MPN-12-NH2 shows potent antinociception effects by targeting opioid, cannabinoid, and neuropeptide FF receptors.

The neuropeptide FF (NPFF) system plays an important role in regulating the analgesic effects of opioids and cannabis. Incorporating NPFF pharmacophores at the C-terminus of opioid or cannabinoid peptides has emerged as a strategy to enhance analgesic potency while mitigating adverse effects. However, the development of such multi-target compounds has been limited to two systems, with no successful commercialization to date. Hence, we developed the chimeric peptide MPN-12-NH2 by incorporating the opioid peptide morphiceptin, the core sequence of pepcans NFKL, and the NPFF analog PFR(Tic)-NH2. In the mouse tail flick test, intracerebroventricular (i.c.v.) injection of MPN-12-NH2 demonstrated dose-dependent analgesic effects (ED50 = 0.71 nmol/mouse). Furthermore, molecular docking and molecular dynamics (MD) simulations were employed to elucidate the binding patterns of MPN-12-NH2 with opioid receptors, cannabinoid receptors, and neuropeptide receptors. In vitro studies revealed that MPN-12-NH2 is a multifunctional agonist of mu opioid receptors (MOR), delta opioid receptors, cannabinoid 1 receptors (CB1), CB2, and NPFF2 receptors. Subsequently, receptor antagonism studies suggested that the central analgesic effects of MPN-12-NH2 were primarily mediated by MOR, CB1, and NPFF receptors. Furthermore, we found that i.c.v. injection of MPN-12-NH2 dose-dependently alleviated pathological pain responses in the SNI, carrageenan, and acetic acid writhing models. However, MPN-12-NH2 (3 nmol/mouse, i.c.v.) did not result in addiction, analgesic tolerance, or microglial cell alterations in the PAG region. Additionally, MPN-12-NH2 effectively differentiated its analgesic effects on acute pain from its inhibitory effects on gastrointestinal motility, with an ED50 ratio of 14.21-fold. In summary, the multi-target compound MPN-12-NH2 exhibited potent analgesic activity mediated by MOR, CB1, and NPFF receptors in mice, along with a favorable side-effect profile. These findings pave a new way for the development of opioid/cannabinoid/NPFF agonists.