Interactions between atypical opioid agonists and MOR

Endomorphins (EMs) are endogenous peptides with high selectivity for MOR; they induce strong antinociception by binding to both central and peripheral MOR but, unlike morphine, they are effective in reducing neuropathic pain and their analgesic effect seems to be dissociated by immunomodulatory, cardiovascular and respiratory effects. As native EMs show poor bioavailability and rapid degradation in vivo, we designed and assayed novel EM-1 (YPWF-NH2) derivatives bearing chemical modification aimed to improve their application as analgesics. The ionic bond between a protonated amine and a conserved Asp in the third TMH of the opioid receptor is considered the driving force for ligand-receptor interaction of all opioid agonists, being the amine of Tyr the key pharmacophore for opioid peptides. The removal or derivatization of this pharmacophore usually transforms agonists into inactive compounds or antagonists, with only few compounds maintaining an agonist behaviour when deprived of such amino group. Recently, we discovered the novel EM-1 derivative c[YpwFG]; it displayed good affinity to MOR (Ki 34 nM), is an effective and potent analgesic for visceral pain when administered peripherally (i.p ED50 1,25 mg/kg; s.c. ED50 2,7 mg/kg), and retains central analgesic effects (tail-flick test) only at high doses (20 mg/kg). Interestingly, it triggers MOR internalization similarly to DAMGO but displayed an opposite effect on MOR transcription. This cyclopeptide is a structurally atypical opioid agonist, being deprived of the key pharmacophore, therefore we performed investigations by 2D-NMR, conformational analysis, and molecular docking to provide insights into its interaction with and activation of MOR. The resulting receptor-bound structure served as a general model to design new MOR-active compounds containing the sequence wF, to optimize ligand-receptor interactions. This search lead to c[YGwFG], which showed a 10- fold higher affinity for the MOR (Ki 3,6 nM) as well as good analgesic properties in vivo. Finally, we verified the predictive power of the general model by designing a non-EM-like opioid compound, the cyclic tetrapeptide c[d(1-NH2)beta-AwF]. In summary, these results suggest that alternative interactions might duly replace the electrostatic interaction of the protonated nitrogen with the Asp residue, which has not to be considered a conditio sine qua non for opioid receptor activatio