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Analogues of the argininamide-type NPY Y1 receptor (Y1R) antagonist BIBP3226, bearing carbamoyl moieties at the guanidine group, revealed subnanomolar Ki values and caused depression of the maximal response to NPY (calcium assay) by up to 90% in a concentration- and time-dependent manner, suggesting insurmountable antagonism. To gain insight into the mechanism of binding of the synthesized compounds, a tritiated antagonist, (R)-N(α)-diphenylacetyl-N(ω)-[2-([2,3-(3)H]propionylamino)ethyl]aminocarbonyl-(4-hydroxybenzyl)arginin-amide ([(3)H]UR-MK299, [(3)H]38), was prepared. [(3)H]38 revealed a dissociation constant in the picomolar range (Kd 0.044 nM, SK-N-MC cells) and very high Y1R selectivity. Apart from superior affinity, a considerably lower target off-rate (t1/2 95 min) was characteristic of [(3)H]38 compared to that of the higher homologue containing a tetramethylene instead of an ethylene spacer (t1/2 3 min, Kd 2.0 nM). Y1R binding of [(3)H]38 was fully reversible and fully displaceable by nonpeptide antagonists and the agonist pNPY. Therefore, the insurmountable antagonism observed in the functional assay has to be attributed to the extended target-residence time, a phenomenon of relevance in drug research beyond the NPY receptor field.

The cloning of several receptors activated by either CC or CXC chemokines and belonging to the G protein-coupled family of receptors has been reported recently. In the present work, we describe the cloning of a human gene, named ChemR13, encoding a new CC-chemokine receptor. The gene encodes a protein of 352 amino acids with a calculated molecular mass of 40 600 Da and displaying a single potential site for N-linked glycosylation. Using a set of overlapping lambda clones, the genomic organisation of the locus was investigated, demonstrating that the ChemR13 gene is physically linked, and in the same orientation, as the CC-CKR2 gene that encodes a receptor for the monocyte chemoattractant protein-1 (MCP-1). A distance of 17.5 kb separates the two coding regions, which share 75% identity in nucleic acid and amino acid sequences. Human ChemR13 was functionally expressed in a stably transfected CHO-K1 cell line. Physiological responses to chemokines were monitored using a microphysiometer. Macrophage inflammatory protein 1 alpha (MIP-1 alpha) was the most potent agonist. MIP-1 beta and RANTES were also active at physiological concentrations. The other CC-chemokines, MCP-1, MCP-2 and MCP-3, as well as CXC-chemokines (IL-8, GRO alpha) had no effect. ChemR13 receptor transcripts were detected by Northern blotting in the promyeloblastic cell line KG-1A, suggesting a potential role in the control of granulocytic lineage proliferation or differentiation. ChemR13 is thus a new member of the growing family of chemokine receptors that mediate the recruitment of cells involved in immune and inflammatory processes. Being the fifth functionally identified receptor in his class, this new CC-chemokine receptor (CC-CKR) is tentatively designated CC-CKR5.

The synthesis of aryldiazonium and arylazido derivatives of carfentanil, etonitazene, and naltrexone and of a triazaspirodecane derivative is described. The chemical stability and the spectral characteristics of these compounds were verified, and their binding affinity constants for the different opioid receptor classes were determined, in the absence of light, from competition experiments. With the exception of the naltrexyl derivatives, which remained nonselective, all compounds tested displayed a pronounced mu-binding selectivity with mu/delta and mu/kappa ratios ranging from 12 to 1000. After irradiation, only the arylazido probes led to an irreversible mu-binding-site inactivation. This inactivation fulfilled the criteria for photoaffinity labeling such as protection against inactivation by other opiate ligands and absence of an effect of scavengers on the extent of the inactivation. Most of the photoactivatable probes formed long-lasting reversible complexes with the opioid binding sites: an efficient dissociation procedure was thus required to discriminate between pseudoirreversible and covalent complexes. The marked differences in labeling efficacy between aryldiazonium salts and their corresponding arylazido derivatives are discussed.