > 120 min versus t₁/₂ = 12.5 min, n=3; t-test, P<0.05).
Conclusions: BVE7-523 demonstrate an increase in potency compared to BE1-7, achieving potency to the fentanyl. The rational bivalent design approach enhances local effective concentration and promotes synergistic activity, increasing the probability of activating MOP. BVE7-523D retained potency while demonstrating improved stability. In summary, these results support the bivalent design alongside D-amino acid substitution as viable strategies to optimize pharmacological activity and stability in the design of novel pain management candidates.