Fluorescence-labeled neuropeptide Y (NPY) has been used in flow cytometric binding assays for the determination of affinity constants of NPY Y1, Y2, and Y5 receptor ligands. Because the binding of fluorescent NPY is insufficient for competition studies at the human Y4 receptor (hY4R), we replaced Glu-4 in hPP with Lys for the derivatization with cyanine-5. Because cy5-[K(4)]hPP has high affinity (Kd 5.6 nM) to the hY4R, it was used as a probe in a flow cytometric binding assay. Specific binding of cy5-[K(4)]hPP to hY4R was visualized by confocal microscopy. The hY(4)R, the chimeric G protein G(qi5) and mitochondrially targeted apoaequorin were stably coexpressed in CHO cells. Aequorin luminescence was quantified in a microplate reader and by a CCD camera. By application of these methods 3-cyclohexyl-N-[(3-1H-imidazol-4-ylpropylamino)(imino)methyl]propanamide (UR-AK49) was discovered as the first nonpeptidic Y4R antagonist (pKi 4.17), a lead to be optimized in terms of potency and selectivity.

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The quantification of pharmacological parameters at G protein-coupled receptors (GPCRs) is indispensable in drug research but costly and time-consuming when conventional methods are sequentially applied. With neuropeptide Y (NPY) Y(1), Y(2), and Y(5) receptors as model systems, a homogenous flow cytometric method for the simultaneous determination of the affinity, selectivity, and activity of GPCR ligands was developed. Mixtures of cells expressing the receptors of interest and cyanine-labeled NPY as a universal fluorescent Y(1), Y(2), and Y(5) receptor agonist were used. Calcium mobilization was measured in different channels with the aid of fluo-4 and fura red. A combination of dye-loaded HEL-Y(1) and CHO-Y(2)-Galpha(qi5) cells with unloaded HEC-1B-Y(5) cells allowed the simultaneous determination of Y(1), Y(2), and Y(5) receptor selectivity preceded by the Y(1) and Y(2) receptor-mediated response with one and the same sample. The data are in good agreement with those determined by radioligand binding and spectrofluorimetry. The convenient, robust, and inexpensive multiparametric procedure offers a broad range of applications in the pharmacological characterization of GPCR ligands.

Strongly basic groups such as guanidine moieties are crucial structural elements, but they compromise the drug-likeness of numerous biologically active compounds, including ligands of G-protein-coupled receptors (GPCRs). As part of a project focused on the search for guanidine bioisosteres, argininamide-type neuropeptide Y (NPY) Y₂ receptor (Y₂R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, N(G) -acylated argininamides were obtained by guanidinylation with tailor-made mono-Boc-protected N-acyl-S-methylisothioureas. The compounds were investigated for Y₂R antagonism (calcium assays), Y₂R affinity, and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the N(G) -substituted (S)-argininamides showed Y₂R antagonistic activities and binding affinities similar to those of the parent compound, whereas N(G)-acylated or -carbamoylated analogues with a terminal amine were superior (Y₂R: K(i) and K(B) values in the low nanomolar range). This demonstrates that the basicity of the compounds, although 4-5 orders of magnitude lower than that of guanidines, is sufficient to form key interactions with acidic amino acids of the Y₂R. The acylguanidines bind with high affinity and selectivity to Y₂R over the Y₁, Y₄, and Y₅ receptors. As derivatization of the amino group is tolerated, these compounds can be considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y₂R. The results support the concept of bioisosteric guanidine-acylguanidine exchange as a broadly applicable approach to retain pharmacological activity despite decreased basicity.