The diastereomeric mixture of d/l-2,7-diaminooctanedioyl-bis(YRLRY-NH2) (BVD-74D, 2) was described in the literature as a high affinity Y4 receptor agonist. Here we report on the synthesis and pharmacological characterization of the pure diastereomers (2R,7R)- and (2S,7S)-2 and a series of homo- and heterodimeric analogues in which octanedioic acid was used as an achiral linker. To investigate the role of the Arg residues, one or two arginines were replaced by Ala. Moreover, N(ω)-(6-aminohexylaminocarbonyl)Arg was introduced as an arginine replacement (17). (2R,7R)-2 was superior to (2S,7S)-2 in binding and functional cellular assays and equipotent with 17. [(3)H]Propionylation of one amino group in the linker of (2R,7R)-2 or at the primary amino group in 17 resulted in high affinity Y4R radioligands ([(3)H]-(2R,7R)-10, [(3)H]18) with subnanomolar Kd values.

Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

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 cross-linked pentapeptides (2R,7R)-diaminooctanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) ((2R,7R)-BVD-74D, (2R,7R)-1) and octanedioyl-bis(Tyr-Arg-Leu-Arg-Tyr-amide) (2) as well as the pentapeptide Ac-Tyr-Arg-Leu-Arg-Tyr-amide (3) were previously described as neuropeptide Y Y4 receptor (Y4R) partial agonists. Here, we report on a series of analogues of (2R,7R)-1 and 2 in which Arg2, Leu3, or Arg4 were replaced by the respective aza-amino acids. The replacement of Arg2 in 3 with a carbamoylated arginine building block and the extension of the N-terminus by an additional arginine led to the high-affinity hexapeptide Ac-Arg-Tyr-Nω-[(4-aminobutyl)aminocarbonyl]Arg-Leu-Arg-Tyr-amide (35), which was used as a precursor for a d-amino acid scan. The target compounds were investigated for Y4R functional activity in assays with complementary readouts: aequorin Ca2+ and β-arrestin 1 or β-arrestin 2 assays. In contrast to the parent compounds, which are Y4R agonists, several ligands were able to suppress the effect elicited by the endogenous ligand pancreatic polypeptide and therefore represent a novel class of peptide Y4R antagonists.

The neuropeptide Y (NPY) Y receptor (YR) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e., radiolabeled and fluorescent ligands, for the investigation of the YR were reported. Previously, [Lys]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the ε-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys]hPP by norleucine and prepared the corresponding [H]propionylated ([H]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y, Y, Y, and Y receptors) and functional (luciferase gene reporter, beta-arrestin-1,2) YR assays. Both molecular probes proved to be useful in radiochemical and flow cytometric saturation and competition YR binding experiments. Most strikingly, there was a different influence of the composition of buffer on equilibrium binding and kinetics: [H]12 affinity (K in Na-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and YR-mediated internalization of 13 (K in Na-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [H]12 and 13 revealed a tendency to higher apparent affinities for a set of reference peptides in hypotonic (Na-free) compared to isotonic buffers. The differences were negligible in the case of hPP but up to 270-fold in the case of GW1229 (GR231118). By contrast, no relevant influence of Na on YR affinity became obvious, when the radioligands [H]12 and [H]propionyl-pNPY were investigated in saturation binding and competition binding.