Maria Sbraccia

Morphine-like Opiates Selectively Antagonize Receptor-Arrestin Interactions

The addictive potential of opioids may be related to their differential ability to induce G protein signaling and endocytosis. We compared the ability of 20 ligands (sampled from the main chemical classes of opioids) to promote the association of μ and δ receptors with G protein or β-arrestin 2. Receptor-arrestin binding was monitored by bioluminescence resonance energy transfer (BRET) in intact cells, where pertussis toxin experiments indicated that the interaction was minimally affected by receptor signaling. To assess receptor-G protein coupling without competition from arrestins, we employed a cell-free BRET assay using membranes isolated from cells expressing luminescent receptors and fluorescent Gβ1. In this system, the agonist-induced enhancement of BRET (indicating shortening of distance between the two proteins) was Gα-mediated (as shown by sensitivity to pertussis toxin and guanine nucleotides) and yielded data consistent with the known pharmacology of the ligands. We found marked differences of efficacy for G protein and arrestin, with a pattern suggesting more restrictive structural requirements for arrestin efficacy. The analysis of such differences identified a subset of structures showing a marked discrepancy between efficacies for G protein and arrestin. Addictive opiates like morphine and oxymorphone exhibited large differences both at δ and μ receptors. Thus, they were effective agonists for G protein coupling but acted as competitive enkephalins antagonists (δ) or partial agonists (μ) for arrestin. This arrestin-selective antagonism resulted in inhibition of short and long term events mediated by arrestin, such as rapid receptor internalization and down-regulation.

Mutations inducing divergent shifts of constitutive activity reveal different modes of binding among catecholamine analogues to the β2-adrenergic receptor

We compared the changes in binding energy generated by two mutations that shift in divergent directions the constitutive activity of the human β2 adrenergic receptor (β2AR). A constitutively activating mutant (CAM) and the double alanine replacement (AA mutant) of catechol‐binding serines (S204A, S207A) in helix 5 were stably expressed in CHO cell lines, and used to measure the binding affinities of more than 40 adrenergic ligands. Moreover, the efficacy of the same group of compounds was determined as intrinsic activity for maximal adenylyl cyclase stimulation in wild‐type β2AR. Although the two mutations had opposite effects on ligand affinity, the extents of change were in both cases largely correlated with the degree of ligand efficacy. This was particularly evident if the extra loss of binding energy due to hydrogen bond deletion in the AA mutant was taken into account. Thus the data demonstrate that there is an overall linkage between the configuration of the binding pocket and the intrinsic equilibrium between active and inactive receptor forms. We also found that AA mutation‐induced affinity changes for catecholamine congeners gradually lacking ethanolamine substituents were linearly correlated to the loss of affinity that such modifications of the ligand cause for wild‐type receptor. This indicates that the strength of bonds between catechol ring and helix 5 is critically dependent on the rest of interactions of the β‐ethanolamine tail with other residues of the β2‐AR binding pocket.

Different Structural Requirements for the Constitutive and the Agonist-induced Activities of the β2-Adrenergic Receptor

We converted Ser-207, located in helix 5 of the β2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to Gs activation, we expressed the mutants in a Gαs-tethered form. GTP exchange in such constructs is restricted to the fused α-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.

Synthesis and structure-activity relationship studies in serotonin 5-HT4 receptor ligands based on a benzo[de][2,6]naphthridine scaffold.

A small series of serotonin 5-HT4 receptor ligands has been designed from flexible 2-methoxyquinoline compounds 7a,b by applying the conformational constraint approach. Ligands 7a,b and the corresponding conformationally constrained analogues 8a-g were synthesized and their interactions with the 5-HT4 receptor were examined by measuring both binding affinity and the ability to promote or inhibit receptor-G protein coupling. Ester derivative 7a and conformationally constrained compound 8b were demonstrated to be the most interesting compounds showing a nanomolar 5-HT4R affinity similar to that shown by reference ligands cisapride (1) and RS-23,597-190 (4). The result was rationalized by docking studies in term of high similarity in the binding modalities of flexible 7a and conformationally constrained 8b. The intrinsic efficacy of some selected ligands was determined by evaluating the receptor-G protein coupling and the results obtained demonstrated that the nature and the position of substituents play a critical role in the interaction of these ligands with their receptor.

