Despite extensive characterization of the -opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A -opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [35 S]guanosine 5-(␥-thio)triphosphate binding to G␣ i , and is allosterically regulated by coupled G i protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys 7 , Cys 8 ]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the -opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.Opioid receptors are members of the G protein-coupled receptor (GPCR) 2 superfamily and are clinical mainstays for inducing analgesia. Three isoforms of opioid receptors, , ␦, and , have been cloned and are known to couple to G i/o proteins to regulate adenylyl cyclase and K ϩ /Ca ϩ ion channels(1-3). An ever growing amount of data suggests that many GPCRs oligomerize (4, 5), and several studies have suggested that -opioid receptors (MORs) and ␦-opioid receptors heterodimerize to form unique ligand binding and G protein-activating units (6 -10). Although intriguing, these studies utilize cellular overexpression systems where it is difficult to know the exact nature of protein complexes formed between the receptors.To study the function of isolated GPCRs, our laboratory and others have utilized a novel phospholipid bilayer reconstitution method (11-16). In this approach purified GPCRs are reconstituted into the phospholipid bilayer of a high density lipoprotein (HDL) particle. The reconstituted HDL (rHDL) particles are monodispersed, uniform in size, and preferentially incorporate a GPCR monomer (14, 15). Previous work in our lab has shown that rhodopsin, a class A GPCR previously proposed to function as a dimer (17)(18)(19), is fully capable of activating its G protein when reconstituted as a monomer in the rHDL lipid bilayer (15). Moreover, we have demonstrated that agonist binding to a monomeric ␤ 2 -adrenergic receptor, another class A GPCR, can be allosterically regulated by G proteins (14). This led us to determine whether a monomer of MOR, a class A GPCR that endogenously binds peptide ligands, is the minimal functional unit required to activate coupled G proteins. We ...

We have previously described opioid peptidomimetic, 1, employing a tetrahydroquinoline scaffold and modeled on a series of cyclic tetrapeptide opioid agonists. We have recently described modifications to these peptides that confer a mu opioid receptor (MOR) agonist, delta opioid receptor (DOR) antagonist profile, which has been shown to reduce the development of tolerance to the analgesic actions of MOR agonists. Several such bifunctional ligands have been reported, but none has been demonstrated to cross the blood brain barrier. Here we describe the transfer of structural features that evoked MOR agonist/DOR antagonist behavior in the cyclic peptides to the tetrahydroquinoline scaffold and show that the resulting peptidomimetics maintain the desired pharmacological profile. Further, the 4R diastereomer of 1 was fully efficacious and approximately equipotent to morphine in the mouse warm water tail withdrawal assay following intraperitoneal administration and thus a promising lead for the development of opioid analgesics with reduced tolerance.

AAH8 may be a better alternative than traditional opioid analgesics, producing antinociception with less development of tolerance and dependence and may be less rewarding than morphine.

We have previously reported a small series of mixed efficacy μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist opioid peptidomimetics featuring a tetrahydroquinoline (THQ) scaffold, and showed the promise of this series as effective analgesics after intraperitoneal administration in mice. We report here expanded SAR of the pendant region of these compounds and focus in particular on the incorporation of heteroatoms into this side chain. These analogues provide new insight into the binding requirements for this scaffold at MOR, DOR, and the κ opioid receptor (KOR), and several (10j,k,m,n) significantly improve upon the overall MOR agonist/DOR antagonist profile of our previous compounds. In vivo data for 10j,k,m,n are also reported, and show the antinociceptive potency and duration of action of compounds 10j and 10m to be comparable to morphine.

In a previously described peptidomimetic series, we reported the development of bifunctional µ-opioid receptor (MOR) agonist and δ-opioid receptor (DOR) antagonist ligands with a lead compound that produced antinociception for 1 h after intraperitoneal administration in mice. In this paper, we expand on our original series by presenting two modifications, both of which were designed with the following objectives: 1) probing bioavailability and improving metabolic stability, 2) balancing affinities between MOR and DOR while reducing affinity and efficacy at the Κ-opioid receptor (KOR), and 3) improving in vivo efficacy. Here we establish that through N-acetylation of our original peptidomimetic series, we are able to improve DOR affinity and increase selectivity relative to KOR while maintaining the desired MOR agonist/DOR antagonist profile. From initial in vivo studies, one compound (14a) was found to produce dose-dependent antinociception after peripheral administration with an improved duration of action of longer than 3 h.