In Search of NPY Y₄R Antagonists: Incorporation of Carbamoylated Arginine, Aza-Amino Acids, or d-Amino Acids into Oligopeptides Derived from the C-Termini of the Endogenous Agonists

Abstract

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.

Highly Selective Y4 Receptor Antagonist Binds in an Allosteric Binding Pocket

“…Aiming at the development of small-molecule probes to selectively target the human Y 4 R, approximately 77 000 compounds were tested by high-throughput screening (HTS). We recently reported on the discovery of Y 4 R positive allosteric modulators (PAM) from this experiment. , In comparison to PAMs and agonists, the hit rate of negative allosteric modulators (NAM) and antagonists was very low (0.022%, Table S1, Supporting Information), reflecting the challenges in the development of Y 4 R antagonists. ,, …”

An Alkyne-functionalized Arginine for Solid-Phase Synthesis Enabling “Bioorthogonal” Peptide Conjugation

“… a Determined by competition binding with [ 3 H] 4 at intact HT-29 colon carcinoma cells. b Determined by competition binding with [ 3 H] 6 at intact CHO-hY 4 R-mtAEQ-G qi5 cells. Presented are mean p K i values ± SEM and corresponding mean K i values from three ( 19 , 20 , 27 , and 28 ) or four ( 21 and 22 ) independent experiments (performed in triplicate). c K i value reported by Schindler et al d K i value reported by Kuhn et al n.a. : not applicable. …”

“…Recently, we reported on a labeling strategy for peptidic receptor ligands based on the N ω -carbamoylated amino-functionalized arginine building blocks 1 and 2 (Figure ), which were incorporated into peptide ligands of G-protein coupled receptors (GPCRs) of different GPCR families. , Unlike recently reported N ω -alkylated arginines, also tolerated in biologically active peptides, the incorporation of 1 and 2 into peptides is feasible by standard Fmoc-strategy solid-phase peptide synthesis (SPPS), leading to amino-functionalized peptides, which are suited, e.g., for conjugation with amino-reactive fluorescent dyes or radionuclide-bearing moieties. , Two examples of radiolabeled peptidic receptor ligands ([ 3 H] 4 and [ 3 H] 6 ), obtained from the parent peptides 3 and 5 by replacement of arginine with an N ω -carbamoylated arginine derived from 1 , are shown in Figure . The radioligands [ 3 H] 4 and [ 3 H] 6 proved to be useful molecular tools. ,,− It should be noted that the weakly basic (p K a : 7–8) carbamoylguanidine moiety of N ω -carbamoylated arginines exhibits, in contrast to N -acylated guanidines, high chemical stability . Peptide conjugation or labeling based on amino-functionalized N ω -carbamoylated arginines, derived from arginine derivatives such as 1 or 2 (cf.…”

“…Aim of the present study: synthesis of the alkyne-functionalized, SPPS-compatible arginine building block 7 and incorporation of 7 into peptide ligands ( 8 – 11 ) of different GPCRs ( 8 , 9 , NTS 1 R; 10 , NPY Y 4 R; 11 , CXCR4). Note: 8 and 9 represent analogs of neurotensin(8–13) (NT(8–13), 3 ), which corresponds to the C-terminal hexapeptide sequence of the tridecapeptide neurotensin, the physiological agonist of neurotensin receptors.…”

“…The use of NMM as allyl scavenger in combination with acetic acid was reported, e.g., by Horne et al 17 Here, for the deprotection of 18 by the use of Pd(PPh 3 ) 4 and NMM, acetic acid was omitted. In low (8−11) of different GPCRs (8, 9, NTS 1 R; 6 10, NPY Y 4 R; 4 11, CXCR4 10 ). Note: 8 and 9 represent analogs of neurotensin(8−13) (NT(8−13), 3), which corresponds to the C-terminal hexapeptide sequence of the tridecapeptide neurotensin, the physiological agonist of neurotensin receptors.…”