Synthesis and pharmacological evaluation of bivalent antagonists of the nociceptin opioid receptor

Bivalent ligands constituted by two identical pharmacophores structurally related to the Nociceptin Opioid Receptor (NOPr) antagonist JTC-801 were synthesized and their binding affinities for NOPr were evaluated. The novel ligands are formed by two modified JTC-801 units linked by di-iminic and di-aminic spacers with length ranging from three to ten methylene units. Moreover, the synthesis and the pharmacological characterization were extended to the corresponding univalent ligands. The latter compounds consisted in a single modified JTC-801 unit and an alkyl or alkylamino or alkylimino tail. The purpose of this study is to feature the location and surroundings of the allosteric binding site(s) of pharmacophores containing the 4-aminoquinoline structure. Most important, the bivalent ligands were exploited to reveal the eventual occurrence of a supramolecular receptorial architecture of the NOPr. All the bivalent derivatives 4 and 5 proved to be active in the nanomolar range with no outstanding dependence on the chain length. They showed potencies from three to ten times higher than the corresponding monomers. Consequently, results clearly indicated a positive role of the second pharmacophore in the ligand-protein interaction. The pharmacological profile of the monomers 7 and 8 clarified the contribution of the linker chain to NOP receptor affinity and suggested the presence of a lipophilic acidic site neighbouring the binding site of the JTC-like ligands. Selectivity of saturated compounds 5, 7, and 8 was tested by binding experiments on δ, κ and μ opioid receptors. Results indicated a general loss of selectivity as compared to JTC-801. In the [(35)S]GTPγS binding assay, all the compounds revealed antagonistic properties at the NOP Receptor. In conclusion the present study set the basis for a systematic investigation on the structural modifications that can be introduced into novel ligands for NOPr and helped to feature the surrounds of the allosteric site of NOPr.

In vitro quantitative determination of phospholipid adducts of chloroform intermediates in hepatic and renal microsomes from different rodent strains.

We have comparatively studied in vitro the oxidative and reductive pathways of chloroform metabolism in hepatic and renal microsomes of rodent strains used for carcinogenicity testing (B6C3F1 mice, Osborne Mendel and Sprague Dawley rats). To this aim we exploited the regioselective binding of phosgene to phospholipid (PL) polar heads and of dichloromethyl radical to PL fatty acyl chains, using a method based on the chemical transmethylation of PL adducts, followed by phase partitioning of the resulting products (De Biasi et al., 1992). The analysis of results let us to conclude at first that a (14)C label partitioning by 89.2 (±6.5)% or 13.7 (±5.0)% in the aqueous phase is typical of the PL adduct with phosgene (PL-PHOS) or with dichloromethyl radical (PL-RAD), respectively. Metabolism of 0.1 mM CHCl(3) was mainly oxidative in all the samples, being hepatic microsomes more active than renal ones by about one order of magnitude and levels of CHCl(3)-derived PL adducts in B6C3F1 mouse liver microsomes higher than in rat samples. At 5 mM CHCl(3), total levels of PL adducts in renal microsomes reached levels almost similar to those found in liver microsomes. However, while B6C3F1 mouse kidney microsomes produced both reactive metabolites, similarly as the hepatic samples, Osborne Mendel rat kidney microsomes bioactivated CHCl(3) only reductiveiy, producing the radical. The relevance of this finding depends on the fact that phosgene is known to be the major cause of CHCl(3) toxicity, based on data with the rat liver and mouse liver and kidney, while nephrotoxicity in rats occurs with minimal production of COCl(2). Chloroform reductive bioactivation may therefore provide a reasonable explanation for the toxicity of chloroform to the rat kidney. The same finding may be of interest in elucidating the metabolic reasons of the chloroform-induced kidney tumors in Osborne Mendel rats.

Comparative characterization of CHCl3 metabolism and toxicokinetics in rodent strains differently susceptible to chloroform-induced carcinogenicity

A comparative kinetic study in B6C3F1 mice, Osborne-Mendel (OM) and Sprague-Dawley (SD) rats has been undertaken with the major aim to determine the extent of covalent binding of chloroform reactive metabolites produced in vivo through oxidative and/or reductive metabolism in the target organs of chloroform carcinogenicity. Some additional kinetic observations of chloroform biotransformation were also collected comparatively. Expiration of [14C]-CO(2) showed that chloroform metabolism went to saturation in all tested rodent strains. In the B6C3F1 mouse maximal rates of approximately 135 µmol [14C]-CO(2)/kg b.w./h were reached at a dose of approximately 150 mg/kg, while in the two rat strains saturation occurred at a dose of approximately 60 mg/kg, with a maximal rate of approximately 40 µmol [14C]-CO(2)/kg b.w./h. At doses of 150-180 mg/kg b.w., limited differences were found in the distribution and elimination of [14C]-chloroform in the liver and kidney. Species differences have been found in the kinetics of alkali-extractable radioactivity in the blood. The levels of adducts of electrophilic intermediates with the polar heads (PH) of phospholipids (PL) showed a limited variability accross the rodents tested and did not correlate with the species and organ susceptibility to chloroform carcinogenicity. The levels of adducts of radical intermediates with the fatty acyl chains (FC) of PL were much lower than the PH adducts in all the samples analyzed; at the carcinogenicity bioassay doses, statistically significant levels of hepatic FC adducts were present only in the B6C3F1 mouse, where chloroform is hepatocarcinogenic. The observations in the rat kidney were suggestive of the formation of electrophilic reactive metabolites, presumably different from phosgene and associated with an initial chloroform reduction.