Daniela Riitano

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.

Design, synthesis, and structure–affinity relationship studies in NK1 receptor ligands based on azole-fused quinolinecarboxamide moieties

The substituent in position 2 of the quinoline nucleus of NK(1) receptor ligands 5 has been constrained into different five-membered heterocyclic moieties in order to obtain information on the binding site pocket interacting with this apparently critical portion of ligands 5. This structure-affinity relationship study led to the discovery of novel tricyclic NK(1) receptor ligands 6 showing affinity in the nanomolar range to the sub-micromolar one. The systematic structure variation suggests that electronic features of the tricyclic moiety play a role in modulating the interaction of these amide derivatives with their receptor.

A Mutation Changes Ligand Selectivity and Transmembrane Signaling Preference of the Neurokinin-1 Receptor

We studied the biochemical properties of a genetically engineered neurokinin-1 receptor (NK1R) in which two residues lying on the extracellular edge of the fourth transmembrane domain were replaced by equivalently located elements of the neurokinin-2 receptor (G166C, Y167F NK1R mutant). The mutation produced two effects. The first is enhancement of the apparent binding affinity for heterologous tachykinins (substance K and neurokinin B) and for N- or C-terminal modified analogues of substance P, but not for substance P itself, its full-length analogues, and several peptide and nonpeptide antagonists. Only two antagonists, as exceptions, were found to exhibit a diminished affinity for the mutant receptor. The second effect is a shift in NK1R preference for distinct G protein-mediated signaling pathways. NK1R-mediated phosphoinositide hydrolysis was enhanced both in transiently and permanently transfected cells, while stimulation of cAMP accumulation did not change in transient expression experiments and was reduced in permanently expressing cells. The effect of the mutation on ligand affinity was not related to any obvious structural commonality, nor to the selectivity for different neurokinin receptors or the agonistic/antagonistic nature of the ligand. However, all ligands responding to the mutation appear to share the ability to induce phosphoinositide signaling more efficiently than cAMP responses when binding to NK1R. We suggest that the mutation shifts the internal equilibria of different functional forms of NK1R. A theoretical analysis according to a multistate allosteric model suggests that the link between binding and biological changes can result from altered stability constants of substates in the conformational space of the receptor.

Non-peptide NK1 receptor ligands based on the 4-phenylpyridine moiety

The quinoline nucleus of the previously described 4-phenylquinoline-3-carboxamides NK(1) receptor ligands 7 has been transformed into either substituted or azole-(i.e., triazole or tetrazole) fused pyridine moieties of compounds 9 and 10, respectively, in order to obtain NK(1) receptor ligands showing lower molecular weight or higher hydrophilicity. The program of molecular manipulations produced NK(1) receptor ligands showing affinity in the nanomolar range. In particular, 4-methyl-1-piperazinyl derivative 9j showed an IC(50) value of 4.8 nM and was proved to behave as a NK(1) antagonist blocking Sar(9)-SP-sulfone induced proliferation and migration of microvascular endothelial cells. Therefore, compound 9j has been labeled with [(11)C]CH(3)I (t(1/2)=20.4 min, β(+)=99.8%) starting from the corresponding des-methyl precursor 9i using with a radiochemical yield of about 10% (not decay corrected) and a specific radioactivity>1 Ci/μmol in order to be used as a radiotracer in next PET studies.

Different mechanisms of negative efficacy. Distinguishing inverse agonists from negative antagonists

Abstract The subject of negative efficacy at seven transmembrane receptors has attracted increasing interest during the past years. We briefly review how that started and the reasons why this phenomenon is commonly interpreted as an indication that some ligands can inhibit the constitutive association between receptors and G proteins. However, as shown for a cannabinoid receptor antagonist, negative efficacy may also be the outcome of ligands that stimulate receptors to lock G protein in nonactive form. We suggest that inhibition of spontaneous receptor activity and stimulation of receptor-mediated G protein sequestration underlie different mechanisms of negative efficacy and justify the distinction between negative antagonists and inverse agonists, respectively. Using a simplified model to describe ligand-induced shifts in guanine nucleotide binding, we propose that these diverse mechanisms of efficacy may correspond to distinct patterns in the way agonists affect the binding properties of the G protein.

Thrombin Receptor Aromatic Residues for Edge-to-Face CH/π Interaction with Ligand Phe-2-phenyl Group

Serine protease thrombin plays an important role in blood coagulation and possesses a specific receptor in the platelets. When thrombin cleaves the peptide bond between Arg-41 and Ser-42 in the N-terminal segment of receptor, the newly exposed N-terrninal peptide segment binds to the receptor itself as a tethered ligand which activates the receptor [1]. Synthetic heptapeptide Ser-Phe-Leu-Leu-Arg-Asn-Pro (SFLLRNP, one letter amino acid codes) corresponding to this tethered-ligand is able to activate the receptor without thrombin, and the Phe-2 residue of this SFLLRNP peptide was found to be essential for receptor recognition and activation. In the present structure-activity studies to elucidate the role of Phe-2 in receptor activation, the benzene hydrogens in the Phe-2-phenyl group were suggested to be in the edge-to-face CH/π interaction with the receptor aromatic groups. Computer modeling of thrombin receptor indicated that the aromatic amino acid cluster in the fifth transmembrane domain (TM5: YYAYYFSAFSAVFFF) is a binding site of this Phe-2-phenyl. In this study, in order to determine the genuine binding site of Phe-2-phenyl, we prepared mutant receptors in which Tyr in TM5 were replaced by Ala.