Ligand recognition and activation of neuromedin U receptor 2.

Neuromedin U receptor 2 (NMU2), an emerging attractive target for treating obesity, has shown the capability in reducing food intake and regulating energy metabolism when activated. However, drug development of NMU2 was deferred partially due to the lack of structural information. Here, we present the cryo-electron microscopy (cryo-EM) structure of NMU2 bound to the endogenous agonist NmU-25 and Gi1 at 3.3 Å resolution. Combined with functional and computational data, the structure reveals the key factors that govern the recognition and selectivity of peptide agonist as well as non-peptide antagonist, providing the structural basis for design of novel and highly selective drugs targeting NMU2. In addition, a 25-degree rotation of Gi protein in reference to NMU2 is also observed compared in other structures of class A GPCR-Gi complexes, suggesting heterogeneity in the processes of G protein-coupled receptors (GPCRs) activation and G protein coupling.

Prototypic 18F-Labeled Argininamide-Type Neuropeptide Y Y1R Antagonists as Tracers for PET Imaging of Mammary Carcinoma.

The neuropeptide Y (NPY) Y1 receptor (Y1R) selective radioligand (R)-Nα-(2,2-diphenylacetyl)-Nω-[4-(2-[18F]fluoropropanoylamino)butyl]aminocarbonyl-N-(4-hydroxybenzyl)argininamide ([18F]23), derived from the high-affinity Y1R antagonist BIBP3226, was developed for imaging studies of Y1R-positive tumors. Starting from the argininamide core bearing amine-functionalized spacer moieties, a series of fluoropropanoylated and fluorobenzoylated derivatives was synthesized and studied for Y1R affinity. The fluoropropanoylated derivative 23 displayed high affinity (Ki = 1.3 nM) and selectivity toward Y1R. Radiosynthesis was accomplished via 18F-fluoropropanoylation, yielding [18F]23 with excellent stability in mice; however, the biodistribution study revealed pronounced hepatobiliary clearance with high accumulation in the gall bladder (>100 %ID/g). Despite the unfavorable biodistribution, [18F]23 was successfully used for imaging of Y1R positive MCF-7 tumors in nude mice. Therefore, we suggest [18F]23 as a lead for the design of PET ligands with optimized physicochemical properties resulting in more favorable biodistribution and higher Y1R-dependent enrichment in mammary carcinoma.

Influence of different chelators on the radiochemical properties of a 68-Gallium labelled bombesin analogue.

UNLABELLED: The radiolabelled bombesin analogue AMBA shows high potential for diagnosis and treatment of prostate and breast cancer, but the influence of different chelators, which differ in terms of radiochemical reactivity and stability, have not been explored so far. In order to find the best suitable chelator for labelling of AMBA, we synthesized AMBA analogues linked to the most commonly used chelators DOTA, NOTA and NODAGA and compared their reactivity and stability after labelling with 68-Gallium. METHODS: For the synthesis of DO3A-, NO2A- and NODAGA-AMBA, a solid-phase synthesis approach was used. The influence of concentration, pH and temperature on the radiolabelling was analysed. The in vitro stability of all complexes in saline, human serum, human whole blood and against transchelation and transmetallation was analysed. RESULTS: The peptides were synthesised in high yield and purity. Purity and identity of products and impurities were confirmed using UHPLC coupled to ESI-MS. Radiolabelling of these peptides was optimal at elevated temperature, although room temperature labelling was reported previously for NOTA and NODAGA chelators. The highest reactivity was observed for NODAGA-AMBA. On preparation of NO2A-AMBA, the formation of a by-product was detected with HPLC. More detailed analysis revealed the formation of an isomer with the same mass to charge ratio which led to the conclusion that a coordination isomer was formed. All complexes showed high stability in saline, human serum or when challenged with DTPA, transferrin and varying metals (Fe(3+), Cu(2+), Zn(2+)). Conversely, the stability in human blood was low, and varying metabolites were detected and identified by ESI-MS. CONCLUSION: All three precursors are available in high yields suitable for routine production. NODAGA-AMBA showed the most favoured features when labelled with 68-gallium, but a further comparison in vivo should be performed in order to confirm the superior features found in vitro.

