Two-Step Design of Triazole-Stapled Opioid Agonists Exhibiting Potent and Prolonged Peripheral Analgesic Activity

Abstract

This study establishes a two-step strategy for developing triazole-stapled multifunctional agonists that integrate opioid and neuropeptide FF (NPFF) pharmacophores. Initially, calcium mobilization assays, enzymatic stability tests, and analgesic activity screens identified triazole-stapled opioid peptide 16, derived from the opioid pharmacophore of the multifunctional opioid/NPFF agonist DN-9. Subsequently, chimeric conjugation of 16 with the NPFF pharmacophore yielded the novel multitarget analgesic agonist, 16a. Functional assays demonstrated that 16 functions as a μ-opioid receptor (MOR) agonist and a δ-opioid receptor partial agonist, while 16a exhibits multitarget activity at MOR, κ-opioid receptor, and NPFF receptors, with G-protein-biased signaling at MOR. Pharmacological evaluations revealed that subcutaneous administration of both compounds elicited sustained dose-dependent antinociceptive effects across acute and visceral pain models. Notably, these compounds exhibited diminished propensity for addiction and respiratory depression and furthermore produced nontolerant antinociception via an NPFF receptor-independent pathway. This strategy offers a promising framework for developing long-lasting analgesics.