Jeffrey D. Daubert

Endomorphins fully activate a cloned human mu opioid receptor.

Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin‐1 and endomorphin‐2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin‐stimulated increase of cAMP in a dose‐dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein‐activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose‐dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [d‐Ala2,N‐Me‐Phe4,Gly‐ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G‐protein‐activated inwardly rectifying potassium (GIRK) channels.

Antipruritic and antihyperalgesic actions of loperamide and analogs

Loperamide and three of its analogs were evaluated for their ability to inhibit binding to cloned human opioid receptor subtypes and to produce antipruritus and antinociception following local s.c. administration to rodents. All four compounds were fully efficacious agonists with affinities of 2 to 4 nM for the cloned human mu opioid receptor. Local s.c. injection of loperamide, ADL 01-0001 or ADL 01-0002 at the same site as the introduction of the pruritogenic compound 48/80 resulted in antipruritic activity in a mouse model of itch. Similarly, i.paw or i.pl. administration of compounds ADL 01-0001, ADL 01-0002 and ADL 01-0003 to inflamed paws caused potent antinociception, inhibiting late phase formalin-induced flinching, Freunds adjuvant-induced mechanical hyperalgesia and tape stripping-induced mechanical hyperalgesia. Loperamide and its analogs were efficacious in animal models of itch and inflammatory pain, and may have potential therapeutic utility as antipruritic and antihyperalgesic agents.

Arylacetamides as peripherally restricted kappa opioid receptor agonists.

Analogues of the kappa (kappa) opioid receptor agonist, ICI 199441, were prepared. Ki values for these analogues at the cloned human kappa opioid receptor ranged from 0.058 to 25 nM. Trifluoromethylaryl derivatives were potent analgesics when administered subcutaneously in the rat and were more peripherally restricted than the parent compound, ICI 199441.

Structure-activity relationships of dynorphin a analogues modified in the address sequence.

The peptide [Pro3]Dyn A(1-11)-NH2 2 exhibits high affinity (K(i) = 2.4 nM) and over 2000-fold selectivity for the opioid receptor. Stepwise removal of the C-terminal residues from this ligand demonstrated that its positively charged Arg residues, particularly Arg6 and Arg7, were crucial for binding to the kappa receptor. Analogues shorter than seven amino acids lacked significant affinity for opioid receptors. Comparison with a series of truncated analogues of Dyn A showed that the relative losses in binding potency differed only slightly between the two series. The neutral residues Ile8 and Pro10 could be removed without significant loss in affinity for the kappa receptor. Their replacement, in the Pro3 analogue, with additional Arg residues led to analogues with improved kappa affinity (e.g., [Pro3,Arg8]Dyn A(1-11)-NH2 20: K(i)(kappa) = 0.44 nM). This type of modification did not compromise the high kappa selectivity of the Pro3 analogues. These findings support the view that a negatively charged domain in the putative second extracellular loop of the kappa receptor selectively recognizes residues 6-11 of dynorphin through electrostatic interactions. As with parent compound 2, analogue 20 and related compounds displayed kappa antagonist properties.

Pharmacological Characterization of Human κ / μ Opioid Receptor Chimeras that Retain High Affinity for Dynorphin A

Arylacetamide analgesics that stimulate k opioid receptors in the central nervous system mediate dysphoria and psychosis as well as analgesia. However, the naturally occurring peptide agonist, dynorphin A, is analgesic in the absence of dysphoria and psychosis, indicating that the therapeutic effects of k opioid agonists may be separated from their side effects. As part of our effort to discover κ opioid receptor analgesics lacking side effects, we designed and constructed two μ/κ chimeric receptors, composed primarily of amino acid residues derived from the μ opioid receptor, that were expected to bind dynorphin A with high affinity. In one, extracellular loop 2 and transmembrane domain 4 were derived from the κ opioid receptor and in the other, only extracellular loop 2 was derived from the κ opioid receptor. Most competitors of [3H]diprenorphine binding from a variety of structural classes bound to the chimeras with affinities similar to those with which they bound to the m opioid receptor. In contrast, dynorphin A analogs bound to the chimeras with affinities similar to those with which they bound to the κ opioid receptor. Pharmacological characterization of [35S]GTPgS binding mediated by the chimera with extracellular loop 2 derived from the κ opioid receptor showed that it behaved as if it were m opioid receptor with high affinity for dynorphin A analogs. These chimeras may be useful in identifying novel κ receptor agonists that bind to the second extracellular loop of the receptor and share the desirable therapeutic profile of dynorphin A.

[3H] 1,3-di(2-tolyl) guanidine binds to a σ2 receptor on Jurkat cell membranes, but σ compounds fail to influence immunomodulatory events in human peripheral blood lymphocytes

Abstract The binding characteristics of the σ ligand [3H] 1,3-di(2-tolyl)guanidine (DTG) were investigated in membranes prepared from the Jurkat T cell line. Binding was saturable with a KD of 56 ± 3 nM and a Bmax of 11706 ± 3173 fmol/mg protein (n = 3). The rank order of potency for σ reference compounds to inhibit binding in the Jurkat cell line was ifenprodil > 1,3-di(2-tolyl)guanidine > haloperidol > carbetapentane > (+)3-(3-hydroxyphenyl)-N-propylpiperidine ((+)3-PPP) > (−)pentazocine > caramiphen > (+)pentazocine, and significantly correlated with potency at σ2 binding sites in guinea pig brain (r= 0.90, p

Use of receptor chimeras to identify small molecules with high affinity for the dynorphin A binding domain of the κ opioid receptor

A series of 2-substituted sulfamoyl arylacetamides of general structure 2 were prepared as potent kappa opioid receptor agonists and the affinities of these compounds for opioid and chimeric receptors were compared with those of dynorphin A. Compounds 2e and 2i were identified as non-peptide small molecules that bound to chimeras 3 and 4 with high affinities similar to dynorphin A, resulting in K(i) values of 1.5 and 1.2 nM and 1.3 and 2.2 nM, respectively.