Functional reconstitution of human neuropeptide Y (NPY) Y(2) and Y(4) receptors in Sf9 insect cells.

The four functionally expressed human neuropeptide Y receptor subtypes (hY(1)R, hY(2)R, hY(4)R, hY(5)R) belong to class A of the G-protein-coupled receptors (GPCRs) and interact with pertussis toxin-sensitive G(i/o)-proteins. The number of small molecules described as ligands for hY(1)R and hY(5)R exceeds by far those for hY(2)R. Potent non-peptidergic ligands for the hY(4)R are not available so far. Here, we report on the functional reconstitution of the hY(2)R and the hY(4)R in Sf9 insect cells using the baculovirus system. Sf9 cells were genetically engineered by infection with up to four different baculoviruses, combining the receptors with G-proteins of the G(i/o) family and regulators of G-protein signaling (RGS) proteins to improve signal-to-noise ratio. In steady-state GTPase assays, using pNPY (Y(2)) and hPP (Y(4)), the GPCRs coupled to various G(i)/G(o)-proteins and both, RGS4 and GAIP, enhanced the signals. Co-expression systems hY(2)R + G?(i2) and hY(4)R + G?(i2)/G?(o) + RGS4, combined with G?(1)?(2), yielded best signal-to-noise ratios. hY(2)R function was validated using both agonistic peptides (NPY, PYY, NPY(13?36)) and selective non-peptidergic antagonists (BIIE0246 and derivatives), whereas the hY(4)R model was characterized with peptidergic agonists (PP, NPY, GW1229, and BW1911U90). Tunicamycin inhibited receptor N-glycosylation diminished NPY signals at hY(2)R and abolished hY(4)R function. Investigations with monovalent salts showed sensitivity of hY(4)R toward Na(+), revealing moderate constitutive activity. After validation, an acylguanidine (UR-PI284) was identified as a weak non-peptide Y(4)R antagonist. In summary, the established steady-state GTPase assays provide sensitive test systems for the characterization of Y(2) and Y(4) receptor ligands.

Application of the guanidine-acylguanidine bioisosteric approach to argininamide-type NPY Y2 receptor antagonists.

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.

Synthesis and characterization of the first fluorescent nonpeptide NPY Y1 receptor antagonist.

Cyanine-5-labelled neuropeptide Y (NPY) was demonstrated to be an ideal universal fluorescent ligand for the combined investigation of NPY Y(1), Y(2) and Y(5) receptors. With respect to improved stability, detection of receptor subtypes in cells and tissues, and prevention of receptor internalization, small nonpeptidic fluorescent antagonists should be superior. Here we present a set of four fluorescent nonpeptide NPY Y(1) receptor (Y(1)R) antagonists. The highest affinity was obtained by labelling an N(G)-(6-aminohexanoyl)argininamide derived from the Y(1)R antagonist BIBP 3226, with Py-1, a small pyrylium dye. The fluorescent pyridinium-type Y(1)R antagonist, compound 4 had K(i) values of 29 nM and 2.7 nM, which were determined by radioligand binding and flow cytometry under equilibrium conditions, respectively; 4 had a K(b) value of 0.6 nM (Ca(2+) assay). The large Stoke's shift (541 vs. 615 nm) in buffer (PBS, pH 7.4) in the presence of 1% BSA and the red emission (quantum yield 56%) are advantageous with respect to the signal-to-noise ratio. The new probe was successfully used in fluorescence-based binding experiments evaluated by flow cytometry and confocal microscopy; this demonstrates the potential of pyrylium dyes for the preparation of fluorescent ligands that are applicable for the study of G protein-coupled receptors on living cells.

Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands.

With respect to the discovery and characterization of neuropeptide Y(2) receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY(2) receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gq(i5) enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration-response curves of porcine neuropeptide Y in the presence of the Y(2)-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y(2) receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.