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Opiate alkaloids, such as morphine, are powerful analgesic agents that are the drugs of choice for the treatment of severe pain. The pharmacological effects of opiates are mediated through the binding and activation of membrane-bound opioid receptors that are found in the central and peripheral nervous systems. Opioid receptors have been classified into three different types, mu, delta and kappa, and are activated by the specific ligands. It has been demonstrated that the most potent antinociceptive effects are mediated by the mu-receptor. However, until 1997 no endogenous ligand for this receptor was known. The identification of endomorphins opened a new era in the research of the mu-opioid system. They are the first reported brain peptides that label mu-receptor with high affinity and selectivity and therefore are proposed as the endogenous mu-opioid receptor ligands. Morphine and endomorphins act as agonists at the same mu-opioid receptor, but the latter are thought to inhibit pain without some of the undesired side-effects of plant opiates. This observation encouraged extensive studies on the possible use of endomorphin analogs as analgesics instead of morphine. This review summarizes a decade of research on structure-activity relationship studies of endomorphin analogs, aimed at obtaining compounds with increased bioavailability, in particular with better barrier penetration and resistance against enzymatic degradation. Chemical modifications that led to obtaining potent and selective agonists and antagonists based on the structure of endomorphins are discussed.

Novel endomorphin-2 (EM-2) analogs have been synthesized, incorporating unnatural amino acids with six-membered heterocyclic rings, such as piperidine-2-, 3- and 4-carboxylic acids (Pip, Nip and Inp, respectively) instead of Pro in position 2. [(R)-Nip(2)]EM-2 displayed an extremely high affinity for the mu-opioid receptor with IC(50) = 0.04 +/- 0.01 nM in comparison with IC(50) = 0.69 +/- 0.03 nM for EM-2. This analog was also very potent in the aequorin luminescence-based functional calcium assay and showed significantly enhanced stability in rat brain homogenate.

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The aim of the present study was to characterize the binding selectivity of the mu-opioid receptor ligands, endomorphin-1, endomorphin-2, and DAMGO, in the in vitro functional assay, based on the changes in intracellular calcium levels. For the experiments Chinese hamster ovary cells, stably expressing human mu-receptor, were used. The mu-agonist-induced calcium responses were significantly inhibited by naloxone, an opioid antagonist with high preference for the mu-opioid receptors. Naloxonazine, a mu1-non-peptide antagonist, inhibited the effect of all tested mu-agonists. However, there was no significant difference in the antagonist effect of naloxonazine on the calcium response induced by mu1- (endomorphin-2) and mu2-agonists (endomorphin-1, DAMGO). [D-Pro2]endomorphin-1 and [D-Pro2]endomorphin-2, putative peptide mu2- and mu1-antagonists, respectively, which had been shown in vivo to inhibit the antinociception induced by mu-agonists, produced no inhibitory effect in our in vitro experiments. Our results demonstrated that there is only one population of the mu-opioid receptors expressed in the Chinese hamster ovary cells. We suggest that the mu-opioid receptors form a homogenous population in the in vitro systems. However, the existence of mu-receptor subtypes in vivo is still pharmacologically possible.